Karine Beaugelin-Seiller

Rodolphe Gilbin

Sophie Reygrobellet

Jacqueline Garnier-Laplace

A single indicator of noxiousness for people and ecosystems exposed to stable and radioactive substances

Keywords: noxiousness, integration, human health, ecosystem, chemotoxicity, radiotoxicity

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

INTRODUCTION

It is today consensually acknowledged that stressors occur usually in mixture in the environment, and may impact simultaneously human and ecological health. This reality generates for any living organism complex and uncertain exposure situations for which potential health and ecological consequences are difficult to assess. Most often, the risk assessment for human health is conducted independently, as well as the ecological risk assessment, ignoring other risks (pyscho-social, politic, etc.). The awareness of this situation by the general public result in a growing demand for risk integration that should be reflected in regulatory developments. The notions underlying risk assessments are changing to integrate this new dimension. This can only be done progressively, as it supposes to concily concepts and methods specific to each domain involved as for example within the TRIAD approach, the procedure recommended by the ISO for sitespecific ecological risk assessment of soil contamination (ISO, 2015). At present, it is possible to deal operationally with the complex question of the assessment of a global impact of pollutant mixtures. Effect models exist that would make it possible with some improvements to grasp risks associated with the releases of complex mixtures and subsequent exposure of plural targets in the environment. The approach we propose here should be seen as a proof of concept for a framework to characterize complex source terms for the environment through a unique noxiousness indicator. This indicator aims to integrate in an harmonized way human health and ecological aspects for two categories of toxicity, chemotoxicity and radiotoxicity. The final objective is to obtain a unique operational indicator of the noxiousness of these complex source terms that would be a first-rate help for the management and communication of the covered risks.

The Life Cycle Impact Asessessment (LCIA) approach provides a source of inspiration with regard to integration in terms of risks, and more specifically of ecological risks in freshwaters (ISO 2006a(ISO , 2006b)). The corresponding indicator of ecosystem damages is the Potentially Affected Fraction (PAF) of species in the ecosystem, mostly obtained from a Species Sensitivity Distribution (SSD) [START_REF] Pennington | Aquatic ecotoxicological indicators in life-cycle assessment[END_REF]. The ms-PAF was developed as the next step to express the ecotoxic pressure of mixtures of substances [START_REF] De Haes | Life-Cycle Impact Assessment: Striving Towards Best Practice[END_REF][START_REF] Van De Meent | Calculating life-cycle assessment effect factors from potentially affected fraction-based ecotoxicological response functions[END_REF][START_REF] Solomon | Extrapolation Practice for Ecotoxicological Effect Characterization of Chemicals[END_REF]. Based on a common toxic unit, this concept opens the door to ambitious integrative approaches. We took a first step in that direction in 2007 [START_REF] Garnier-Laplace | A Screening Level Ecological Risk Assessment and ranking method for liquide radioactive and chemical mixtures released by nuclear facilities under normal operating conditions[END_REF], looking for the assesment as a whole of the radioactive and chemical risks for fauna and flora exposed to liquid releases from nuclear facilities. We have recently deepened the principles of this approach [START_REF] References Beaumelle | Ecological risk assessment of mixtures of radiological and chemical stressors : methodology to implement an msPAF approach[END_REF], with an investigation of the combined use of SSDs and additivity models. On the strength of these developments, we propose an operational method that produces additive indicators for both chemotoxicity and radiotoxicity, applied to human and ecological health. Our objective was to demonstrate the feasibility of an integrated single indicator for the global noxiousness of different stressors to which people and wildlife can be simultaneously exposed. We present first the construction of this indicator. We defined a toxicity factor Ftox as a simplification of the Effect Factor used in Life Cycle Impact Assessment, which relies for a given substance on its hazardous concentration for 50% of the species. This Ftox parameter is then combined with an information on the substance quantity to obtain a basic Noxiousness Indicator (NI). This has to be done without prior selection for each substance, each exposure pathway, each toxicity endpoint. Basic NIs can be summed in different ways to produce intermediate indicators, relevant for a category of toxicity, an exposure pathway or a toxicity endpoint. A second level of integration is proposed to obtain the final indicator, the global noxiousness indicator. In a second part we applied our approach to a High Level radioactive Waste, a mixture of radionuclides and stable metals ideal to test our indicator. The selection of substances was then done according to the availability of the data required by the indicator calculation, our guideline being to privilege recognized and homogeneous sources of data.

The framework presented in this paper to address the risk assessment of mixtures of stable and radioactive substances for both man and the environment was conceived to meet an operational need for comparison between complex source terms, i.e. in a purely comparative perspective between related sources of toxicity. This main goal allowed simplification in the processes taken into account; it required robust but overconservative assumptions in order to achieve the integration which are not compatible with a realistic risk assessment, but mainly answer the need for comparison. To enhance realism in the selected scenarios, our method as presented here should be seen as a starting point to develop a more consistent and homogeneous approach, within a collaborative process associating experts from each domain.

MATERIAL AND METHODS

The method described hereafter was initially developed for ecosystems. Its extension to human health was logically considered in a second time but is less accomplished. We considered its achievement will require the collaboration of experts and specialists in the field of human toxicity. We decided to briefly present what we though to be the required associated concepts and basics to invite beyond this feasability exercise the most suitable audience to contribute to its improvement.

The detailed mathematical formulations of the factors and indicators presented below, as well as the fully referenced data sources, are provided as supplemental data.

Elementary bricks: Fate and Effect Factors

Most of the method was described in a previous work [START_REF] Garnier-Laplace | A Screening Level Ecological Risk Assessment and ranking method for liquide radioactive and chemical mixtures released by nuclear facilities under normal operating conditions[END_REF].

Directly inspired from the LCIA methodology, it combines a fate-analysis step and an effect-analysis step respectively described by a fate factor (FF) and an effect factor (EF) [START_REF] Pennington | Risk and regulatory hazard-based toxicological effect indicators in life-cycle assessment (LCA)[END_REF]. These factors allow to express for each substance one-by-one the change in exposure from a given release and the change in effect per unit change of exposure. In summary, FF is defined per substance as the equilibrium ratio between the concentration within the receiving compartment and the concentration in the donor. EF is defined as the ratio ∆PAFi/∆Ci, also equal to the ratio 0.5/HC50i, where HC50i is the Hazardous Concentration of the substance i affecting 50% of species at their 50% effect, as recommended to compare impact categories [START_REF] Pennington | Risk and regulatory hazard-based toxicological effect indicators in life-cycle assessment (LCA)[END_REF]. The variation in ∆PAF is expressed as 0.5Σi(/∆Ci/HC50i). Theories, assumptions and options related to the ∆PAF, EF, their relationship and their use in a multi-stressor context have been presented and discussed elsewhere (e.g. [START_REF] Van De Meent | Calculating life-cycle assessment effect factors from potentially affected fraction-based ecotoxicological response functions[END_REF][START_REF] Garnier-Laplace | A Screening Level Ecological Risk Assessment and ranking method for liquide radioactive and chemical mixtures released by nuclear facilities under normal operating conditions[END_REF].

