Brendan Evano

Gilles Le Carrou

Geneviève Almouzni

Shahragim Tajbakhsh
email: shahragim.tajbakhsh@pasteur.fr.

Differential cell fates of muscle stem cells are accompanied by symmetric segregation of canonical H3 histones in vivo

Stem cells are maintained through symmetric or asymmetric cell divisions. While various mechanisms initiate asymmetric cell fates during mitosis, possible epigenetic control of this process has emerged recently. The asymmetrical distribution of a canonical histone H3 variant during mitosis in fly germline has suggested a role for partitioning old and new nucleosomes in asymmetric cell fates. Here, we provide resources for single cell assays and show the asymmetric segregation of transcription factors along with old and new DNA in mouse muscle stem cells ex vivo and in vivo. However, these differential fate outcomes contrast with a symmetric distribution of the canonical H3.1 vertebrate variant. These findings point to different evolutionary mechanisms operating in fly germline stem cells and vertebrate somatic stem cells to mitigate epigenetic regulation of asymmetric cell fates.

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A long-standing question in multicellular organisms is how epigenetic information is transmitted or changed through cell division. The nucleosome is the basic unit of chromatin and notably contributes to gene expression regulation through its composition (histone variants and modifications). It is therefore particularly critical to determine how, and where in the genome, the choice of histone variants, their turnover and post-translational modifications contribute to the maintenance or switch of a particular cell fate.

Histone dynamics has been addressed with a variety of approaches, on bulk chromatin or at specific loci. Stable isotope labeling followed by mass spectrometry analysis notably revealed differences in turnover rates of the histone H3 variants H3.1 and H3.3 [START_REF] Xu | Partitioning of histone H3-H4 tetramers during DNA replicationdependent chromatin assembly[END_REF] . Fluorescent tags were used to assess global incorporation or inheritance patterns of H3 variants in D. melanogaster [START_REF] Ahmad | The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly[END_REF][START_REF] Tran | Asymmetric division of Drosophila male germline stem cell shows asymmetric histone distribution[END_REF] . Additionally, photo-bleaching, photo-activation and photo-conversion have been used to measure histone dynamics on short timescales [START_REF] Kimura | Kinetics of core histones in living human cells: little exchange of H3 and H4 and some rapid exchange of H2B[END_REF] . Complementary approaches were developed to measure locus-specific histone dynamics, by sequential expression of tagged histones followed by chromatin immunoprecipitation and DNA sequencing [START_REF] Dion | Dynamics of Replication-Independent Histone Turnover in Budding Yeast[END_REF][START_REF] Verzijlbergen | Recombination-induced tag exchange to track old and new proteins[END_REF] , which have poor temporal resolution, or by metabolic labeling of histone proteins, which has a better resolution but does not distinguish between different histone variants [START_REF] Deal | Genome-Wide Kinetics of Nucleosome Turnover Determined by Metabolic Labeling of Histones[END_REF] . Although these methods provided significant insights into chromatin dynamics, they suffer from specific downsides, from lack of positional information to delays in expression of inducible reporters.

In addition, current knowledge about histone dynamics arises predominantly from in vitro models where cell fate cannot be manipulated.

Histone H3 variants have a major role in epigenetic maintenance during development and disease [START_REF] Filipescu | Histone H3 Variants and Their Chaperones During Development and Disease: Contributing to Epigenetic Control[END_REF][START_REF] Yadav | Chromatin plasticity: A versatile landscape that underlies cell fate and identity[END_REF] , but tools to measure histone dynamics in vivo either globally or at specific loci are lacking. Here, we sought to investigate histone H3 variants dynamics in vivo in mouse, using SNAP tags as reporters and skeletal muscle stem cells as a model of changes in cell fates. SNAP tags react specifically and irreversibly with benzylguanine derivatives [START_REF] Keppler | A general method for the covalent labeling of fusion proteins with small molecules in vivo[END_REF] . A number of cell-permeable SNAP substrates are available (fluorescent, biotinylated or amenable to custom-probe synthesis through benzylguanine building blocks), with low toxicity and allowing fast and quantitative labeling [START_REF] Hinner | How to obtain labeled proteins and what to do with them[END_REF] . SNAP tags were reported to work efficiently and specifically in vivo in mice [START_REF] Yang | Genetic targeting of chemical indicators in vivo[END_REF] , and proved to be superior to classical fluorescent proteins in terms of spectral flexibility, signal-to-noise fluorescence and resistance to histological sample preparations [START_REF] Yang | Genetic targeting of chemical indicators in vivo[END_REF][START_REF] Kohl | Ultrafast tissue staining with chemical tags[END_REF] . Further, SNAP tags allow the development of functionalities via synthetic substrates that cannot be achieved with classical genetically encoded reporters [START_REF] Keppler | Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro[END_REF] . Finally, SNAP tags have been used extensively to measure histone H3 variants dynamics on bulk chromatin [START_REF] Bodor | Assembly in G1 phase and long-term stability are unique intrinsic features of CENP-A nucleosomes[END_REF][START_REF] Ray-Gallet | Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity[END_REF][START_REF] Adam | Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA[END_REF][START_REF] Clément | High-resolution visualization of H3 variants during replication reveals their controlled recycling[END_REF] , and were adapted recently to measure specific histone variants turnover genome-wide [START_REF] Deaton | Enhancer regions show high histone H3.3 turnover that changes during differentiation[END_REF] .

In most tissues, stem cells self-renew and give rise to daughter cells committed to differentiation, thereby ensuring both tissue homeostasis and maintenance of the stem cell pool [START_REF] Morrison | Asymmetric and symmetric stem-cell divisions in development and cancer[END_REF] . This can be achieved through different modes of cell division, either symmetric (Symmetric Cell Division, SCD), self-renewing or differentiating, or asymmetric (Asymmetric Cell Division, ACD) divisions. Fine-tuning the balance between these different modes is critical for proper tissue development, homeostasis and regeneration, and to prevent tumorigenesis [START_REF] Morrison | Asymmetric and symmetric stem-cell divisions in development and cancer[END_REF] .

A variety of subcellular constituents, including transcripts, organelles, centrosomes or sister chromatids can distribute asymmetrically between daughter cells during cell division [START_REF] Knoblich | Mechanisms of asymmetric stem cell division[END_REF] .

A major challenge is to understand how their biased segregation dictates asymmetric cell fates [START_REF] Tajbakhsh | Biased segregation of DNA and centrosomes: moving together or drifting apart?[END_REF] . The hypothesis of a role for replication-coupled nucleosome assembly in determining asymmetric cell fates in C. elegans nervous system has raised much interest [START_REF] Nakano | Replication-coupled chromatin assembly generates a neuronal bilateral asymmetry in C. elegans[END_REF] . Furthermore, recent studies reported non-equivalent inheritance of specific histone variants during ACD of D. melanogaster male germline stem cells (GSCs) [START_REF] Tran | Asymmetric division of Drosophila male germline stem cell shows asymmetric histone distribution[END_REF][START_REF] Xie | Histone H3 threonine phosphorylation regulates asymmetric histone inheritance in the Drosophila male germline[END_REF] . These findings along with the importance of chromatin in cell plasticity [START_REF] Yadav | Chromatin plasticity: A versatile landscape that underlies cell fate and identity[END_REF] raised the possibility of an epigenetic mechanism for maintaining the identity of self-renewed stem cells while resetting the identity of their differentiating sibling cells.

Here we sought to investigate whether the non-equivalent inheritance of specific histone variants is conserved in mammals, using mouse skeletal muscle stem cells as a model.

Adult skeletal muscle satellite (stem) cells are mostly quiescent during homeostasis.

Following muscle damage, they proliferate and differentiate into muscle fibres by cell-cell fusion [START_REF] Reznik | Thymidine-3H uptake by satellite cells of regenerating skeletal muscle[END_REF][START_REF] Gayraud-Morel | Skeletal muscle as a paradigm for regenerative biology and medicine[END_REF] . A fraction of the population self-renews and reconstitutes a de novo satellite cell pool. This is accompanied by a temporal expression of cell fate markers, such as the transcription factors Pax7 (stem), Myod (commitment) and Myogenin (differentiation).

Satellite cells can divide symmetrically and asymmetrically during muscle regeneration [START_REF] Kuang | Asymmetric self-renewal and commitment of satellite stem cells in muscle[END_REF][START_REF] Rocheteau | CC-BY-NC-ND 4.0 International license It is made available under a ([END_REF][START_REF] Yennek | Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells[END_REF] , which is critical for proper muscle regeneration and maintenance of the satellite cell pool [START_REF] Kuang | Asymmetric self-renewal and commitment of satellite stem cells in muscle[END_REF][START_REF] Lukjanenko | Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice[END_REF][START_REF] Dumont | Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division[END_REF] .

Results

To address whether the mechanism uncovered in Drosophila GSCs operates in a somatic mammalian stem cell, we developed a pulse-chase approach to follow histone variants using SNAP tags. SNAP-tagged histones have been used for addressing human H3.1 and H3.3 turnover during DNA replication and repair [START_REF] Ray-Gallet | Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity[END_REF][START_REF] Adam | Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA[END_REF][START_REF] Clément | High-resolution visualization of H3 variants during replication reveals their controlled recycling[END_REF] . The asymmetric inheritance of H3, but not H3.3, reported in D. melanogaster GSCs 3 prompted us to generate mouse reporter lines expressing SNAP-tagged H3.1 and H3.3 to follow their inheritance patterns during ACD of satellite cells.

H3.1-SNAP and H3.3-SNAP transgenes were expressed in several tissues (Supplementary Fig. 1a), at low levels (< 20%) compared to endogenous histone expression (Supplementary Fig. 1b). SNAP labeling of embryonic fibroblasts derived from reporter mice showed ubiquitous H3.1-SNAP expression, whereas H3.3-SNAP expression was lower and variegated (data not shown). Furthermore, as reported [START_REF] Ray-Gallet | Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity[END_REF] we find that newly deposited H3.1-SNAP histones overlapped with sites of DNA replication, while newly deposited H3.3-SNAP histones were uncoupled from DNA replication (Supplementary Fig. 1c). These results indicate that the Tg:H3.1-SNAP and Tg:H3.3-SNAP lines are faithful reporters of H3.1 and H3.3 turnover in mouse primary cells.

To examine the distribution of parental H3.1 histones in satellite cells, adult Tibialis anterior (TA) muscles were injured to induce satellite cell activation and muscle regeneration. Parental H3.1 histones were SNAP-labeled prior to satellite cell isolation (labeling efficiency ≈ 80%, Fig. 1a and Supplementary Fig. 1d). Satellite cells were isolated from compound transgenic Tg:Pax7-nGFP [START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF] and Tg:H3.1-SNAP mice by fluorescence-activated cell sorting (FACS) where GFP intensity can be used to fractionate subpopulations with distinct properties, such as Pax7-nGFP Hi cells previously reported to divide asymmetrically 5 days post-injury (dpi) [START_REF] Rocheteau | CC-BY-NC-ND 4.0 International license It is made available under a ([END_REF] . In vivo activated/SNAP-labeled Pax7-nGFP Hi cells were isolated by FACS and the ongoing cell division was monitored on micropatterns (Fig. 1a). Micropatterns provide a controlled microenvironment that allows confinement of single cells and their progeny [START_REF] Théry | The extracellular matrix guides the orientation of the cell division axis[END_REF] . We and others have shown that their geometry impacts on the frequency of ACD of stem cells [START_REF] Yennek | Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells[END_REF][START_REF] Freida | Human bone marrow mesenchymal stem cells regulate biased DNA segregation in response to cell adhesion asymmetry[END_REF] . If the parental histones are partitioned equally following mitosis, the fluorescent SNAP signal will be inherited equally between daughter cells. However, if the parental histones are inherited asymmetrically, the fluorescent SNAP signal will be detected only in a subset of daughter cells (Fig. 1b).