The adaptation of the method to radionuclides needs to convert radiotoxicity data expressed in µGy/h into an HC50 expressed in mol/L, as for stable substances. The conversion process described by Garnier-Laplace et al (2009) is replicated.

The toxicity factor Ftox

An additional step is taken toward the operational aspect of the method, by further simplifying the EF expression. Focusing on comparisons, any proportionality factor independent of the substance is useless and therefore removed. As such, we define a new EF for ecosystems, called Toxicity Factor (Ftox) and simply equal to 1/HC50i.

Recognized practices in LCIA supported by available data prompt us to suggest the extension of the approach to human health, considering ingestion and inhalation exposures. The corresponding Ftox takes the form of the inverse of the ED50 , , .

This is the life dose inducing a 50% increase of the probability to develop the pathology e for individuals exposed to the substance i via the pathway v (kg internalized/ whole life).

This concept is shared between human radiotoxicity and chemotoxicity and should be easily expressed in a common unit. But its use will require some care. For example, the duration of what is similarly called "whole life", a shared notion used in the derivation of ED50 values, differs slightly between chemotoxicity (70 years) and radiotoxicity (86 years). The issue of how comparable the various types of toxicity benchmarks (i.e. for radioactive or stable substance) are, must also be solved. Some homogenization work would also be probably necessary to obtain consistent datasets regardless the exposure pathway (ingestion, inhalation) and the pathology (cancer, non-cancer).

The Noxiousness Indicator NI

The notion of Ftox lacks the quantitative dimension attached to the source term, which introduction leads to the definition of the Noxiousness Indicator (NI). NI corresponds, for a given substance s, to the product of its Ftox by its concentration Ci in the source term under consideration i.e. the ratio of the substance concentration for example in water to its HC50. This dimensionless indicator can be summed for ecosystem on substances to give two basic indicators, either the radiological (NI-Erad) or chemical (NI-Echem) ecological noxiousness indicator. Summation over toxicity categories produces the final global indicator NI-E (Table 1). These three indicators allow dealing with two of the six impact categories selected (chemo-and radio-ecotoxicity).

The remaining four impact categories (human cancer and non-cancer effects due to chemotoxicity or radiotoxicity), remain to treat consistently. The previous approach has then been applied to human health in a similar however a bit more complex way. In addition to the category of effects (cancer; non-cancer) and type of toxicity (radiological; chemical) the noxiousness has to be characterised also with regard to exposures pathways. Indeed the ED50 values for people required by the method are usually reported for a given exposure pathway. A look in some relevant databases showed that data are easily available mainly for ingestion or inhalation. We ackownledged that other exposure pathways such as irradiation at distance or dermal adsorption could be of importance.

However we considered that an objective of feasibility demonstration as pursued here could be satisfied by the immediate availability of ingestion and inhalation data.

Moreover reducing the application to these two exposures pathways allowed a certain homogeneity in the data required for human and ecosystem health by using a same internationaly recognised source. That means finally that eight basic indicators with the same unit have to be produced to cover all the possible combinations (Table 1). These The second level of integration produces two indicators, one per category of toxicity (i.e.

NI-hhchem the indicator related to chemotoxicity including all effects and pathways). The third and last level of integration gives the final single expected indicator by summing the two previous ones.

The difficulty relies on ensuring the homogeneity of significance of all these indicators and expressing them in a same unit. The proposed method does not aim to assess risk realistically, but rather to offer a comparison tool able to put on a same scale different impact categories initially not comparable. It relies on oversimplification of environmental transfer processes, exposure and effect characterizations (for example, use of HC50 rather than HC10 as usually in ERA). The numerical values of the calculated indicators have no meaning in themselves, they do not indicate a level of detriment or damage. They are only the product of a standardised rigourous approach applicable to any set of substances in a perfectly similar way, authorizing comparisons of noxiousness of various inventories of multiple substances (or source-term) or for a given inventory of multiple substances, the time evolution of the indicators.

Data sources

Toxicity data were collected from aknowledged sources for both human and ecosystems, ensuring consistence by using as long as possible common supports. For chemicals, the databases associated with the version 2.0 of the tool USETOX were then consulted for humans (ED50; Fantke et al, 2017) and ecosystems (EC50; Hugonnet et al, 2014). For radionuclides, the FREDERICA database [START_REF] Copplestone | The development and purpose of the FREDERICA radiation effects database[END_REF] and the derived dose-effect relationships (Garnier-Laplace et al, 2010) underpinned the reasoning for ecosystems. For humans, data were retrieved from the UNSCEAR report (UNSCEAR, 1982) and the paper from [START_REF] Edwards | Risks from ionising radiation: deterministic effects[END_REF].

Data regarding the parameterisation of transfer in the environment were found in the ERICA Tool [START_REF] Brown | The ERICA Tool[END_REF] specifically for wildlife and in some IAEA reports (IAEA, 2001[START_REF] Iaea | Handbook of Parameter Values for the Prediction of Radionuclide Transfer in Terrestrial and Freshwater Environments[END_REF] for humans. Additional information and details are provided as supplemental data.

APPLICATION AND DISCUSSION

The proposed approach was applied to a well characterised source-term (High Level radioactive Waste -HLW), including stable and radioactive substances, and its evolutionary composition at the short-, medium-and long-term (0, 10 3 and 10 4 years).

Due to radioactive decay, the radiological toxicity of radionuclides initially present decreases with time, by generating new substances that may themselves be radiologically or chemically toxic.

According to Table 1, basic, intermediate and final noxiousness indicators were calculated both for human health and ecosystems. The scarcity in (eco)toxicity data is a well-known weakness of risk assessment methods since it concerns the vast majority of chemical substances with the exception mainly of some metals (e.g. Pb, Hg, Ni). Due to the anticipated lack of data, especially for stable substances, the application focused on the need for demonstration on the basis of freshwater ecosystems for which the greatest number of substances are characterised in terms of toxicity. For the human health aspect, the already identified limitations were not considered at this stage of method development, but will have to be solved for an operational application.

Intrinsic vs. In Situ Noxiousness

The objective was to evaluate comparatively the global noxiousness of the time dependent compositions of the HLW (each of the three was considered as a different waste for the study) . We defined an intrinsic noxiousness, which is a property of a given composition of the waste itself (inner waste toxicity), apart from any consideration of dispersion in the environment. We defined additionally the in situ noxiousness, to integrate the filter effect of environmental dispersion. This was done at first by introducing in the calculation the liquid-solid partition coefficient. Both components of noxiousness allow a more complete characterisation of the waste under investigation.

Use of Noxiousness Indicators in isolation

Ecological indicators

The ecological noxiousness indicators were calculated for any substance which ecotoxicity was sufficiently documented. The resulting total intrinsic ecological NI decreases by about two orders of magnitude over a million years (Figure 1).

Radiotoxicity is the major contributor over the full time period, the total chemotoxicity being less by two to four orders of magnitude. Taking into account the chemotoxicity of radionuclides and their stable decay products does not significantly alter the total chemotoxicity, almost entirely due to stable substances initially present. The contribution of stable substances generated by radioactive decay is not quantitatively significant. It significantly increases over time, by two orders of magnitude over the studied period.

To illustrate the additional information possibly retrieved from such an approach, we focused on the hierarchization of toxics, which may support management decision. We limited arbitrarily the toxic classification to the top ten substances in each category, radiotoxic and chemotoxic substances. They have been prioritized with regard to their contribution to the intrinsic ecological noxiousness of HLW from their noxiousness indicators as a function of time. Initially from far the most harmful for wildlife (Figure 2A), radionuclides lose ranking places over time and their order or even their nature is modified (e.g. 243 Am rises from ninth to second place, disappareance of 137 Cs), due to radioactive decay. After 1000 years, four metals (Al, Ni, Zn and Cr) integrate the first half of the ranking (Figure2B), without change in their ranking. After 10 000 years, the top ten places are equitably occupied by radioactive (always the most harmful) and stable substances (Figure2C), with order modification only for radionuclides. Intrinsic noxiousness indicators range almost over two orders of magnitude, while they varied initially over five orders of magnitude.

Human health indicators

When looking for completeness to calculate ecological noxiousness indicators, the determination of human health indicators focused on a reduced number of substances.

The objective was clearly to demonstrate feasibility of an approach that would produce consistent noxiousness indicators for humans and wildlife integrating chemo-and radiotoxicity. The substances were selected among those for which both types of indicators could be calculated.

Within the limit of the selected substances and under the assumptions made, the total intrinsic chemical noxiousness of HLW does not evolve for human health over the studied period of time (Figure 3). This indicator is mainly driven by cancer effects linked to ingestion of substances, (inhalation is of minor contribution -results not shown). This noxiousness, initially up to four orders of magnitude lower than that of radiological harm, remains lower for about 10,000 years. Effects mainly responsible for radiotoxicity would be non-cancer effects, but differences between the two types of effect are much less marked than for chemotoxicity (results not shown). As expected, results in terms of in situ noxiousness would be similar, with a shift toward lowest values of indicators due to the self-time filter effect. These observations have no absolute value; they depend closely on the substances taken into consideration. This exercise has been done for illustrative purposes only.

Use of Noxiousness Indicators in combination

Indicators were calculated for human health and ecosystems for the same source term according to a harmonized approach, making them consistent. The next step would The decrease over time of the total intrinsic noxiousness indicators for the selection of stable and radioactive substances from the HLW, already discussed, moves the waste noxiousness from category XIII in the short term to category XI then VIII in the medium and long terms respectively.

CONCLUSIONS

The proposed method was applied to the time changing composition of a High Level radioactive Waste. Such a material includes both radionuclides and toxic metals, but is today regulated only with regard to its radiotoxicity. Within the acknowledged limitations of the approach, the global Noxiousness Indicator we calculated confirmed as expected the dominance of this radiotoxicity through time over a period of 1 million years, for both people and the environment. The use in combination of human health and ecological indicators allowed to obtain a clear and synthetic view of the temporal evolution of the global noxiousness of the waste, which could be a powerful communication and decision support. Lower levels of integration are also rich in insights, notably in terms of comparison between substances. This method is however largely perfectible, for example due to some "dead ends", data gaps, over simplification or huge uncertainties.

The concept of a single indicator of noxiousness for both human health and ecosystems, explicitly embedding radio-and chemotoxicity, is attractive and the related approach we presented appears promising. It illustrates the feasibility of full integration into a consistent approach related to human and ecosystem health. The reverse of the method is for chemicals at worst the well known lack of toxicity data, at best their heterogeneity.

SUPPLEMENTAL DATA

The Supplemental Data are available on the Wiley Online Library at DOI:10.1002/etc.xxx.

FIGURE CAPTIONS

  indicators might be aggregated to express the noxiousness in an integrated manner by summing some or all the individual indicators (all substances per effect category, all effects categories …) or even the global noxiousness. Basically, each indicator NIhhtoxicity,effect,pathway is calculated for each substance, stable or radioactive. They are then summed for all subtances in a toxicity category (chemo-or radiotoxicity) for a given exposure pathway and a given effect category to produce the eight basic indicators (i.e. NI-hhchem,c,ing indicator related to the cancer effect linked ot the ingestion of all the chemical substances). The first level of integration produces by summing over effects (cancer and non-cancer) four intermediate indicators that express the noxiousness related to an exposure pathway for a given category of toxicity (i.e.NI-hhchem, ing indicator related to the toxicity by ingestion of all chemicals), and by summing over exposure pathways four other indicators that express the noxiousness related to an effect category for the same category of toxicity (i.e. NI-hhchem,c indicator related to cancer due to all chemicals).

  logically to combine them to express the total noxiousness of this source term and ultimately to compare several such terms. Multiple combinations are possible. The simplicity of reading and interpretation guided the choice presented here. Both indicators are reported on a single graph to allow categorization and immediate comparison of the source terms. Each source-term is positioned according to its combination of indicators: the human health indicator is plotted on the x-axis, the ecological indicator on the y-axis, even though their values are not necessarily bound. Classes of noxiousness are defined as the bands delimited by two successive pairs of identical values on the x and y axes (i.e. band defined by x1=y1=a and x2=y2=a+1 increment). Two source terms in the same class (i.e. same band) have a comparable total intrinsic noxiousness. Two source terms in different bands have different noxiousness the one in the right-most band being considered more harmful. This representation offers the advantage of a great readibility, but supposes the implicit assumption of the equivalence between noxiousness for humans and ecosystems for the same value of their respective indicators. This is questionable and should be considered on a case-by-case basis, depending on the objective and context of the noxiousness characterization. This issue could be solved by weighting one or the other of the indicators. Then the question arises as to which weight values to apply… Indicators were retrieved from the sets of results acquired for substances common to both previous steps. We defined 14 classes of noxiousness covering the range of values of total intrinsic noxiousness indicators for humans and ecosystems (Figure4).