Modes of cell division were scored based on absolute presence or absence of cell fate markers. Their expression level and SNAP signal intensity were further measured quantitatively. We observed self-renewing and differentiating (Fig. 1c left and middle) SCDs, in addition to ACDs (Fig. 1c right), as reported [START_REF] Rocheteau | CC-BY-NC-ND 4.0 International license It is made available under a ([END_REF][START_REF] Yennek | Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells[END_REF] . Interestingly, we observed clones containing 4 cells with two self-renewed cells (Pax7 + ) and two differentiated cells (Myog + ), indicating a switch from self-renewing SCD to ACD (Supplementary Fig. 1e). Among all observed clones, the SNAP signal intensity was equivalent between daughter cells, irrespective of their mode of cell division (Fig. 1c). The maximal SNAP intensity difference between daughter cells was less than 8% (Fig. 1c left), while transcription factors displayed differences in expression up to 80% between daughter cells in case of ACD (Fig. 1c right).

These results indicate that parental H3.1 histones were essentially inherited symmetrically during both SCD and ACD of satellite cells.

H3-Thr3 phosphorylation (H3T3 P ) was reported to distinguish pre-existing and newlydeposited H3 in prophase, and to be required for asymmetric inheritance of old vs. new histones in D. melanogaster GSCs 24 . To further address the issue of histone inheritance, we investigated the distribution of H3T3 P in in vivo activated/SNAP-labeled Pax7-nGFP Hi cells on micropatterns. We readily detected H3T3 P signals in mitotic cells, colocalizing with SNAP signals yet not restricted to a subset of chromatids in early mitosis (Fig. 1d). These observations indicate that H3T3 P did not point to a differential distribution of old vs. new histones in early mitosis of satellite cells. Within adult muscles, satellite cells reside in a specific niche and a complex balance between extrinsic and intrinsic cues is required for proper satellite cell proliferation and cell fate determination [START_REF] Dumont | Intrinsic and extrinsic mechanisms regulating satellite cell function[END_REF] . As with other stem cell niches [START_REF] Voog | Stem cells and the niche: a dynamic duo[END_REF] , the composition of the satellite cell microenvironment is dynamic during muscle regeneration [START_REF] Bentzinger | Cellular dynamics in the muscle satellite cell niche[END_REF] , raising the possibility that some niche factors that cannot be modelled ex vivo might drive ACDs. Although ACDs have been reported extensively ex vivo, to the best of our knowledge, only one study has demonstrated asymmetry involving transcription factors in a vertebrate model system in vivo [START_REF] Kim | Cell cycle-dependent differentiation dynamics balances growth and endocrine differentiation in the pancreas[END_REF] .

Therefore, we established a clonal tracing strategy to examine two-cell clones in regenerating muscles in vivo (Fig. 2a and Fig. 2b), together with SNAP-labeling of parental H3.1 histones (in vivo labeling efficiency ≈ 60%, Supplementary Fig. 2b). As previously, modes of cell division were scored based on absolute presence or absence of cell fate markers.

Their expression level and SNAP signal intensity were further measured quantitatively. We observed self-renewing (66.7%, n=98 of 147 cell pairs, Fig. 2c Notably, the relative frequencies of the different modes of cell division observed in vivo correspond to those on micropatterns ex vivo [START_REF] Yennek | Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells[END_REF] . Among all clones analysed, we observed an equivalent SNAP signal between daughter cells (Fig. 2d). Interestingly, a difference in SNAP signal intensity (up to 40%) between daughter cells was noted for a limited number (≈ 5%) of divisions (Fig. 2d left and right, Fig. 2g right). However, the differences in SNAP intensity were lower than the observed differences in expression of cell fate markers, resulting from fluctuations in expression in case of SCD (Fig. 2d left) or asymmetric fate decisions (Fig. 2d right). Further, we did not detect examples showing exclusive distribution of parental histones to one daughter. These results point to a symmetric inheritance of parental H3.1 histones, irrespective of the mode of cell division in vivo. Notably, differences in SNAP signals between daughter cells were not observed ex vivo on micropatterns (Fig. 1c), suggesting variable H3.1 turnover rates between daughter cells in vivo and ex vivo. These observations highlight the importance of addressing epigenetic identity and plasticity in vivo. Finally, the SNAP signal was readily detected after histological preparation (strong fixation and heat-induced epitope retrieval), while the mGFP fluorescence was lost and required immunodetection, confirming the superiority of synthetic SNAP fluorophores over classical fluorescent proteins.

The asymmetric segregation of stem (Pax7) and differentiation (Myogenin) transcription factors was previously reported to correlate in part with non-random DNA segregation (NRDS) ex vivo, where old DNA strands are inherited by the stem cell and new DNA strands by the committed cell [START_REF] Rocheteau | CC-BY-NC-ND 4.0 International license It is made available under a ([END_REF][START_REF] Yennek | Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells[END_REF] . NRDS has been hypothesized to be a consequence of epigenetic mechanisms, possibly involving distribution of old and new histones or nucleosome pools [START_REF] Tajbakhsh | Biased segregation of DNA and centrosomes: moving together or drifting apart?[END_REF] ; however NRDS has not been shown robustly in vivo largely due to technical challenges. To examine parental H3.1 histone distribution and NRDS in vivo, we adopted the same strategy as above, together with a 5-ethynyl-2'-deoxyuridine (EdU) pulsechase labeling (Fig. 2a and Fig. 2e previously ex vivo where parental DNA strands are labeled and inherited by the Pax7expressing cell [START_REF] Rocheteau | CC-BY-NC-ND 4.0 International license It is made available under a ([END_REF][START_REF] Yennek | Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells[END_REF] . Among all the clones analysed, the SNAP signal was equivalent between daughter cells (Fig. 2g), indicating a symmetric parental H3.1 inheritance, independent of the mode of DNA segregation.

Discussion

Here we demonstrate that SNAP tags are powerful tools to investigate histone dynamics in mice in vivo. Our results indicate that parental H3.1 histones are equally inherited in the vast majority of dividing mouse satellite cells, ex vivo as in vivo, during asymmetric cell fate decisions with defined stem cell and differentiated cell transcription factors, as well as non-random segregation of DNA. Asymmetric histone H3 inheritance has uniquely been reported in D. melanogaster GSCs to date and was correlated with the execution of different cell fate programs following cell division [START_REF] Tran | Asymmetric division of Drosophila male germline stem cell shows asymmetric histone distribution[END_REF][START_REF] Xie | Histone H3 threonine phosphorylation regulates asymmetric histone inheritance in the Drosophila male germline[END_REF] . A two-step mechanism was proposed where old and new histones are incorporated into different sister chromatids during S phase, followed by differential recognition and segregation of old and new sister chromatids by the mitotic apparatus [START_REF] Tran | Asymmetric division of Drosophila male germline stem cell shows asymmetric histone distribution[END_REF][START_REF] Xie | Histone H3 threonine phosphorylation regulates asymmetric histone inheritance in the Drosophila male germline[END_REF] . This requires both an asymmetric distribution of histones at the replication fork, and strictly unidirectional DNA replication throughout the genome. Recently, two reports highlighted that while histone partitioning in the wake of the replication fork is inherently asymmetric between leading and lagging DNA strands, components of the replication machinery compensate to ensure an equal distribution of parental histones between sister chromatids [START_REF] Yu | A mechanism for preventing asymmetric histone segregation onto replicating DNA strands[END_REF][START_REF] Petryk | MCM2 promotes symmetric inheritance of modified histones during DNA replication[END_REF] . Asymmetric histone distribution in Drosophila GSCs might then result from retention of parental histones on the leading strand and preferential unidirectional DNA replication [START_REF] Wooten | Asymmetric Histone Incorporation During DNA Replication in Drosophila Male Germline Stem Cells[END_REF] . While our results address global inheritance of parental histones and cannot exclude local limited asymmetry, notably at the replication fork, the discrepancy observed between D. melanogaster GSCs and our analysis of mouse satellite cells might indicate that the molecular mechanisms of asymmetric histone inheritance are not generally conserved through evolution, or alternatively between germline and somatic stem cells.

Our results show unambiguously for the first time NRDS in vivo and indicate that this process is not driven by a global asymmetric inheritance of old and new pools of H3.1.

Nevertheless, according to the silent sister hypothesis [START_REF] Lansdorp | Immortal strands? Give me a break[END_REF] , epigenetic differences might exist between sister chromatids due to their relative difference in age. Asymmetric recognition of centromeres would then be the basis for NRDS, which would in turn lead to asymmetric gene expression and cell fate decisions. In this context, it is interesting to note that CENP-A, the centromeric H3 variant, was shown to be inherited asymmetrically during stem cell divisions in the fly midguts [START_REF] Del Arco | In vivo analysis of centromeric proteins reveals a stem cell-specific asymmetry and an essential role in differentiated, non-proliferating cells[END_REF] , a possibility to explore in the future.

Finally, while our study investigated inheritance of bulk histones, the tools and methodologies reported here, combined with the recent advent of time-ChIP [START_REF] Deaton | Enhancer regions show high histone H3.3 turnover that changes during differentiation[END_REF][START_REF] Siwek | time-ChIP: A Method to Determine Long-Term Locus-Specific Nucleosome Inheritance[END_REF] , will allow genome-wide tracking of histone variant turnover rates. This can be done in a wide variety of mouse primary cell types in their in vivo environment thereby facilitating investigations of how epigenetic identity is maintained in stem cells, and remodeled in their differentiating progeny.

Methods

Mouse strains

Tg:Pax7-nGFP [START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF] , Pax7 CreERT2 45 and R26 mTmG 46 were described previously. Breeding was performed on an F1:C57BL6/DBA2 background. 6-8 weeks-old male littermates were used in this study, heterozygous for each allele used. Animals were handled according to national and European community guidelines, and protocols were approved by the ethics committee at Institut Pasteur.

Cell lines and culture conditions

Primary fibroblasts were derived at E13. 1.

RT-qPCR

Organs were homogenized in TRIzol (ThermoFisher) and RNA extracted following manufacturer's instructions. gDNA was eliminated with Ambion DNAseI (ThermoFisher) and reverse transcription performed with SuperScript III Reverse Transcriptase (ThermoFisher). RNA was eliminated with RNAseH endonuclease (Roche) for 20 min at 37°C. mRNAs level was assessed with SYBR green master mix (Roche) and analysis was performed using the 2 -ΔCt method [START_REF] Livak | Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCT method[END_REF] . RT-qPCR analyses have been normalized with Tbp.

Specific forward and reverse primers used for RT-qPCR are listed in Supplementary Table 1.

Western blot

Histones were extracted following [START_REF] Rumbaugh | Epigenetic changes in the brain: measuring global histone modifications[END_REF] and protein concentration measured with the Pierce BCA Protein Assay Kit (ThermoFisher). 1 µg of histones was run on 4-12% Bis-Tris Gel NuPAGE (ThermoFisher) in NuPAGE MES SDS Running Buffer (ThermoFisher) and transferred on a nitrocellulose membrane (Hybond ECL, Sigma) in 1X Tris-Glycine-SDS buffer (Euromedex) with 20% ethanol. Equivalent loading was evaluated from Ponceau staining of the membrane. The membrane was then blocked with 5% non-fat milk in Tris Buffer Saline (TBS) Tween 0.05% (TBS-T) for 20 min at RT and probed with specific primary antibodies overnight at 4°C in TBS-T 5% milk. After three washes in TBS-T, the membrane was incubated with HRP-conjugated secondary antibodies in TBS-T for 30 min at RT, washed three times in TBS-T, and revealed by chemiluminescence (Pierce ECL Plus Western Blotting Substrate, ThermoFisher) with autoradiography films (Hyperfilm ECL, GE Healthcare).