  Effect endpoints, species, and other characteristics of lab tests devoted to ECx determination are extremely variable. Methods to obtain metadata from these basic information are no longer homogeneous. The next step in the development of such indicators would necessarily require signficant work to homogenize all the underlying data. Additional consideration should be payed to bioavailability that conditions the substance toxicity. The treatment of uncertainties has to be added. Solving these aspects will produce truly consistent indicators, which use in combination will need an extra step of normalization and weigthing. This finalization requires the involvment of all stakeholders, especially when combining human and environmental health indicators.

Fig. 1 1 Figure 2 Figure 3 Figure 4

 11234 Fig.1 temporal evolution of indicators of intrinsic ecological noxiousness of HLW,

Table 1 .

 1 Indicators proposed to characterize the noxiousness of complex source terms for human health (hh) and ecosystems (E), depending on the level of aggregation

	Human health	Ecosystem

c third level of aggregation (all inclusive)


Philippe J R Cohen
email: philippe.cohen@treefrog.fr.

Elisa Luquet

Justine Pletenka

Andrea Leonard

Elise Warter

Basile Gurchenkov

Jessica Carrere

Clement Rieu

Jerome Hardouin

Fabien Moncaubeig

Clément Rieu

Michael Lanero

Eddy Quelennec

Helene Wurtz

Emilie Jamet

Maelle Demarco

Celine Banal

Paul Van Liedekerke

Pierre Nassoy

Maxime Feyeux

Nathalie Lefort

Kevin Alessandri

Affiliations

C-STEM: ENGINEERING NICHE-LIKE MICRO-COMPARTMENTS FOR OPTIMAL AND SCALE-INDEPENDENT EXPANSION OF HUMAN PLURIPOTENT STEM CELLS IN BIOREACTORS

research documents, whether they are published or not. The documents may come

INTRODUCTION

Following pioneering works targeting Parkinson's disease [START_REF] Lindvall | Human fetal dopamine neurons grafted into the striatum in two patients with severe parkinson's disease: A detailed account of methodology and a 6month follow-up[END_REF][START_REF] Parmar | The future of stem cell therapies for Parkinson disease[END_REF], diabetes [START_REF] Shapiro | Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen[END_REF][START_REF] Sneddon | Stem Cell Therapies for Treating Diabetes: Progress and Remaining Challenges[END_REF], macular degeneration [START_REF] Mandai | Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration[END_REF] or heart failure [START_REF] Menasché | Myoblast transplantation for heart failure[END_REF][START_REF] Murry | Stem cells and the heart -The road ahead[END_REF], cell therapies are now addressing an increasing diversity of indications [START_REF] Deinsberger | Global trends in clinical trials involving pluripotent stem cells: a systematic multi-database analysis[END_REF][START_REF] Yu | Cancer cell therapies: the clinical trial landscape[END_REF]. Therapeutic cells represent a hope for millions of patients worldwide with chronic diseases or unmet medical needs. For each of those patients, the number of required cells range between 10 5 and 10 10 cells. While academic pre-clinical studies or small-scale clinical trials have already been proved to be successful, a transition to true clinical scale is now urgently needed. To enable treatment of thousands to millions of patients, production capacity must scale while maintaining high-quality cells compatible with transplantation.

Due to their unlimited self-renewal capacity and potency to give rise to any cell type in the body, human pluripotent stem cells (hPSCs) hold great promise to provide the required quantities of therapeutic cells. In vivo, a few embryonic stem cells can give rise to the 30×10 12 cells of the adult human body [START_REF] Sender | Revised Estimates for the Number of Human and Bacteria Cells in the Body[END_REF] while maintaining their genome integrity. As a consequence, much effort has been dedicated to the isolation and in vitro culture of these cells in physiological conditions.

Historically, the first stem cell lines were established by micro-dissecting embryos and manual passaging as twodimensional (2D) adherent epithelial colonies on a layer of feeder cells [START_REF] Evans | Establishment in culture of pluripotential cells from mouse embryos[END_REF][START_REF] Martin | Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[END_REF][START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF]. Cell proliferation and pluripotency maintenance were thus achieved in vitro. The need for an embryonic source was then alleviated with the discovery that differentiated cells could be reprogrammed into induced pluripotent stem cells (iPSC) [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors[END_REF]. This breakthrough led to pioneer works in developmental cell biology and catalyzed the development of cell therapy applications. Even though a lot of work has been devoted to the development of media and substrate compatible with clinical use, topology of hiPSCs cultures has not been gathering the same attention. Nevertheless, 2D in vitro cultures suffer high mortality rates, spontaneous differentiation and genetic drift [START_REF] Adil | Expansion of human pluripotent stem cells[END_REF]. An estimated 40-fold increase in the number of mutations compared to in vivo conditions [START_REF] Kuijk | Mutational impact of culturing human pluripotent and adult stem cells[END_REF] often results in rejection of clinical batches due to safety concerns [START_REF] Garber | RIKEN suspends first clinical trial involving induced pluripotent stem cells[END_REF][START_REF] Yamanaka | Pluripotent Stem Cell-Based Cell Therapy-Promise and Challenges[END_REF]. In addition, 2D culture systems have limited "scale-out" possibilities: for example, generating 1 trillion pluripotent stem cells, would require almost 1000 m 2 of plastic . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (which this version posted July 5, 2021. ; https://doi.org/10.1101/2021.07.05.451086 doi: bioRxiv preprint dishes and countless handmade passaging [START_REF] Adil | Expansion of human pluripotent stem cells[END_REF]. The non-scalability and the inability to conserve high quality cells remain the main limitations of 2D culture systems for clinical applications [START_REF] Yamanaka | Pluripotent Stem Cell-Based Cell Therapy-Promise and Challenges[END_REF].