SNAP labeling

In vitro SNAP quenching (Supplementary Fig. 1c) was performed with 10 µM SNAP Cell Block (New England Biolabs) for 30 min at 37°C, followed by three washes with culture medium. In vitro SNAP labeling was performed with 3 µM SNAP Cell TMR Star (New England Biolabs) in culture medium for 20 min at 37°C, followed by three washes with culture medium, and washed in culture medium for 30 min at 37°C. Sites of DNA replication were labeled with 1 µM EdU (ThermoFisher) for 10 min at 37°C. Soluble histones were extracted with CSK buffer prior to fixation as in [START_REF] Ray-Gallet | Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity[END_REF] . EdU incorporation was detected with the Click-iT Plus Alexa Fluor 488 Imaging Kit (ThermoFisher) as recommended.

Ex vivo SNAP labeling (Fig. 1a) was performed by incubating dissected and minced TA muscles in 1 ml satellite cell medium containing 3 µM SNAP Cell SiR 647 (New England Biolabs, S9102S) for 30 min at 37°C with gentle agitation. Muscles were centrifuged at 500g for 5 min, supernatant was discarded and muscles resuspended in 1 ml DMEM. Muscles were centrifuged at 500g for 5 min, supernatant was discarded and muscles resuspended in collagenase D/tryspin mix for satellite cell isolation (see below).

In vivo SNAP labeling was performed by injecting 10 µl of 30 µM SNAP Cell SiR 647 (New England Biolabs) in NaCl 0.9% per TA at 4 dpi under isofluorane anesthesia, 8 h before tamoxifen administration (Fig. 2a). In vivo H3.1-SNAP labeling at 4 dpi enables specific labeling of ≈ 60% of satellite cells as early as 2 h post SNAP substrate injection (Supplementary Fig. 2b).

Muscle injury, tamoxifen and EdU delivery

Muscle injury was done as described previously [START_REF] Gayraud-Morel | A role for the myogenic determination gene Myf5 in adult regenerative myogenesis[END_REF] . Briefly, mice were anesthetized with 0.5% Imalgene/2% Rompun. The Tibialis anterior (TA) muscle was injected with 15 µl of 10 µM notexin (Latoxan) in NaCl 0.9%. Tamoxifen (Sigma) was reconstituted at 25 mg/ml in corn oil/ethanol (5%) and stored at -20°C. Before use, tamoxifen was diluted at appropriate concentration (1 mg/ml or 0.25 mg/ml) with corn oil, and administered (8 µl per g of mouse) by intragastric injection at 4 days post-injury and 14 h prior to sacrifice to allow for a maximum of two consecutive cell divisions in vivo (in vivo satellite cells doubling time about 8 h 28 ). We optimized conditions for clonal tracing leading to two-cell clones shortly after (14 h) tamoxifen administration.

Clones were defined when mGFP-labeled cells were in close proximity (< 150 µm apart, Supplementary Fig. 2a and Supplementary Fig. 2c) and isolated from any other mGFPlabeled cell. In addition, the definition of criteria for clonality was based on an extremely low dose of tamoxifen (2 µg/g of mouse, recombination frequency < 0.5%). We then used a tamoxifen dose (8 µg/g of mouse) inducing a recombination frequency about 10% to increase the representation of the myogenic population. For each mouse, one TA muscle was used for immunohistochemistry analysis, and the contralateral TA was used for determining the recombination efficiency following dissociation and immunostaining. EdU (ThermoFisher) was dissolved in NaCl 0.9% and injected intraperitoneally (0.3 µg/g of mouse) 9 and 7 h before tamoxifen administration.

Satellite cells isolation and live-imaging

Dissections were done essentially as described previously [START_REF] Gayraud-Morel | A role for the myogenic determination gene Myf5 in adult regenerative myogenesis[END_REF] . Injured TA muscles were removed from the bone in cold DMEM, minced with scissors, and then digested with a mixture of 0.08% Collagenase D (Sigma), 0.1% Trypsin (ThermoFisher), 10 µg/ml DNase I (Sigma) in DMEM for five consecutive cycles of 30 min at 37°C. For each round, the supernatant was filtered through a 70 µm cell strainer (Miltenyi) and collagenase and trypsin were blocked with 8% FBS on ice. Pooled supernatants from each digestion cycle were centrifuged at 500g for 15 min at 4°C. The pellet was resuspended in 2 ml cold PBS (ThermoFisher) and placed on top of cold Percoll layers (Sigma)(5 ml of 20% Percoll and 3 ml of 60% Percoll). After centrifugation at 500g for 15 min at 4°C, cells were collected from the 20%-60% interphase, while dense debris were concentrated below the 60% layer. The collected fraction was diluted in 40 ml DMEM 8% FBS and centrifuged at 500g for 15 min at 4°C. The pellet was resuspended in 500 µl satellite cell medium and filtered through a 40 µm cell strainer (Falcon). Cells were plated on glass coverslips coated with Matrigel or sorted (Supplementary Fig. 3) using a FACS Aria III (BD Biosciences) and collected in cold 250 µl of satellite cell medium. Sorted cells were plated on micropatterns (see below) and incubated at 37°C, 5% CO 2 , and 3% O 2 in a Zeiss Observer.Z1 microscope connected to an LCI PlnN 10x/0.8 W DICII objective and Hamamatsu Orca Flash 4 camera piloted with Zen (Zeiss).

Cells were filmed and images were taken every 9 min with bright-field filter to ensure that single cells were plated on the micropatterns and that their progeny remained confined. The raw data were analysed with TrackMate 50 , transformed and presented as videos.

Micropatterns

Micropatterns were manufactured essentially as described [START_REF] Yennek | Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells[END_REF][START_REF] Vignaud | Polyacrylamide hydrogel micropatterning[END_REF] . Briefly, glass coverslips (24 x 60 mm #1, Thermo Scientific Menzel) were cleaned serially in H 2 0/acetone/isopropanol, activated with an oxygen plasma treatment (Harrick Plasma) for 5 min at 30W and incubated with poly-L-lysine polyethylene glycol (PLL(20)-g[3.5]-PEG(2), SuSoS) at 100 µg/ml in 10 mM HEPES pH 7.4 at room temperature (RT) for 30 min, washed twice with H 2 0 and stored dried at 4°C. PLL-PEG-coated slides were placed in contact with an optical mask containing transparent micropatterns (Toppan Photomask) using H 2 O (0.6 µl/cm 2 ) to ensure tight contact between the mask and the coverslip and then exposed to deep UV light (Jelight). Micropatterned slides were subsequently incubated with 40 µg/ml fibronectin (Sigma) and 5 µg/ml fibrinogen Alexa Fluor 488 (ThermoFisher) in 100 mM NaHCO 3 pH 8.3 for 25 min at RT and rinsed three times in NaHCO 3 , three times in H 2 0 and dried and transferred immediately on a 12 kPa polyacrylamide (PAA) gel on silanized glass coverslips, to match the substrate rigidity of skeletal muscles [START_REF] Urciuolo | Collagen VI regulates satellite cell self-renewal and muscle regeneration[END_REF] . 81 mm 2 -square glass coverslips were cut out larger coverslips (22 x 22 mm #1, Thermo Scientific Menzel), cleaned serially with H 2 O/EtOH 70%/EtOH 100%, deep-UV-activated, silanized with 7.1% (vol/vol) bind-silane (Sigma) and 7.1% (vol/vol) acetic acid in EtOH 99% for 10 min at RT, washed twice with EtOH 99% and dried. Transfer of micropatterns on PAA gel was performed with 7 µl/cm 2 of a solution containing 7.5% acrylamide (Sigma), 0.16% bis-acrylamide (Sigma), 0.05% TEMED (Sigma), 0.05% APS (Sigma, A3678) in HEPES 10 mM pH 7.4 for 45 min at RT. Gels were rehydrated in NaHCO 3 100 mM pH 8.3 for 15 min at RT, detached from patterned coverslips and washed with PBS three times before cell seeding.

Micropatterned PAA gels were transferred in an in-house-made glass-bottom 6 well Petri dish to allow live-imaging and PAA embedding before immunostaining (see below). Briefly, 32 mm diameter glass coverslips were cleaned and silanized, a 14 x 14 x 0.45 mm piece of silicone (Smooth-on) was put at the center, and coverslips were layered with a 12 kPa PAA gel ('surrounding' PAA). After PAA polymerization, the coverslips were sealed (silicone) to the bottom of a 6 well plate (TPP) drilled out to 30 mm diameter. The silicone at the center of the PAA gel was removed and replaced with a 16 x 16 x 3 mm silicone isolator with a 10 x 10 x 3 mm window at the center to fit micropatterned PAA gels (9 x 9 mm) after filling with PBS. The apparatus was washed several times and equilibrated with satellite cell medium.

Medium was removed completely and cells (250 µl) seeded within the silicone isolator on micropatterns and, 90 min after plating, nonattached cells were washed with medium and medium volume was adjusted to 5 ml.

Immunocytochemistry and Immunohistochemistry

Cells on micropatterns were fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) in PBS containing 0.9 mM CaCl 2 and 0.5 mM MgCl 2 (PBS Ca 2+ Mg 2+ ) for 5 min at RT and washed in PBS Ca 2+ Mg 2+ for 5 min at RT. To prevent cell loss during subsequent immunostaining steps, cells were embedded in PAA. Cells were equilibrated in a solution containing 3.82% acrylamide, 0.13% bis-acrylamide and 0.1% TEMED in PBS for 30 min at RT, and the solution was replaced with 3.82% acrylamide, 0.13% bis-acrylamide, 0.1% TEMED and 0.05% APS in PBS. The silicone isolator was carefully detached and a parafilmcovered 20 x 20 mm glass coverslip (20 x 20 mm #1, Thermo Scientific Menzel) was adjusted on top of the micropatterns, sitting on the 'surrounding' PAA. Excess polymerization solution was removed and PAA allowed to polymerize for 1 h at RT. After rehydration in PBS for 15 min, the parafilm-covered coverslip was detached, PAA-embedded micropatterns were recovered and washed several times in PBS. Cells were permeabilized in 0.5% Triton X-100 (Sigma) for 20 min at RT, washed three times 10 min in PBS, blocked in 10% goat serum (GS, Gibco) for 4 h at RT, incubated with primary antibodies overnight at 4°C in 2% GS, washed four times 1 h in PBS at 4°C, incubated with Alexa Fluor-conjugated secondary antibodies and Hoechst when required overnight at 4°C in 2% GS and washed four times 1 h in PBS at 4°C. For Pax7/Myod/Myog triple staining, cells were further blocked with Pax7 primary antibody overnight at 4°C in 2% GS, washed four times 1 h in PBS at 4°C, incubated with Alexa Fluor 488-conjugated anti-Myogenin and DyLight 405-conjugated anti-Myod antibodies (see below) in 2% GS overnight at 4°C and washed four times 1 h in PBS at 4°C. Cells on glass coverslips were fixed in 4% PFA for 5 min at RT, washed twice in PBS, permeabilized in 0.5% Triton X-100 for 5 min, blocked in 10% GS for 30 min at RT, incubated with primary antibodies in 2% GS for 2 h at RT, washed in PBS three times 5 min, incubated with Alexa Fluor-conjugated secondary antibodies and Hoechst for 45 min at RT, washed in PBS three times 5 min and mounted in PBS Glycerol 75%.

For immunohistochemistry, TA muscles were fixed for 24 h in 4% PFA at 4°C, washed in cold PBS four times 1 h at 4°C, equilibrated in 20 % sucrose (Sigma) overnight at 4°C, embedded in Tissue Freezing Medium (Leica) and snap-frozen on liquid nitrogen. 12 µm cryosections were allowed to dry at RT for 30 min and rehydrated in PBS for 15 min. Antigen retrieval was performed in Tris 10 mM EDTA 1 mM pH 9 for 10 min at 95°C, followed by three short PBS washes and an additional wash in PBS for 30 min at RT. Sections were permeabilized in 0.5% Triton X-100 for 20 min at RT, washed three times in PBS, blocked in M.O.M. Mouse Ig Blocking Reagent (Vector Laboratories) for 1 h at RT, washed three times in PBS, incubated in M.O.M. Diluent (Vector Laboratories) for 5 min at RT, incubated with primary antibodies (Pax7 (Fig. 2d) or 5FD (Fig. 2g Immunostainings were analysed with a Zeiss Observer.Z1 and a Zeiss LSM800 confocal microscopes. Images were processed with Imaris 7.2.1 software. For each cell pair, the daughter cells were named 'Cell A' and 'Cell B'. Fluorescence intensity was measured on segmented nuclei and expressed as a fraction of the maximum intensity between Cell A and Cell B. In case of absence of signal in both daughter cells (eg. symmetric absence of Pax7), the normalized fluorescence intensity was not determined (nd value).

Antibody fluorescent labeling

Anti-myogenin antibody (5FD, DSHB) was purified with protein G sepharose (Sigma) as recommended. Briefly, hybridoma supernatant was incubated with protein G sepharose overnight at 4°C and packed into polypropylene columns (Qiagen), washed five times with PBS and antibody was eluted twice with 1.5 ml glycine 100 mM pH 2.7, neutralized with 150 µl Tris 1M pH 9. Elution fractions were pooled and dialyzed (ThermoFisher) against PBS overnight at 4°C. Antibody was concentrated with Amicon Ultra centrifugal filters (Merck) to 1 mg/ml. 100 µl of purified F5D antibody at 1 mg/ml (resp. anti-Myod at 0.75 mg/ml (BD Bioscience)) were dialysed (Thermofisher) against NaHCO 3 100 mM pH 8.

Materials and correspondence

Correspondence

and material requests should be addressed to shahragim.tajbakhsh@pasteur.fr

  and Fig. 2d left) and differentiating (6.8%, n=10 of 147 cell pairs, Fig. 2c and Fig. 2d middle-left) SCDs, in addition to ACDs (23.1%, n=34 of 147 cell pairs, Fig. 2c and Fig. 2d middle-right and right).

  ) to identify both old DNA strands (EdU pulse) and new DNA strands (EdU chase). We observed clonal examples of random DNA segregation (23.1%, n=12 of 52 cell pairs, Fig. 2f and Fig. 2g left) and non-random DNA segregation (76.9%, n=40 of 52 cell pairs, Fig. 2f and Fig. 2g middle and right). Interestingly, 22.5% of the cell pairs with NRDS showed asymmetric Myogenin expression (n=9 of 40 NRDS cell pairs, Fig. 2f and Fig. 2g right), with Myogenin being expressed in the EdU-negative daughter cell, as reported

  5 and cultured in DMEM GlutaMAX (ThermoFisher) 10% Fetal Bovine Serum (FBS) 1% Penicillin-Streptomycin (PS, ThermoFischer) at 37°C 5% CO 2 3% O 2 . Satellite cells were plated on glass coverslips coated with Matrigel (Corning) for 30 min at 37°C or on micropatterns in satellite cell medium (40% DMEM 40% MCDB (Sigma) 20% FBS 2% Ultroser (Pall) 1% PS). Establishment of H3.1/H3.3-SNAP mouse strains Mouse H3.1 (resp. H3.3) coding sequence was PCR amplified (Phusion Taq polymerase, New England Biolabs) from mouse primary fibroblasts cDNA (SuperScript III Reverse Transcriptase, ThermoFisher) with NotI_mH3.1_F (resp. NotI_mH3.3_F) and DPPVAT_mH3.1_R (resp. DPPVAT_mH3.3_R) primers, PCR-fused to a SNAP tag amplified from pSNAPm (New England Biolabs) with DPPVAT_SNAP_F and BamHI_SNAP_R primers, and cloned in pGEM-T easy (Promega) with T4 DNA ligase (New England Biolabs). A DPPVAT linker was included between histone coding sequences and the SNAP tag as in 16,17 . H3.1-SNAP (resp. H3.3-SNAP) EcoRI/BamHI (New England Biolabs) fragment was subcloned in a pCAG SV40 Puro plasmid between the EcoRI and BamHI sites downstream the CAG promoter and upstream an SV40 early polyA termination sequence. CAGG H3.1/H3.3-SNAP polyA SpeI/KpnI (New England Biolabs) DNA fragments were microinjected in fertilized oocytes to derive transgenic lines. Genotyping was performed by PCR from ear punch biopsies with CAG_For and DPPVAT_mH3.1_R or DPPVAT_mH3.R_R primers. H3.1-SNAP transgene insertion was mapped to chromosome 7 by inverse PCR (gDNA digestion with SacI (New England Biolabs), intramolecular ligation with T4 DNA ligase, PCR amplification with SV40 PolyA_F and SNAP qPCR_R primers). H3.3-SNAP transgene insertion was mapped to chromosome 10 by inverse PCR (gDNA digestion with BamHI, intramolecular ligation with T4 DNA ligase, PCR amplification with SNAP qPCR_F and CAG_R3 primers). Primers used are listed in Supplementary Table

  ), GFP, in M.O.M. Diluent) for 1 h at RT, washed three times 5 min in PBS, incubated with M.O.M. Biotinylated Anti-Mouse IgG Reagent (Vector Laboratories) for 10 min at RT, washed three times 5 min in PBS, incubated with secondary antibodies (Streptavidin-Alexa Fluor 405, Donkey anti-Chick Alexa Fluor 594, in M.O.M. Diluent) for 30 min at RT, washed three times 5 min in PBS. For Pax7/Myod/Myog triple staining, sections were further blocked with anti-Pax7 antibody (in M.O.M. Diluent) for 1 h at RT, washed three times 5 min in PBS, incubated with Alexa Fluor 488-conjugated anti-Myogenin and DyLight 550-conjugated anti-Myod antibodies (see below, in M.O.M. Diluent) overnight at 4°C, washed in PBS three times 5 min and mounted in PBS Glycerol 75%. EdU incorporation was detected with the Click-iT Plus Alexa Fluor 488 Imaging Kit (ThermoFisher) as recommended.

  3 (resp. Na 2 B 4 O 7 50 mM pH 8.5) overnight at 4°C, incubated with 120 µM Alexa Fluor 488 NHS ester (ThermoFisher)(resp. 60 µM DyLight 405 NHS ester (ThermoFisher) or 70 µM DyLight 550 NHS ester (ThermoFisher)) at 23°C for 1 h. NaCl concentration was adjusted to 150 mM and excess fluorescent dye was removed with Pierce Dye Removal Columns (ThermoFisher) following manufacturer's recommendations. Desalted labeled antibodies were further dialysed (ThermoFisher, Cat#69570) against PBS overnight at 4°C and stored at -20°C with 1 mg/ml bovine serum albumin (Sigma) and 50% glycerol.

Fig. 1

 1 Fig. 1 Parental H3.1 histones are symmetrically distributed during asymmetric cell fate

Fig. 2

 2 Fig. 2 Parental H3.1 histones are symmetrically distributed during asymmetric cell fate

Acknowlegments

This work was supported by Institut Pasteur and grants from Agence Nationale de la Recherche (Laboratoire d'Excellence Revive, Investissement d'Avenir; ANR-10-LABX-73, ANR-16-CE12-0024) and Association Française contre les Myopathies, CNRS, and the European Research Council (Advanced Research Grant 332893). We would like to thank the Mouse Genetics Engineering Platform (Institut Pasteur), the Flow Cytometry and Photonic Bioimaging Platforms of the Center for Technological Resources and Research (Institut Pasteur), Johan van der Vlag (Radboud University, Netherlands) for providing the H3.1 antibody, Glenda Comai (Institut Pasteur) for initial assistance with confocal imaging, Dan Filipescu for his advice to establish the mouse H3-SNAP variants, and Tom Cheung (Hong Kong University of Science & Technology) for advice on antibody labeling. G.A. received support from ANR.

Data availability

All data supporting the findings of this study are available from the corresponding author on reasonable request.

Author contributions

B.E. and S.T. proposed the concept, designed the experiments, and wrote the paper. B.E. performed and analysed the experiments. G.L.C. provided technical support. G.A. contributed to application for the project, advised on experiments and edited the paper. All authors read and agreed on the manuscript.

Competing interests

The authors declare no competing interests. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . http://dx.doi.org/10.1101/499913 doi: bioRxiv preprint first posted online Dec. 18, 2018;

Statistical information

Bar charts represent the mean ± standard error of the mean (SEM) or standard deviation of the mean (SD) as specified.

Supplementary information

Supplementary figures

Supplementary Fig. 1 Supplementary Fig. Videomicroscopy of a Pax7-nGFP Hi cell isolated from TA muscle at 5 dpi and plated on an asymmetric micropattern (not visible), corresponding to Fig. 1c left. The movie was started 3 h after plating and continued for 40 h; images were taken every 9 min (bright-field, 10× magnification).

Supplementary Movie 2.

Videomicroscopy of a Pax7-nGFP Hi cell isolated from TA muscle at 5 dpi and plated on an asymmetric micropattern (not visible), corresponding to Fig. 1c middle. The movie was started 3 h after plating and continued for 40 h; images were taken every 9 min (bright-field, 10× magnification).

Supplementary Movie 3.

Videomicroscopy of a Pax7-nGFP Hi cell isolated from TA muscle at 5 dpi and plated on an asymmetric micropattern (not visible), corresponding to Fig. 1c right. The movie was started 3 h after plating and continued for 40 h; images were taken every 9 min (bright-field, 10× magnification).