More recently, 3D hPSCs cultures have been developed to model developmental processes. After seeding stem cells in bulk extracellular matrix (ECM) [START_REF] Taniguchi | Lumen Formation Is an Intrinsic Property of Isolated Human Pluripotent Stem Cells[END_REF][START_REF] Shahbazi | Pluripotent state transitions coordinate morphogenesis in mouse and human embryos[END_REF] or using microfluidic chips [START_REF] Zheng | Controlled modelling of human epiblast and amnion development using stem cells[END_REF], cell clusters self-organize into a monolayered epithelium recapitulating numerous features of an epiblast that generates in vivo all tissues in an amniote embryo through gastrulation. Even though these elegant approaches allowed to gain much insight into morphogenesis mechanisms [START_REF] Shahbazi | Mechanisms of human embryo development: from cell fate to tissue shape and back[END_REF], they are not designed for the production of hPSCs per se.

From a bioproduction perspective, significant advances have been achieved by developing 3D suspension cultures and bioreactors. Whether hPSCs are grown as floating aggregates or adherent at the surface of microcarriers, those systems provide increased surface-to-volume ratio and scale-up potentialities. Additionally, the use of stirred-tank bioreactors (STBR) allowed better control of the culture parameters without direct human intervention. Indeed, stirring-mediated mechanical agitation avoids sedimentation of the aggregates or microcarriers onto the bottom of the culture vessel and heterogeneities in the culture medium compositions.

Nonetheless, one intrinsic drawback of agitation is that hydrodynamic shear induces cellular damages [START_REF] Kinney | The multiparametric effects of hydrodynamic environments on stem cell culture[END_REF]. This effect is all the more dramatic as the vessel increases in size: the larger the volume of the bioreactor is, the more power per unit of volume needs to be injected for effective homogenization. Even though expansion rates with hPSC aggregates are regularly increased and could reach up to 70-fold within 7 days [START_REF] Manstein | High density bioprocessing of human pluripotent stem cells by metabolic control and in silico modeling[END_REF][START_REF] Borys | Optimized serial expansion of human induced pluripotent stem cells using low-density inoculation to generate clinically relevant quantities in vertical-wheel bioreactors[END_REF], the ability of scaling up the volume of the production is still restricted. Typically, this mechanical upper limit makes it difficult to reach batches larger than 1 liter or equivalently few billion cells. Finally, the current bottleneck for industrial scalability is the difficulty to fulfill simultaneously high amplification rate, volume and physiological quality.

In this work, by bridging the gap between two distinct communities, namely developmental biology and stem cells bioprocessing, we show that recapitulation of hPSC niche-like micro-environments allows to optimize the suspension culture of hPSC in STBRs. More precisely, we propose a system that utilizes a high throughput microfluidic encapsulation technology compatible with suspension culture of stem cells in a bioreactor and amenable to the production of large volume batches without any degradation of cell survival in contrast with 2D colony-based systems for instance. Briefly, iPSCs are encapsulated in alginate hollow microcapsules internally coated with ECM components at low cell seeding concentration. We assess the maintenance of stemness and pluripotency upon 3D culture in suspension. We then characterize the cell growth inside the capsules before showing that upscaling to 10 liter stirred-tank bioreactor allows to reach unrivalled amplification factors.

Altogether, the C-STEM technology overcomes the scale-up bottleneck faced in cell therapy bioproduction. We discuss that the origins of this performance could be related to unprecedented cell viability of 3D lumenized colonies within these capsules.

RESULTS

High-throughput microfluidic encapsulation of hiPSCs

Using a microfluidic technique to generate hollow hydrogel spheres (Fig. 1A, Movie S1, and detailed description in the Materials and Methods section) previously developed by us and others [START_REF] Ma | Core-Shell Hydrogel Microcapsules for Improved Islets Encapsulation[END_REF][START_REF] Zhang | A novel core-shell microcapsule for encapsulation and 3D culture of embryonic stem cells[END_REF][START_REF] Alessandri | Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro[END_REF][START_REF] Alessandri | A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC)[END_REF] we encapsulated human induced pluripotent stem cells (hiPSC) in liquid core capsules. Briefly, the working principle is the following: a three-layered cylindrical flow is generated using a co-extrusion microfluidic device: the cell suspension is surrounded by an alginate solution. Both solutions are separated by a sorbitol solution to prevent diffusion of calcium released from the cell suspension towards the alginate solution. Upon exiting the microfluidic device at high flow rate (on the order of 120 ml/h for all three solutions), the liquid jet is fragmented into small droplets due to the Plateau-Rayleigh instability. By contrast with the dripping regime obtained at lower flow rate that gives rise to droplets of size in the order of the capillary length (i.e. ~mm), the droplet radius is here dictated by the extruder's nozzle size (~200 microns diameter) [START_REF] Tomotika | On the instability of a cylindrical thread of a viscous liquid surrounded by another viscous fluid[END_REF]. This reduced size, which is below the distance over which oxygen and nutrients supply is limited within a tissue, allows to avoid the formation of a necrotic core [START_REF] Freyei | Regulation of Growth Saturation and Development of Necrosisin EMT6/R0 Multicellular Spheroids by the Glucose and Oxygen Supplyl[END_REF]. Once they fall in a calcium bath, the alginate solution droplets undergo gelation and trap the cell suspension in their interior. Our routine protocol produces capsules at a rate of about 3 kHz (Fig. S1), meaning that a 30 seconds operation generates 100,000 capsules. Morphological analysis shows that capsules are monodisperse in size with mean external radius R = 205 µm±39 µm. and that their shape is close to spherical, with a circularity parameter C= 0.84 ± 0.04 (Fig. 1B).

In order to provide a niche-like environment to hiPSCs, Matrigel, an ECM mixture, is co-injected with the cell suspension [START_REF] Alessandri | Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro[END_REF]. Empirically, we found that a minimal volume fraction of 25% was required to form a continuous matrix layer anchored to the inner wall of the capsule, with the excess (if any) being found as floating gel pieces inside the capsule [START_REF] Alessandri | A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC)[END_REF]. Most of the experiments reported in these works were performed with 50% of Matrigel in volume fraction. The granularity seen in the core of the capsule (Fig. 1E) thus corresponds to small floating aggregates of ECM. Most encapsulations reported hereafter were performed with a density of 0.4×10 6 cells/ml in the cell/matrix suspension, unless otherwise stated, and led to a mean number of cells per capsule right after encapsulation (day 0) of ~2.5 (Fig. 1C), meaning that ~10% of the capsules are empty, consistently with a Poisson distribution. After 6 to7 days of culture, practically defined as the harvest time under these seeding conditions, 3D colonies of hiPSCs were observed, suggesting, not only that hiPSCs survive, but also that they could proliferate (Fig. 1D). Higher magnification reveals the presence of a lumen (Fig. 1E).