Yong He

Boxin Huang

Elrade Rofaani

Jie Hu

Yuanhui Liu

Gabriele Pitingolo

Li Wang

Jian Shi

Carole Aimé

Yong Chen
email: yong.chen@ens.fr

Fabrication of micro-cages and caged tumor spheroids for microfluidic chip-based assays

Keywords: Tumor spheroid, Microfluidics, Cage device

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Despite decades of intensive research, cancer remains one of the worldwide leading causes of mortality and more important efforts are expected, including both in-vivo and in-vitro assays [START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF][START_REF] Collaboration | Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 29 cancer groups, 1990 to 2016: a systematic analysis for the global burden of disease study[END_REF][START_REF] Rodrigues | Emerging tumor spheroids technologies for 3D in vitro cancer modeling[END_REF][START_REF] Katt | In vitro tumor models: advantages, disadvantages, variables, and selecting the right platform[END_REF][START_REF] Costa | 3D tumor spheroids: an overview on the tools and techniques used for their analysis[END_REF]. In this regard, robustly producing and handling of tumor spheroids using cancer cell lines, dissociated cancer cell and cutting tumor samples are necessary for detailed analysis. Previously, Hanging-Drop method [START_REF] Tung | High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array[END_REF][START_REF] Foty | A simple hanging drop cell culture protocol for generation of 3D spheroids[END_REF] and Liquid-Overlay method [START_REF] Achilli | Advances in the formation, use and understanding of multicellular spheroids[END_REF][START_REF] Loessner | Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells[END_REF][START_REF] Ozawa | Alginate gel microwell arrays using electrodeposition for three-dimensional cell culture[END_REF][START_REF] Torisawa | A multicellular spheroid array to realize spheroid formation, culture, and viability assay on a chip[END_REF] were widely used for spheroid formation. These methods are simple, but the resulted spheroids have to be transferred to another culture platform. Rotating bioreactor [START_REF] Martin | The role of bioreactors in tissue engineering[END_REF][START_REF] Lin | Recent advances in three-dimensional multicellular spheroid culture for biomedical research[END_REF] and magnetic levitation [START_REF] Souza | Three-dimensional tissue culture based on magnetic cell levitation[END_REF][START_REF] Haisler | Three-dimensional cell culturing by magnetic levitation[END_REF] are also frequently used to increase the production yield but the fabricated spheroids generally lack the size uniformity. More recently, patterned micro-well arrays [START_REF] Karp | Controlling size, shape and homogeneity of embryoid bodies using poly (ethylene glycol) microwells[END_REF][START_REF] Tang | Agarose multi-wells for tumour spheroid formation and anti-cancer drug test[END_REF][START_REF] Mirab | Preparation and characterization of size-controlled glioma spheroids using agarose hydrogel microwells[END_REF] were used to improve the uniformity of the tumor spheroids but the applications of these methods are limited since they are too simple to take into account the complexity of the tumor niche, which is a specific microenvironment where tumors are formed and regulated.

To overcome the above limitations, we developed a cage method which allows not only the formation of uniform tumor spheroids but also the integration of the fabricated spheroids into a microfluidic device for different assays. Our cage device is in the form of a patch with honeycomb frame sandwiched by a monolayer of agarose coated nanofibers and a mesh of relatively large openings. Both frame and mesh were defined by UV-lithography and soft-lithography and the nanofibers were produced by electrospinning, in a similar way of culture patch fabrication [START_REF] Tang | Induction and differentiation of human induced pluripotent stem cells into functional cardiomyocytes on a compartmented monolayer of gelatin nanofibers[END_REF][START_REF] Tang | Patch method for culture of primary hippocampal neurons[END_REF][START_REF] Tang | Effective motor neuron differentiation of hiPSCs on a patch made of crosslinked monolayer gelatin nanofibers[END_REF]. Single tumor cells could be dispersed through the mesh holes into each of the honeycomb compartments. Upon incubation, cells aggregated to form single spheroids in each of the compartments (Fig. 1). Since the size of the spheroids is generally larger than that of the mesh holes, they could be efficiently caged, allowing robust handling and manipulation. While both mesh and nanofiber monolayer are highly permeable and the patch can be quasi suspended in the culture medium, the spheroid growth conditions should be better than other culture platforms. We thus demonstrate the formation of uniform tumor spheroids and their integration into a microfluidic chip. Interestingly, we observed a significant deflection of the fiber layer due to mechanical forces of the growing spheroids. We also performed a preliminary drug test under both conventional and microfluidic culture conditions. Other types of stresses such as hypoxia could also be applied and the cage device could operate with other devices such as microfilters [START_REF] Tang | Microfluidic device with integrated microfilter of conical-shaped holes for high efficiency and high purity capture of circulating tumor cells[END_REF], therefore holding high potential of this approach.

Materials and methods

Fabrication of micro-cage device

The cage device was fabricated by photolithography, soft-lithography and electrospinning. Firstly, a double layer SU-8 mold was fabricated by photolithography. The mesh layer was patterned on a silicon wafer using a 50 µm thick SU-8 negative photoresist (Fig. 2a). Then, the honeycomb frame of height in the range of 200 to 300 µm was directly patterned on the mesh layer (Fig. 2b). This double layer SU-8 mold was then exposed in trimethylchlorosilane (TMCS, Sigma, France) vapor during 10 min for anti-sticking surface treatment. Afterward, a mixture of PDMS (GE RTV 615) pre-polymer and its cross-linker at ratio of 10:1 was casted on the SU-8 mold (Fig. 2c). After curing at 75℃ for 2 hours, the PDMS layer was peeled off and treated with TMCS anti-sticking agent. Secondly, the replicated PDMS structure was placed on a glass plate and a solution of polyethylene glycol diacrylate (PEGDA Mw=250, Sigma) mixed with 1v/v% Irgacure 2959 (Ciba Specialty Chemicals, France) was injected in the free space of the PDMS-glass assembly, followed by UV exposure for 2 min (Fig. 2d and2e).

Thirdly, a monolayer of gelatin nanofibers was electrospun and cross-linked on the opposite side of the mesh. This has been done by using a similar fabrication protocol of our previous work [START_REF] Tang | Induction and differentiation of human induced pluripotent stem cells into functional cardiomyocytes on a compartmented monolayer of gelatin nanofibers[END_REF][START_REF] Tang | Effective motor neuron differentiation of hiPSCs on a patch made of crosslinked monolayer gelatin nanofibers[END_REF]. Briefly, the electrospinning was performed with a gelatin solution of 10 wt% gelatin powder (G2625, Sigma) in DI water, ethyl acetate, and acetic acid at a volume ratio of 10 : 14 : 21. The ejection of the gelatin solution through a needle (23-gauge) to the collector (cage) was controlled with at a speed of 0.2 ml/h, a distance of 10 cm and bias voltage was 11 kV, respectively (Fig. 2f). The sample was then dried overnight in a desiccator and the gelatin nanofibers was soaked in an ethanol solution containing 2 M N-hydroxysuccinimide (NHS, Sigma) and 0.2 M 1ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC, Sigma) for 4 h. The sample was then washed three times in ethanol and dried in a vacuum overnight. Finally, the nanofiber layer was treated with a solution of agarose (Fisher Scientific, France) 0.2 w/v% in DI water and the cage device was dehydrated in oven at 60º C for 2 hours.

Cell culture

U87-MG cells (human glioblastoma cells) were grown in T75-flask inside an incubator of 37°C and 5% CO2. The culture medium was Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were passaged using TrypLE express enzyme when they reached 80-90% confluency. The cells were resuspended in complete DMEM medium and dispersed at density 1.5x10 5 cells per device in the cage through the mesh. Then, the device was placed in the culture dish for incubation under conventional culture conditions for up to 10 days and the medium was exchanged each two days. Prior to cell seeding, the cage devices were sterilized in 70% ethanol with exposure to UV during 30 min and equilibrated in complete culture medium for 1 hour.

SEM observation

Caged tumor spheroids were fixed in PBS containing 4% formaldehyde for 15 minutes. Then, they were rinsed twice with PBS buffer, and merged in 30% ethanol (in DI water) for 30 min. Afterward, the samples were dehydrated in a graded series of ethanol solutions with concentrations of 50%, 70%, 80%, 90%, 95%, and 100%, respectively, and in each for 10 min. Before observation, a 2 nm thick gold layer was deposited on the samples by sputtering. The images were captured with a SEM machine (Hitachi TM3030) operated at 15 kV.

Microfluidic culture

After incubation for 3 days, the device with caged tumor spheroids was reversibly integrated into a microfluidic chip (MesoBioTech, France) and a tangent flow was applied at a flow rate of 0.1 ml/h in the mesh side using a syringe pump. After incubation for another 3 days, the chip was dismounted and the cage device was placed in a 35 mm dish with complete culture medium for further observation.

Drug test and live/dead assay

The cage device with tumor spheroids was placed in another culture medium containing 1 µM anticancer drug Combretastatin A4 (CA4) [START_REF] Tron | Medicinal chemistry of combretastatin A4: present and future directions[END_REF]. After incubation for 24 hours, disassembling of the tumor spheroids were observed and live/dead assay was performed with PBS containing 4 μM Calcein AM (Invitrogen, L3224) and 5 μM propidium iodide (Sigma-Aldrich, P4170) solution, respectively. After incubation for 30 min, the cage device was rinsed twice with fresh PBS to remove any residual staining molecules and kept in PBS for observation. Finally, fluorescence images were recorded with an inverted fluorescence microscope (Axio Observer Z1, Zeiss).

Results and discussion

Cage device

The fabrication process of the cage device is straightforward by using different lithography methods, including UV lithography, soft lithography and micro-aspiration assisted lithography. To study the tumor growth in different size of space, different height 200 (H200), 250 (H250), 300 (H300) μm of cage devices were fabricated as shown in Fig 3a -3c. Fig. 3d and 3e shows respectively the top-view of a microframe with honeycomb compartments with 200 μm mesh size and a 1.2 µm thick agarose layer with gelatin nanofibers backbone on another side of the frame. Thus, single cells could be introduced on the agarose layer through the mesh and then gave rise to the formation of caged tumor spheroids. It is also interestingly to note that the tumor size increased when they were in a larger height of cage device. However, the final size always remained stable in all three heights of cage devices, due probably to the limitation of device height because spheroid size in H200 and H250 cage devices both exceeded the cage height. Besides, there could be not enough nutrition diffusion in center area of the spheroids, preventing cell proliferation. The above experiments have been repeated for five times, showing the same results.

Caged tumor spheroids

Microfluidic integration

The tumor spheroids formed in the cage were stable and could be easily handled and subjected to different stimuli. The cage device was taken out from the dish at day 3 and was integrated into a microfluidic culture device, for another 3 days diffusive culture, as shown in Fig 5a and5b. The culture medium flowed in the space outside the cage and passed by the honeycomb mesh side at a flow rate of 0.1 ml/h. Figure 4c shows dead/live image of tumor spheroid cultured in a cage device under cis-flow or diffusive culture conditions, showing the feasibility of culturing them under flow conditions. Clearly, the tumor spheroid could be maintained with few dead cells, and large variety of perturbation can thus be applied.

Anti-cancer drug effects

In order to investigate the drug effect on the spheroids formed in cage devices, combretastatin A4 (CA4) was used for anti-cancer study at day 6. This drug binds much better to the colchicine site on tubulin to inhibit polymerization and further prevent cell division, thus it is highly cytotoxic to a variety of human cancer cells. As expected, the caged spheroids taken out from a culture dish at day 6 were then immersed for 2 days in 1 µM CA4 supplemented culture medium and showed a clear disassembling effect, as shown in Fig 6 . The live/dead images showed that there were more dead cells in center area of smaller spheroids, while less in larger spheroids, also due to the drug permeability to the center core, especially in larger height of cage devices. Therefore, these images clearly demonstrated the drug resistance of larger spheroids and strong cancer cell-cell interaction.

Finally, we would like to mention that the deflection of the fiber layer due to the growing tumor spheroid might be used to study the mechanical properties of the tumor. Previously, Stylianopoulos et al have addressed this issue and found that evolution of stress and growth rate both depend strongly on the mechanical interactions with the surrounding host tissue [START_REF] Voutouri | Role of constitutive behavior and tumor-host mechanical interactions in the state of stress and growth of solid tumors[END_REF][START_REF] Kalli | Defining the role of solid stress and matrix stiffness in cancer cell proliferation and metastasis[END_REF]. They suggested that the solid stress on tumor growth involved not only the inhibitory effect of stress on cancer cell proliferation and the induction of apoptosis, but also the resistance of the surrounding tissue to tumor expansion. By studying the mechanic interaction of stem cell colonies with a 3D patterned elastomeric substrate, a contractile force of the order of 1 mN was deduced. In the present case, the solid stress causing the deflection of the fiber layer can be estimated by considering an edge-clamped thin disk. Based on the theory of plates and shells [START_REF] Timoshenko | Theory of plates and shells[END_REF], the pressure ∆p on the disk is given by ∆𝑝 = 16𝐸ℎ 3 3(1 -𝑣 2 )𝑅 4 (𝛿 + 0.488

𝛿 3 ℎ 2 )
where h and R and the thickness and radius of the disk, E and 𝑣 are the Young's module and Poisson ratio of disk material, 𝛿 is the deflection of the disk center point. Assuming a disk of 0.1 µm thickness and 50 µm radius with a Young module of 10 MPa and a Poison ratio < 0.3, a pressure of ~3.4 kPa is obtained, which is in the range of previous findings [START_REF] Kalli | Defining the role of solid stress and matrix stiffness in cancer cell proliferation and metastasis[END_REF]. More detailed analyses are needed for quantitative assessments.

Conclusions

We have developed a fabricated method to produce caged tumor spheroids. This cage device is robust and easy to use. Uniform tumor spheroids could be generated in the cages by single cell seeding and static culture. If the height of the cage is too small, the underneath fibers were deflected due to mechanical stress of the growing tumors. If the height of the case is too large, tumors grew freely and their size was limited by diffusion. The caged tumor spheroids were also integrated in a microfluidic chip and they could be cultivated under tangent flow conditions but dissembled after drug introduction.

More systematic studies will be carried out by considering other types of stress such as hypoxia, ischemia, compression, etc. and isolation of the released cells [START_REF] Tang | Microfluidic device with integrated microfilter of conical-shaped holes for high efficiency and high purity capture of circulating tumor cells[END_REF], thereby faciliating in-vitro cancer modeling.

Fig

  Fig 4a shows the tumor spheroids in the cages of different height at day 1, 4, 5, 6. Cell aggregation and spheroid formation could be observed at day 2. At day 6 after cell seeding, caged spheroids were fixed and dehydrated for SEM observation. Despite the decreased spheroid volume after dehydration, remarkably, we observed an important nanofiber deflection underneath of the tumor spheroids as shown in Fig 1c and 1d. Considering the fact that the final tumor size (about 220 µm) is comparable to the height of the cage (200 µm), the tumor spheroids trended to keep their spheroid shape by deflecting the

Fig

  Fig 4bshows clearly that the tumor size increased until day 4 and started to decrease at day 5. This is probably due to the change from single cells to cell aggregations and the strong effect of cell-cell interaction. It is also interestingly to note that the tumor size increased when they were in a larger height

Figure 1 .

 1 Figure 1. (a) Schematic of a cage, caged tumor spheroid formation, and tumor growth induced deformation of the underneath nanofiber monolayer; (b) Photograph of a cage device; (c-d) Scanning electron microscopy image of a caged tumor spheroid and tumor growth induced deflection of the nanofiber monolayer; (e) Microphotograph of caged tumor spheroids in each of the honeycomb compartments.

Figure 2 .

 2 Figure 2. Schematic diagram of the fabrication process of the cage device: (a) Cage layer patterning of the mold by UV lithography and SU8 resist on a silicon wafer; (b) Microframe patterning of the mold by UV lithography and another SU8 resist layer; (c) Replication of the mold pattern into PDMS by soft lithography; (d) Secondary replica of the mold patterning by micro-aspiration assisted UV curing of PEGDA; (e) Microframe with mesh structures released from glass substrate; (f) Electrospinning, crosslinking and agarose coating of the gelatin nanofiber layer.

Figure 3 .

 3 Figure 3. Scanning electron microscopy images of the fabricated cages: Sideview of the cages with height 200 µm (a, H200), 250 µm (b, H250) and 300 µm (c, H300), respectively; (d) Top-view of the mesh of hole-size 200 µm (d) and agarose coating nanofibers (e).

Figure 4 .

 4 Figure 4. Cage height and incubation time dependences of the caged tumor spheroids: (a) Microphotograph of tumor spheroids in H200, H250 and H300 cages at day 1, 4, 5, 6; (b) Size variation of the spheroids in different cages up to 6 days.

Figure 5 .

 5 Figure 5. Schematic (a) and photograph (b) of a microfluidic chip for the cage device integration; (c) Live/dead fluorescence images of tumor spheroids after incubation for 3 days in a H200 cage device.

Figure 6 .

 6 Figure 6. Bright field and live/dead fluorescence images of tumor spheroids confined in H200, H250 and H300 cages after days in CA4 supplemented medium (day 6-day 8).

Figure 1 Figure 2

 12 Figure 1

Figure 3

 3 Figure 3

Figure 4

 4 Figure 4

Figure 5

 5 Figure 5

Figure 6

 6 Figure 6

Acknowledgements

This work was supported by Agence de Recherche Nationale under contract ANR-17-CE09-0017 (AlveolusMimics), European Commission Cost Action BIONECA (CA 16122), DIM ELICIT program of Ile-de-France, and PSL Valorization through Pre-maturation project. Yong He is grateful to the China Scholarship Council for grant of his PhD studies.


Clément Lacoste

José-Marie Lopez-Cuesta

Anne Bergeret

Development of a biobased superabsorbent polymer from recycled cellulose for diapers applications

Introduction

Superabsorbent polymers (SAPs) are functional materials composed of a crosslinked hydrophilic polymer in a loose three-dimensional network used in many applications such as diapers, cosmetics and absorbent pads [START_REF] Ullah | Classification, processing and application of hydrogels: a review[END_REF]. Due to their highly crosslinked structure, SAPs possess an extraordinary capacity to absorb a large quantity of liquid, and can also retain the absorbed liquid during a long time even under compression [START_REF] Adair | Superabsorbent materials derived from hydroxyethyl cellulose and bentonite: preparation, characterization and swelling capacities[END_REF].

The principal superabsorbent polymer commercially used is a crosslinked partially neutralized polyacrylic acid. The polymer is made from acrylic acid and sodium hydroxide (or sodium carbonate), which are inexpensive reagents. Although the SAPs pose no direct threat to human life or health, the disposal of synthetic SAP waste is a source of various environmental pollutants [START_REF] Nandi | Environmentally friendly superabsorbent polymers for water conservation in agriculture lands[END_REF].

Bio-based SAPs are attractive due to their renewability and biodegradability. Among them, carboxymethyl cellulose (CMC), a water soluble and biodegradable cellulose derivative (Fig. 1), could be a good precursor for the synthesis of bio-SAP [START_REF] Gyles | A review of the designs and prominent biomedical advances of natural and synthetic hydrogel formulations[END_REF]. Nowadays, CMC is manufactured in significant amounts and produced by the 2-step reaction of cellulose with alkali and monochloroacetic acid (MCA), using organic solvents under heterogeneous conditions [START_REF] Haleem | Synthesis of carboxymethyl cellulose from waste of cotton ginning industry[END_REF]. In the first step, the cellulose is treated with NaOH in the presence of inert solvent, which acts both as a swelling agent and as a diluent and thus facilitates good penetration of NaOH into the cellulose structure. The alkali cellulose is consequently accessible and reactive toward MCA, which is added to the reaction in the second step. The CMC can be neutralized and dried immediately to give a technical grade or neutralized and washed to give a purified grade and used in many industrial applications (cement, paper and coating industries, adhesives, drug delivery, tissue engineering, wound dressing) [START_REF] Gyles | A review of the designs and prominent biomedical advances of natural and synthetic hydrogel formulations[END_REF][START_REF] Capanema | Superabsorbent crosslinked carboxymethyl cellulose-PEG hydrogels for potential wound dressing applications[END_REF][START_REF] Yeasmin | Synthesis of highly substituted carboxymethyl cellulose depending on cellulose particle size[END_REF].

The properties (viscosity, solubility, biodedradability) of CMC are mainly depending on the degree of substitution (DS). The theoretical maximum DS that can be achieved is 3. CMC is soluble in water when DS is higher than 0.4 and the biodegradability decreased with the increase of the DS. Yeasmean and Mondal [START_REF] Yeasmin | Synthesis of highly substituted carboxymethyl cellulose depending on cellulose particle size[END_REF] have observed the increase of the DS from 0.21 to 2.41 during the carboxymethylation of corn husk cellulose particles when the particles size decreased from 1071 µm to 74 µm respectively. The surface area as well as the availability of the hydroxyl groups are determinant to achieve a high substituted structure. The selection of carboxymethylation conditions (temperature, time, concentration) are also essential to prepare high substituted CMC. For commercial CMC products, the most common DS value is usually between 0.4 and 1.4. Nowadays it exists considerable interest in finding cheaper and environmental-friendly alternative methods and raw materials to produce CMC. Some authors have reported the synthesis of CMC from various cellulosic sources as alternative to virgin softwood pulp. Heydarzadeh et al. [START_REF] Haydarzadeh | Catalyst-free conversion of alkali cellulose to fine carboxymethyl cellulose at mild conditions[END_REF] successfully synthesized CMC from alkali cotton fibers with DS of 0.15-0.70 and excellent purity of 99.3% recommended for pharmaceutical applications. Unlü [START_REF] Ünlü | Carboxymethylcellulose from recycled newspaper in aqueous medium[END_REF] has studied the synthesis of CMC from newspaper in aqueous medium and obtained a DS of 0.3-0.7 with 84-94% CMC content. Joshi et al. [START_REF] Josji | Synthesis and characterization of carboxymethyl cellulose from office waste paper: a greener approach towards waste management[END_REF] have pulped and deinked office waste paper with a yield measured at 80.62 ± 2.0% to prepared CMC with a maximum DS of 1.07. Haleem et al. [START_REF] Haleem | Synthesis of carboxymethyl cellulose from waste of cotton ginning industry[END_REF] have produced CMC from waste cotton grinning and found a maximum DS = 0.874. Mondal et al. [START_REF] Mondal | Preparation of food grade carboxymethyl cellulose from corn husk agrowaste[END_REF] have extracted alpha-cellulose from corn husk to prepare high purity CMC with yield 2.4 g/g, DS 2.41, and water holding capacity of 5.11 g/g. Other authors like Bidgoli et al. [START_REF] Bidgoli | Preparation of carboxymethyl cellulose superabsorbents from waste textiles[END_REF] have produced CMC from cotton and viscose textiles and have obtained a DS between 0.50 and 0.86.

In the first part of this study, the production of CMC from waste cardboards and cellulose fibers was investigated, as precursor for the synthesis of a SAP.

In the recent past, the new class of hydrogels SAP has emerged based on natural derivatives such as CMC having good structure stability and biocompatibilities properties [START_REF] Dorez | Effect of cellulose, hemicellulose and lignin contents on pyrolysis and combustion of natural fibers[END_REF]. However, chemically crosslinked hydrogels might exhibit cytotoxicity due to the chemical nature of the unreacted crosslinkers (i.e. aldehydes). Therefore, there is a need to promote the use of non-toxic and bio-based crosslinker through the entire process. Citric acid is a cost-effective bio-based monomer that has three carboxylic and one hydroxyl group which provides both hydrophilicity and crosslinking sites and it is also known to be non-toxic [START_REF] Kim | Synthesis of super absorbent polymer using citric acid as a bio-based monomer[END_REF].

In this work, bio-SAPs were synthesized by crosslinking citric acid with carboxymethyl cellulose, derived either from cellulose fibers as reference source, or from recovered cardboard. Different particle sizes ⁎ Corresponding author. E-mail address: clement.lacoste@mines-ales.fr (C. Lacoste).