Culture, expansion and stemness of encapsulated 3D hiPSC colonies

Using phase contrast imaging, we observed in more details the growth kinetics of these 3D hiPSC colonies. First, hiPSCs form a small cluster (typically during the first 24h) before self-organizing in a cyst structure around a central lumen (Fig. 2A, Movie S2 & S3). Then, the 3D hiPSC colonies grow within the capsules while keeping the same spherical shape. In the early stages, the cells within the monolayer of the cyst have a cuboid cell shape of about 10 µm side (Fig. 2B left, ~5 days post-encapsulation). Before harvesting, as seen in the confocal image of a representative 7 day-old hiPSC 3D colony immunostained for actin and nucleus, cells exhibit an elongated morphology perpendicular to the surface of the cyst. Yet, the cyst remains monolayered suggesting a transition towards a pseudostratified columnar epithelium with most nuclei being located on the basal side opposing the lumen. In this stage, the cysts are characterized by a thickness of about ~40 µm (Fig. 2B right) and a radius of about ~100 µm Fig. S2,day 7). Note that later stages are ignored. Indeed, if cells are not harvested, cysts become confined by the capsules and further grow inwards leading to a progressive loss of the lumen (Movie S4) and eventually the appearance of "fractures" (Fig S2 The maintenance of the stemness of the encapsulated 3D hiPSC colonies was then checked. The expression of key self-renewal markers such as OCT4 and SOX2 was first assessed after capsule dissolution, fixation and staining.

The alginate shell was dissolved by adapting the protocol of (29): we performed a short rinse of ReLeSR, which serves here as a calcium chelator that gently dissolves the alginate gel. Fixation and immunostaining were carried out following standard protocols (see Materials and Methods section) while preserving the 3D architecture. Image analysis of representative confocal images of individual cysts (Fig. 2C (top row) and Fig. S2) allows to derive that the percentage of cells positive for OCT4 and SOX2 is 97% (Fig. 2D top row). To further assess the consistency of stemness phenotype, we applied the approach pursued in a different context for epiblast-stage hPSCs spheroids by Freedman et al. [START_REF] Freedman | Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids[END_REF]. "Naked" hiPSCs cysts were dissociated and replated into 2D cultures (Fig. 2C). We observed that 2D colonies are readily formed and stemness markers are detected (Fig. 2C-D, bottom row) with a percentage of OCT4 ansd SOX2 positive cells larger than 98%.

Following this characterization at the scale of individual capsules, we sought to assess the potential variability between capsules and between hiPSC lines. We thus dissociated the bulk suspension cultures, extended staining to OCT4, SOX2 and NANOG and performed flow cytometry (Fig. 2E-F). For 4 different cell lines (see Materials and Methods section) by pooling all experiments (n = 42) of each hiPSC line, we found that the mean percentage of positive cells is 93% OCT4, 98% SOX2 and 92% NANOG (Fig. 2F). This finding is in good agreement with the above-described findings at the single 3D colony level, suggesting an overall homogeneity of the stem cell culture.

Note that, if capsules culture was prolonged beyond 7 days, despite drastic changes of topology from cyst to aggregate (Fig. S2 C-E and Movie S4), the stemness of the hiPSCs was not affected, as revealed by OCT4 and SOX2 staining before and after lumen collapse (Fig. S2 B-C), suggesting that harvest timing is not critically stringent with respect to the stemness maintenance.

Pluripotency and genomic integrity of encapsulated 3D hiPSC colonies

While OCT4, SOX2 and NANOG are often considred as pluripotency markers, they actually are stemness markers. To assess more thoroughly the pluripotency and validate the quality of hPSCs upon 3D culture in their ability to differentiate as bona fide pluripotent stem cells, the most commonly used assay is the trilineage differentiation assay [START_REF] Sheridan | Analysis of embryoid bodies derived from human induced pluripotent stem cells as a means to assess pluripotency[END_REF], in which stochastic differentiation is induced. Following a standard protocol [START_REF] Quelennec | Generation of two induced pluripotent stem cell lines IMAGINi004-A and IMAGINi005-A from heathy donors[END_REF] (see Materials and Methods section), decapsulated and dissociated hiPSC (from the three available cell lines) were driven towards early differentiation, as shown by the stainings for specific markers of the 3 germ layers: namely b3-tubulin (TUJI) for ectoderm, smooth muscle actin (α-SMA) for mesoderm and a-fetoprotein (AFP) for endoderm (Fig. 3A). Even though the expression level may differ from one cell line to another, all stainings are positive and clearly reveal a differenciation into the three germ layers.

To further quantify the differentiation potential after 3D culture within capsules, we used qPCR Scorecard TM assay to evaluate the transcription profile of the cells obtained in the trilineage assay (Fig. 3B). The set of 94 previously validated qPCR markers of self-renewal, ectoderm, mesendoderm, mesoderm and endoderm [START_REF] Tsankov | A qPCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells[END_REF][START_REF] Bock | Reference maps of human es and ips cell variation enable high-throughput characterization of pluripotent cell lines[END_REF] was used to compare standard 2D culture and 3D culture-in-capsules. Fig. 3B shows that, for a given marker and a given cell line, there is a striking similarity between the transcription signatures in 2D and 3D culture conditions, indicating that pluripotency assessed as the in vitro differentiation capability of hiPSC clonies is definitely not altered in our encapsulated 3D colonies. A pooled analysis by germ layer (Fig. S4) confirms similar differentiation profile between 2D and 3D stem cells.

Finally, to control genomic integrity of the 3D colonies, we performed high resolution SNP (single nucleotide polymorphism) arrays before and after amplification within the capsules (Fig. 3C and Fig. S5) [START_REF] Assou | Concise Review: Assessing the Genome Integrity of Human Induced Pluripotent Stem Cells: What Quality Control Metrics?[END_REF]. Comparative SNP analysis showed the absence of aneuploidies, deletions or duplications, as evidenced by the superimposable karyotypes. The high degree of SNP concordance (>99.8% for all hiPSC lines) before and after encapsulation confirms cell line identity (Fig. 3D).

Comparative growth of 3D hiPSC colonies at the scale of a single capsule, in a static suspension and in bioreactors

To evaluate whether the strategy to produce 3D colonies in ECM-coated capsules impacts the growth and expansion rates of hiPSC, we performed a series of sytematic experiments to probe the cell growth kinetics. The standard 2D cell cultures were taken as a control. Since the amplification factor is defined as AF=N(t 0 +Dt)/N(t 0 ), where N(t 0 ) and N(t 0 +Dt) are the cell numbers at the initial time t 0 and t 0 +Dt respectively, direct cell counting at day 6 after passaging give a mean AF 2D (t=6 days)~13. Since, by definition, AF(Dt)=2 Dt/PDT , with PDT the cell population doubling time, by pooling amplification factors at different harvesting time, we obtain a mean PDT 2D =34h ±5 hours for iPS C line, which falls within the range of data reported in the literature [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors[END_REF][START_REF] Adil | Expansion of human pluripotent stem cells[END_REF][START_REF] Ludwig | Derivation of human embryonic stem cells in defined conditions[END_REF].