T

were used and their influence on the final properties of the bio-SAPs is described. The synthesized CMC was characterized in terms of degree of substitution, FTIR and thermal properties. The water absorption capacity of the bio-SAP was measured and the influence of the crosslinker concentration was also discussed.

Materials and methods

Materials

Waste cardboards were kindly supplied by a local recycling facility, PAPREC, France. All other reagents (glacial acetic acid, sodium hydroxide, monochloroacetic acid, isopropyl alcohol, ethanol, acetone, methanol, acetic acid, sodium silicate) used for cellulose extraction and carboxymethylation were analytical grades, purchased by Merck (Sigma-Aldrich), France.

Some commercial products (cellulose, CMC, and SAP) were used as reference and their properties were compared to the synthetized products of the present study. For cellulose, the fibers Arbocel® (cellulose content > 99%) were provided by J. RETTENMAIER & SÖHNE, Germany. They are industrially produced from softwood pulp which contain around 45-50% of cellulose extracted through a kraft process and named A1 (fiber length = 300 µm), A2 (fiber length = 23 µm) and A3 (fiber length = 8 µm). Carboxymethyl cellulose (DS = 1.2; molecular weight = 250,000 Da) was provided by Merck, France. A commercial polyacrylate SAP, named HYSORB 9400, was purchased from BASF, Germany.

Isolation of cellulose from waste cardboards

A triple knife mill (Alpine, type 20/10R0, 1500 rpm, and granulator grid of 1 mm) was used to grind the cardboards into particles with average length of 0.9 ( ± 0.2) mm. Purification of cardboards was done using alkaline peroxide treatment. A designed amount of cardboards particles were dispersed in 250 mL aqueous NaOH (20 wt%) and stirred 1 h. Then, addition of NaOH/H 2 O 2 /Na 2 SiO 3 mixture was done and stirred 2 h at room temperature. Hydrogen peroxide (H 2 O 2 ), sodium silicate (NaSiO 3 ) and alkaline (NaOH) concentrations were kept constant at 1% w/w, 2% w/w, and 2% w/w. The pulp obtained was filtered and washed with distilled water and dried in a fume hood at room temperature overnight. The recovered cardboard cellulose (RCC) was then dried 2 h at 60 °C and weighted.

Synthesis of carboxymethyl cellulose

10 g of cellulose (RCC, A1, A2 or A3) was dispersed in 100 mL isopropanol. Isopropanol was used as solvent to provide miscibility and accessibility of the etherifying reagent to the reaction center of the cellulose chains [START_REF] Josji | Synthesis and characterization of carboxymethyl cellulose from office waste paper: a greener approach towards waste management[END_REF]. Synthesis of CMC was carried out in a two stepsreaction: (i) alkalization followed by (ii) etherification. Alkalization was done by drop to drop addition of 20 mL aqueous NaOH (20% w/v) under continuous stirring over a period of 1 h at room temperature. Finally, predissolved monochloro acetic acid (MCA) (10 g) in 10 mL isopropanol was added to the alkali treated mass under continuous stirring. The mixture was heated at 50 °C and stirred 2 h. When reaction period was over, the pH was neutralized with glacial acetic acid and the product was filtered and washed three times with ethanol. The remaining material was dried overnight at room temperature and then 8 h at 60 °C in oven.

Synthesis of bio-based superabsorbent polymers

CMC (2 g) was stirred at room temperature in 100 mL distilled water until complete dissolution occurred. Then, the crosslinking agent (i.e. citric acid) was added in different amount (Table 1) and the CMC solutions were stirred continuously for 1 h. The solutions were poured into aluminum molds and were allowed to dry at room temperature for 24 h to remove water. In the sequence, the samples were cured at 80 °C in oven for 24 h for the cross linking reaction. After curing, the samples were cut with a Retsch® ZM200 grinder at 8000 rpm using a 1 mm grid.

Determination of CMC yield and DS

The CMC yield was calculated according to the following equation (Eq. ( 1)):

= × Yield weigthofthedriedsynthetizedCMC g weigthofthedriedinitialcellulose g (%) ( ) ( ) 100 (1)
The degree of substitution (DS) is the average number of hydroxyl group in the cellulose structure which was substituted by carboxymethyl groups at C2, C3 and C6 sites. It was determined by the following method. Prepared CMC sample (4 g) was stirred in a beaker with 100 mL acetone for 5 min. 12 mL of chloric acid (6 M) was added to this solution and stirred for 30 min in order to obtain acid CMC (H-CMC). The H-CMC obtained was filtered under vacuum and the residue was washed with methanol. The residue is then stirred in acetone for 5 min, filtered, and dried at 60 °C for 2 h.

Aqueous solution of H-CMC (0.5 g) mixed with 20 mL of 0.2 M NaOH solution and 50 mL of distilled water was titrated with 0.05 M HCl by phenolphthalein indicator. To estimate the degree of substitution, following Eqs. ( 2) and (3) were used:

Carboxymethylcellulose (CMC)

Citric acid (CA)

= - A BC DE F (2) = × - × DS A A 0.162 1 (0.058 ) (3)
where A = milli-equivalents of consumed HCl per gram of specimen; B = volume of NaOH added; C = molarity of NaOH; D = volume of consumed HCl; E = molarity of HCl; F = CMC in grams; 162 Da is the molecular weight of the anhydrous glucose unit and 58 Da is the net increment in the anhydrous glucose unit for every substituted carboxymethyl group.

Spectral and thermal characterizations

The thermal properties of the products were studied through differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and Fourier Transform infrared (FTIR) analysis. Each measurement was done in duplicate.

DSC measurements were performed on a Perkin Elmer Diamond operating under a constant flow of nitrogen (30 mL min -1 ). Samples were weighed (around 15 mg) into aluminum crucibles. Analysis in comparison to a blank crucible was performed using a temperature profile (ramp rate of 10 °C.min -1 ) from 0 °C to 250 °C. Thermal transitions were calculated from Perkin software through tangent method.

Measurements of mass loss versus temperature were performed using a Perkin Elmer thermogravimetric analyzer (TGA) module Pyris 1, under N 2 purge (flow rate of 50 mL.min -1 ). Typically, 8-10 mg of sample were placed on an aluminum oxide pan, and heated from 30 °C to 600 °C at 10 °C min -1 .

FTIR spectra of sample pellets in potassium bromide (10 wt%) were recorded with a Bruker Vertex 70 spectrometer in transmission mode. The spectra were recorded in the range of 4000-400 cm -1 with a resolution of 2 cm -1 and 32 scans per sample.

Absorption capacity of the bio-SAPs

SAPs particles (200 mg) were placed in a 50 mL beaker and immersed in excessive amount of distilled water or 0.9% NaCl solution for 1 h. The swollen gels were separated from the water and wiped with a filter paper to remove the excess of liquid and carefully weighted. The absorption capacity was calculated with the following equation ( 5):

= - Q g g m m m ( / ) s 0 0 ( 5
)
where m s and m 0 are the swollen and dry weight of the SAP respectively.

Environmental scanning electron microscopy

SAPs particles were placed in a Peltier module in an environmental scanning electron microscope (ESEM) (FEI Quanta 200). The working temperature was 5 °C and the pressure was 4.9 Torr in order to obtain a relative humidity of 90% in the working chamber. The samples were left in the chamber for 240 min and pictures were taken every minute.

Results and discussion

Characterization of cellulose recovered from cardboards (RCC)

In the first step, cellulose was recovered from waste cardboard by alkaline peroxide treatment. This method is used to fractionate crop wastes into cellulose, hemicellulose, and lignin [START_REF] Ünlü | Carboxymethylcellulose from recycled newspaper in aqueous medium[END_REF]. Hydroperoxide anion radicals and oxygen radicals are generated in the solution from H 2 O 2 and NaOH. These radicals oxidize cellulose-hemicellulose and hemicellulose-lignin bonds to solubilize cell wall components and obtain recovered cellulose. Recovered cellulose was 88% by weight with respect to the starting material.

FTIR analyses were performed to characterize both cellulose fibers (A1, A2 and A3) and recovered product (RCC) (Fig. 2). The spectrum of the RCC showed typical pattern of cellulose. As a general trend common to cellulosic samples, it can be observed the broad band in the 3700-3000 cm -1 region assigned to νO-H vibrations and the peaks at 2905 cm -1 related to νC-H vibrations. The spectrum of RCC shows additional absorption bands located at 1512 cm -1 and in the 1620-1690 cm -1 region compared to cellulose fibers. These bands belong to aromatic ring vibrations of phenylpropane skeleton and nonconjugated and conjugated carbonyl group respectively [START_REF] Capanema | Superabsorbent crosslinked carboxymethyl cellulose-PEG hydrogels for potential wound dressing applications[END_REF]. The presence of these additional absorbance bands reveal the presence of small amount of lignin fragments bonded to cellulose. Complete removal of lignin was not attended in these mild conditions and could be probably achieved by stronger reaction conditions (higher temperature or alkaline peroxide concentration). As residual lignin will not interfere with carboxymethylation [START_REF] Ünlü | Carboxymethylcellulose from recycled newspaper in aqueous medium[END_REF], any further purification was done and the recovered cellulose wad used as it is for carboxymethylation.

Characterization of carboxymethyl cellulose (CMC)

Degree of substitution and yield of the carboxymethylation

The objective of the present study is to use CMC obtained from RCC as precursor for the synthesis of a SAP in water medium. The solubility of the synthetized product is then of major consideration. Mondal et al. [START_REF] Mondal | Preparation of food grade carboxymethyl cellulose from corn husk agrowaste[END_REF] have studied the solubility of CMC and found that with DS lower than 0.4, CMC is insoluble. The reaction time (2 h) and the reaction temperature (50 °C) was then chosen in the present study to achieve a DS higher than 0.4.

The DS determination of the samples was performed using the titration method and the results are reported in Table 2. CMC_A1, CMC_A2 and CMC_A3 have a DS of 1.40 ( ± 0.01), 1.29 ( ± 0.01), and 1.35( ± 0.40) respectively, whereas CMC_RCC has a DS of 1.41 ( ± 0.14). No influence of the particles size on the DS was observed. The DS obtained is quite high considering the mild conditions used in the present study. Haleem et al. [START_REF] Haleem | Synthesis of carboxymethyl cellulose from waste of cotton ginning industry[END_REF] have measured a maximum DS of 0.87 for CMC derived from cotton ginning after strong cellulose extraction (10% of H 2 SO 4 at 80 °C) and carboxymethylation with MCA at 40-60 °C for 3 h. Joshi et al. [START_REF] Josji | Synthesis and characterization of carboxymethyl cellulose from office waste paper: a greener approach towards waste management[END_REF] have obtained a maximum DS value of 1.07 for a carboxymethylation at 50 °C for 3 h. It is possible to increase the DS by increasing the reaction temperature, time, or the NaOH concentration [START_REF] Mondal | Preparation of food grade carboxymethyl cellulose from corn husk agrowaste[END_REF].

The yield of the carboxymethylation in the present study was measured between 1.459 g/g (A2) and 1.619 g/g (A3) for CMC samples synthetized from cellulose fibers. The yield for cellule fibers is higher than for CMC derived from office waster papers [START_REF] Josji | Synthesis and characterization of carboxymethyl cellulose from office waste paper: a greener approach towards waste management[END_REF] with similar synthesis conditions. For CMC_RCC, the yield decrease at 1.120 g/g (Table 2). As the fiber length of RCC is higher (≈0.9 mm ( ± 0.2)), the kinetic of the carboxymethylation was modified. However, the comparison of the carboxymethylation yield with several studies from literature supports the suitability of arbocel fibers and RCC as sustainable sources for CMC production.