Then, in order to characterize the growth of individual encapsulated 3D hiPSC colonies, we cultured them in 35 mm petri dishes (typically as few as 10 capsules in a volume of medium ~5ml, permitting to conserve the same medium for the whole course of the experiment without any risk of nutrient depletion and acidification). We performed time-lapse phase contrast imaging over a one-week period. We assume that cell volume remains constant, which allows us to derive AF capsule (Dt)= V(t 0 +Dt)/V(t 0 ) by measuring the volume of the cyst V(t) from image analysis:

( )

3 3 4 ( ) 3 out in V t R R π = -
, where R in and R out are the average internal and external radii of the cyst (see notations on Fig. 4A). Figure 4B shows the evolution of AF capsule as a function of time for individual encapsulated 3D colonies. One immediately observes that AF capsule (t=7 days)= 212. Additionally, since AF increases exponentially as ሻݐ∆ሺܨܣ ൌ exp ሺ ଶ.∆௧

் ೌೞೠ ሻ , one finds PDT capsule = 22±1 hours.

With the perspective of scaling up the production of hiPSCs, we also investigated how the growth of encapsulated 3D hiPSC colonies was impacted when cultured in conditions of i) static bulk suspension in standard T-Flasks (Fig. 4B) and ii) stirred suspension in a benchtop bioreactor (Fig. 4C). STBRs are the most common bioreactors used to culture biological agents for biotechnological applications. Besides ther capacity to monitor and adjust the pH and oxygen partial pressure and to refresh the medium, the mechanical agitation provided by the impellers allows better fluid mixing and oxygen transfer ability as compared to static suspension [START_REF] Kropp | Progress and challenges in large-scale expansion of human pluripotent stem cells[END_REF]. However, the drawback may also be that the shear stess induced by the impellers was shown to cause deleterious effects such as cell death or decrease in cell growth in aggregate-or microcarrier-based cultures [START_REF] Croughan | Effects of microcarrier concentration in animal cell culture[END_REF][START_REF] Hewitt | Expansion of human mesenchymal stem cells on microcarriers[END_REF]. Practically, we loaded capsules from a same batch in T-flasks and in a STBR at the same initial density. The bioreactor impeller rotational speed was set to 150 rpm, which is sufficient to maintain medium homogeneity and avoid capsule sedimentation. We could not detect any change in the shape of the capsules and 3D colonies under these stirring conditions. Then, after dissolution of the alginate shell and dissociation of the cysts, we counted the stem cells in both static and stirred culture conditions to derive the amplification factors AF in time. We found AF flask (7 days)= 109 ± 6 and AF bioreactor (7 days)= 104 ± 18 (Fig. 4D). From the values of the characteristic times for the exponential variation of AF, we could calculate, as explained above, PDT flask = 25± 6h and PDT STBR =25±3 h (Fig. 4E). Three remarks can be made. First, these PDT values are significantly lower than the ones derived from 2D cultures, indicating again than the expansion is greatly improved in 3D, as evidenced by the low number of dead cells in capsules (Fig. 4F) as compared with 2D colonies (Fig. S6). Second, the AF values are about twice as low as the one derived from the masurements at the single capsule level. Third, the absence of statistical difference between the two culture systems suggests that, while the impeler-induced shear stress does not affect cell viability, stirred suspension culture in a benchtop bioreactor with expected better homogenization does not enhance the expansion under the experimental conditions selected here.

Additionally, we performed flow cytometry analysis and found that more than 92% of the cells are positive for SOX2, NANOG and OCT4. Stemness thus remains high and similar between static and stirred cultures (Fig. S7), indicating that, by contrast with previous reports [START_REF] Kehoe | Scalable Stirred-Suspension Bioreactor Culture[END_REF], shear stress does not trigger the differentiation of hiPSC colonies grown in hollow capsules. Finally, in order to confirm that the amplification factors reported above are not hiPSC line specific, we carried out the same series of experiments for the other 3 cell lines in static culture (Fig. 4E). Not only are the differences between cell lines not significant, but their PDT in 3D is also found very close to the value derived for the commercial hiPSC line that we have extensively investigated in this section, i.e.

PDT flask = 27± 2 h by averaging over all 3 cell lines.

Optimized culture conditions: Impact of capsule size and oxygen tension

As shown above, the static or stirred batch cultures of hiPSC colonies in capsules yielded amplification at day 7 about twice as low as the one measured at the single capsule level. Although this difference only corresponds to a 3h difference in PDT, we sought to address this issue and find out solutions to further improve the amplification of hiPSC in batch for large-scale production. We investigated the impact of two possible parameters.

First, we tested whether hiPSC amplification depends on the cell seeding density. The most obvious way would be to increase the volume fraction of cells in the core solution loaded to the microfluidic injector. However, this would lead to earlier and more frequent harvesting. Instead, we pursued a different strategy. We kept the cell density constant but increased the size of the capsules by changing the size of the nozzle [START_REF] Alessandri | Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro[END_REF]. Doing so, for a given volume of the encapsulation cell suspension, the number of produced capsules is indeed reduced by (R big /R small ) 3 but the mean number of cells per capsule, λ, is increased by the same fold. In the context of sparse distribution, Poisson statistics applies and the generation of 300 µm in radius capsules instead of 200 µm leads to an increase in λ by about 3 fold. The immediate consequence is that the probability to have capsules containing no cell is decreased from about 8% to negligible (~0.03%). But, more importantly, the probability to have capsules with only one cell that may die or exhibit some lag phase before proliferation goes from 20% to 0.3%. High occurrence of capsules loaded with only one cell is expected to lower the effective amplification factor and thus to increase the doubling time of the cell population. Fig. 5A shows representative phase contrast images of 300 µm in radius capsules filled with hPSC colonies. Noteworthily, most capsules contain several cysts, suggesting that cyst growth was nucleated from several cell aggregates. More quantitatively, after monitoring the growth kinetics in benchtop STBR, derivation of the time constant reveals shorter doubling time in big capsules: PDT big =22h±1h

< PDT small =25h±1h (Fig. 5B).