FTIR analysis

The infrared spectra of synthetized CMC from A1, A2, A3, and RCC are shown in Fig. 3. The broad absorption ban at 3335 cm -1 belongs to the stretching frequency of the OH group and the band at 2890 cm -1 is due to C-H stretching vibration. After carboxymethylation, hydroxyl groups of CMC_A1 are still present in a significant amount compared to the other samples. The high specific surface area of A1 fibers (length = 300 µm) can reduce the accessibility of the reactive sites. For CMC_RCC, the quantity of hydroxyl groups is clearly reduced after the reaction. However, the presence of a strong absorption band at 1620 cm -1 confirms the presence of asymmetric elongation of carboxyl groups and the band at and 1425 cm -1 is attributed to carboxyl groups as salt. These bands were also found on the spectra of the other samples but the intensity for CMC_A1 was lower. As the carboxymethylation took place in basic medium, CMC samples were in sodium salt form (eCOO -Na + ) rather than in acid form (eCOOH). The acid form was indeed not detected in the sample due to the absence of the characteristic bands usually observed at 1715-1730 cm -1 . FTIR analyses proved that carboxymethylation reaction was performed successfully for the samples.

Thermal stability

The results of the TGA analysis are given in Table 3. It can be seen that the degradation temperature of the cellulosic samples are between 349 °C (A3) and 361 °C (RCC). The recovered cellulose from cardboards (RCC) is slightly more stable than the others fibers samples. It can be due to the presence of lignin fragments as seen previously. Lignin is more resistant to thermal degradation than cellulose and its thermal degradation is observed on the curves in Fig. 4. The char content is also higher for this sample resulting also from the lack purity and the presence of mineral charges usually observed in cardboards (≈12%) [START_REF] Yáñez | Production of hemicellulosic sugars and glucose from residual corrugated cardboard[END_REF].

CMC samples have a lower thermal stability with a decomposition temperature measured between 294 °C (CMC_A2) and 314 °C (CMC_RCC). One time more the higher thermal stability of CMC_RCC could be due to lignin. The pyrolysis of hemicellulose and cellulose occurs quickly with a weight loss from 220 to 315 °C for hemicellulose and from 315 to 400 °C for cellulose. Lignin decomposes over a wider range of temperature (from 160 to 900 °C) and generates a high char yield [START_REF] Dorez | Effect of cellulose, hemicellulose and lignin contents on pyrolysis and combustion of natural fibers[END_REF]. However, the quantity of char at 900 °C is huge higher than those of cellulose fibers and similar char content values (∼30%) were observed for all CMC samples. The decrease of the decomposition temperature indicates a lower thermal stability of the CMC samples compared to cellulose. But the chemical grafting of carboxyl methyl groups on cellulose backbone induce the formation of a stable char. Many authors have reported a similar phenomenon after the chemical grafting on natural polymers [START_REF] Costes | Bio-based flame retardants: When nature meets fire protection[END_REF].

Characterization of the bio-SAPs

DSC

SAP_ref were prepared with varying amount of crosslinker from 5% to 20% (Table 1) and DSC curves are presented in Fig. 5. Two glass transition temperatures were observed by DSC. The results are given in Table 4. The addition of citric acid from 5% to 20% slightly increases the Tg 1 from 52.8 °C to 64.9 °C respectively. A glass transition temperature was clearly observed for SAP_ref containing 5% and 10% of CA located at 120.9 °C and 115.1 °C respectively indicating the crosslinking reaction between carboxyl groups of CMC and citric acid. At a concentration of CA 15%, the intensity of the second transition decreases (Table 4) and reaches ΔH = 0.35 J/g.°C, and finally no transition was observed for SAP_ref with CA 20%. These DSC results indicate that for a concentration of CA of 5%, 10%, and 15%, CMC is partially crosslinked with citric acid but for a concentration of 20% of CA, the network is completely crosslinked. In Fig. 5(b) no transition was observed and all samples are completely crosslinked during the second run. This result also indicates that the curing temperature during the synthesis of the SAP should be selected carefully. The temperature chosen in this work was 80 °C and was convenient.

Gel swelling

Gel swelling characterizations were performed in distilled water as the swelling media. Fig. 6a shows the absorption capacity (Q) of Table 2 Cellulose content, yield and degree of substitution of the CMC samples compared to literature.

SAP_ref as a function of the citric acid (CA) concentration. It can be observed that without crosslinker, the Q was only 2.5 g/g and at low crosslinker concentration (i.e. 5%), the absorption increased up to 32.9 g/g showing the production of a superabsorbent hydrogel. The presence of hydroxylate groups, as negatively charge groups, allowed the formation of the gel. The polymer is swelling up to a maximum absorption corresponding to a CA concentration of 10%. But the concentration of hydroxylate groups is progressively reduced with the addition of the acid. A decrease of the swelling capacity was observed at higher crosslinker concentrations (> 10%), which evidence the formation of a rigid polymer network with a slow motion. It limits the polymer chains elongation which is a necessary condition of the volume change. The expansion is then limited by the crosslinking density.

It is known that absorption capacities of SAPs depend on the pH and the salt concentration of the solution [START_REF] Kim | Synthesis of super absorbent polymer using citric acid as a bio-based monomer[END_REF][START_REF] Alam | Natural cellulose-chitosan crosslinked superabsorbent hydrogels with superior swelling properties[END_REF][START_REF] Chang | Superabsorbent hydrogels based on cellulose for smart swelling and controllable delivery[END_REF]. This specificity is due to the presence of ionic charges spaced along the polymer chains (carboxylate groups) that are strongly solvated in the presence of water solution. If ionic components are present in the liquid to be absorbed, ion exchange may occur and alter the absorbancy of the liquid However, saline absorption is an important characteristic of a SAP considering it simulates urine. The results showing the water and saline absorption capacities of SAP_ref as a function of the crosslinker concentration is shown in Fig. 6a. The saline absorption capacity is as expected lower than for water, and the mean value was found to be Fig. 4. Evolution of the weight of all cellulose and CMC samples as a function of the temperature.

7.45 g/g. The maximum value was 9.03 g/g for CA 5%. No significant influence of the concentration was observed. However, it can be noticed that the saline absorbency value is only 6 time lower than the water absorbency. Such deviation has already been observed in literature for polysaccharides derived hydrogels [START_REF] Alam | Natural cellulose-chitosan crosslinked superabsorbent hydrogels with superior swelling properties[END_REF]. In Fig. 6b, the absorption capacity of SAP_A1, SAP_A2, SAP_A3 and SAP_RCC crosslinked with 10 wt% of citric acid is shown. The absorbency of the SAP_RCC was 31.5 g/g. The particles size of the raw material seems to influence the absorbency of the final SAP product. Indeed, the water absorption capacities of the SAP_A1, SAP_A2, and SAP_A3 were found to be 33.8 g/g, 36.0 g/g and 38.4 g/g respectively, whereas the particle sizes of these products were 300 µm, 23 µm, and 8 µm respectively. The highest water absorbency was 45.87 g/g measured for SAP_ref. SAP-RCC shows a good absorption capacity in comparison with SAP_ref and confirmed the successful synthesis of a SAP from waste cardboards.

ESEM observation

ESEM pictures (Fig. 7) of the SAP_ref in comparison with a commercial poyacrylate SAP (Hysorb) were taken after 240 min exposition in a Peltier chamber regulated at 90% of relative humidity (RH). In Fig. 7(A) and (C), both SAP particles have not yet absorb the ambient humidity. The edges of the polyhedron particules were cleary observed as the particule is still dried. After 240 min at 90% RH, the volume enhancement of the particles was perceived corresponding to the penetration of the water molecules into the polymer chains leading to a swollen hydrogel. The phenomenon was more evident on the commercial product which has a water absorption capacity of 300 g/g. The bio-SAP obtained from carboxymethyl cellulose and crosslinked with citric acid appeared as a swelling hydrogel.

Conclusion

Cellulose was successfully recovered from waste cardboards under mild oxidizing alkaline conditions with a yield of 0.88. FTIR analyses indicate the presence of some residual lignin fragments bonded to the recovered cellulose which slightly increase the thermal stability of the product. However, the presence of residual lignin fragments does not interfere with the carboxymethylation. The degree of substitution was found to be around 1.4 for all samples without any influence of the cellulose source. The carboxymethylation yield was found to be 1.12 g/ g and with range 1.46-1.60 g/g for the cellulose recovered from cardboard material and from micrometrics cellulose fibers respectively, which confirms the sustainability for industrial production. The results demonstrated that bio-SAPs crosslinked with citric acid were produced. SEM analysis evidenced the formation of the hydrogel. The water absorption capacity was depending on the crosslinker concentration (optimum at 10 wt%) with a maximum measured at 45.87 g/g. The saline solution swelling capacity was 7.45 g/g on average without any significant influence of the crosslinker concentration. The complete production of a bio-SAP was successfully developed from recovered cellulose and could be a promising material for many industrial

Fig. 1 .

 1 Fig. 1. Carboxylmethyl cellulose (CMC) and citric acid chemical structures.

Fig. 2 .

 2 Fig. 2. FTIR spectra of cellulose fibers (a) A1, (b) A2, (c) A3, raw cardboard (d) and recovered cardboard cellulose RCC (e).

Fig. 3 .

 3 Fig. 3. FTIR spectra of the carboxymethyl cellulose samples (a) CMC_A1, (b) CMC_A2, (c) CMC_A3, (d) CMC_RCC.

Fig. 5 .

 5 Fig. 5. DSC curves of SAP_ref containing different quantity of citric acid (a) first and (b) second run.

Fig. 6 .Fig. 7 .

 67 Fig. 6. Water and saline absorption capacity Q (g/g) of (a) SAP_ref as a function of the citric acid concentration and (b) water absorption capacity of all SAPs crosslinked with 10% of citric acid.

Table 1

 1 Cellulose raw material, CMC, and bio-SAP samples.

	Cellulose name	Cellulose origin (median fiber size)	CMC name	Bio-SAP name	Citric acid concentration (%)
	ref	Commercial	CMC_ref	SAP_ref	0 -5 -10 -15 -20
	A1	Softwood (300 µm)	CMC_A1	SAP_A1	10
	A2	Softwood (23 µm)	CMC_A2	SAP_A2	10
	A3	Softwood (8 µm)	CMC_A3	SAP_A3	10
	RCC	Waste cardboard (900 µm)	CMC_RCC	SAP_RCC	10

Table 3

 3 Results of the TGA for the cellulose and CMC samples.

		Decomposition Temperature (°C)	Char 900°C (%)
	Cellulose samples
	A1	356 °C	10.2
	A2	350 °C	8.8
	A3	349 °C	3.2
	RCC	361 °C	17.1
	Carboxymethylcellulose samples
	CMC_A1	312 °C	29.4
	CMC_A2	294 °C	32.9
	CMC_A3	298 °C	28.9
	CMC_RCC	314 °C	29.5

Table 4

 4 Results of the DSC analysis of SAP_ref with varying citric acid concentration.

	Sample Citric acid	Tg 1 (°C) ΔH (Tg 1 ) (J/	Tg 2 (°C) ΔH(Tg 2 ) (J/
	(%)		g.°C)		g.°C)
	SAP_ref 5	52.8	0.05	120.9	5.64
	10	54.5	0.06	115.1	2.04
	15	63.6	0.10	123.8	0.35
	20	64.9	0.08	-	-

Acknowledgement

The research leading to these results is part of the European project POLYBIOSKIN (H2020 -grant agreement N°745839) and all the authors acknowledge the funding support from Bio-Based Industries.