Second, we pursued along our physiomimetic approach. Among all factors that make a stem cell niche, we have already recapitulated interactions with the ECM. However, untill now, we have omitted to consider oxygen tension, which is known to be naturally low in developping embryos [START_REF] Simon | The role of oxygen availability in embryonic development and stem cell function[END_REF]. This low level of oxygen was further shown to be key to reduce mutation rates and epigenetic alterations [START_REF] Thompson | Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions[END_REF][START_REF] Okazaki | Oxygen, epigenetics and stem cell fate[END_REF] as well as to improve the expansion rate (46) while reducing the probability of unwanted differentiation [START_REF] Ezashi | Low O2 tensions and the prevention of differentiation of HES cells[END_REF]. We thus performed the same culture experiments in big capsules (300 µm radius) by decreasing the dissolved oxygen level (DO) from 100% to 20%.

(see Materials and Methods section). Under these hypoxic conditions, the population doubling time derived from the growth kinetics was found to be ܶܦܲ ௬௫ ൌ 20.4݄ (Fig. 5B), i.e, slightly but significantly shorter than in normoxic conditions. Remarkably, this value is better than the one found at the single capsule level in normoxia, suggesting that the optimization of capsule size and oxygen tension allowed to upscale the production of hPSCs in a bench-scale bioreactor without any degradation of the expansion efficiency.

In order to assess whether this protocol is not only theoretically scalable but can be truly upscaled to an industrial level, we carried out a final experiment in duplicate in a 10 L STBR (Fig. 5C, S8 and Movie S5) by keeping all other parameters constant. Figure 5D shows the the expansion-fold as a function of time. The two curves from these two independent experiments are superimposable and we found AF 10L STBR (6.5 days)=277, corresponding to a doubling time PDT 10L STBR =19.6 h.

These data were obtained without passaging. In order to assess the robustness of the approach for a seed train of passaging and demonstrate that the technology can be integrated into a classical cell therapy production, we performed serial passaging. At harvest after about 7 days, the capsules were dissolved, the 3D colonies were dissociated and cells re-encapsulated following the same protocol (Fig. 6). We found that expansion-fold and stemness were preserved over two consecutive encapsulations within 14 days (Fig. S9).

DISCUSSION

In this work, we have developed an in vitro culture system for hiPSCs, that we named C-STEM (Fig. 6) and combines the benefits of biomimetic 3D culture and scalable bioreactor-based production. By contrast with other approaches using scaffold embedding in bulk matrix [START_REF] Lei | A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation[END_REF] or hydrogel beads [START_REF] Swioklo | Process parameters for the high-scale production of alginate-encapsulated stem cells for storage and distribution throughout the cell therapy supply chain[END_REF][START_REF] Serra | Microencapsulation technology: A powerful tool for integrating expansion and cryopreservation of human embryonic stem cells[END_REF], our hollow capsules allow in-situ engineering of stem cell niche-like microenvironment. With biological and topological cues driving 3D self-organization of hiPSCs colonies that are reminiscent of epiblasts [START_REF] Shahbazi | Deconstructing and reconstructing the mouse and human early embryo[END_REF]. While cell-cell interactions are not impaired due to the absence of enwrapping scaffold, the presence of the shell also provides mechanical protection against impeller damage and turbulence-induced so-called Kolmogorov eddies during stirring [START_REF] Ponnuru | Analysis of stem cell culture performance in a microcarrier bioreactor system[END_REF]. Besides exploiting both the biomimetic and protective properties of the developed C-STEM platform, we have finally refined the culture conditions by optimizing the initial mean number of cells per capsule and the oxygen tension.

Combination of all these critical factors allowed us to upscale the production of hPSCs and demonstrate that the amplification efficiency is scale-independent. In particular, we could reach 282-fold amplification in 6.5 days in a 10 L stirred-tank bioreactor.

To the best of our knowledge, this level of both amplification and scalability is unmatched in the field [START_REF] Adil | Expansion of human pluripotent stem cells[END_REF][START_REF] Polanco | Bioprocess Technologies that Preserve the Quality of iPSCs[END_REF].

This technical and quantitative tour de force is actually related to the higher cell viability obtained in our hollow capsules as compared with 2D cultures or other 3D suspension cultures. Since the amplification factor AF(t) of a culture system is given by ሻݐሺܨܣ ൌ 2 ௧/் , where PDT is the population doubling time and accounts both for cell division and cell death, we may rewrite it as ሻݐሺܨܣ ൌ 2 ሺ శ ି ష ሻ௧ , where k + and k -are respectively the division and death rate of cells. Thus, the upper theoretical limit for AF is obtained for k -=0 (i.e. infinite death time), which then yields a minimal PDT min value, equal to k + . Quite surprisingly, the measurement of k + or the duration of the cell cycle t + =1/k + of hiPSC has been overlooked in the literature. The sole report we are aware of arises from a recent work in which cell cycle kinetics was tracked using a rainbow reporter in primed pluripotent stem cells [START_REF] El-Nachef | A Rainbow Reporter Tracks Single Cells and Reveals Heterogeneous Cellular Dynamics among Pluripotent Stem Cells and Their Differentiated Derivatives[END_REF]. The obtained cell cycle duration was found to be t + ~14 h. While the question of "gemellarity" between embryonic stem cells (ESC) and hiPSC is still under debate [START_REF] Narsinh | Comparison of human induced pluripotent and embryonic stem cells: Fraternal or identical twins?[END_REF], this value is also consistent with previous estimates of t + =11-16-h for the cell cycle duration in human and mammalian ESC [START_REF] Liu | The cell cycle in stem cell proliferation, pluripotency and differentiation[END_REF][START_REF] Orford | Deconstructing stem cell self-renewal: Genetic insights into cell-cycle regulation[END_REF][START_REF] Padgett | From clocks to dominoes: lessons on cell cycle remodelling from embryonic stem cells[END_REF]. By assuming that the duration of the cell cycle in the 3D cyst topology is identical to the value obtained in 2D cultures with AF(t=6.5 days)=282 corresponding to PDT=19h, we find that the doubling time of the encapsulated hiPSC colonies is only 5h±2h longer than the intrinsic cell cycle duration. The difference corresponds to a death rate k -=1/53h -1 . By comparison, in the seminal Yamanaka's paper [START_REF] Takahashi | Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors[END_REF], doubling times in 2D hiPSC colonies of about 45 h indicate that k -=1/20h -1 . More meaningful than the death rate k -, the fraction of dead cells can be roughly estimated as

߶ ௗௗ ൎ ଶ ೖ ష ଶ ುವ
=2.6% at t=6.5 days, while cell counting gives 1.30% in bioreactors and 1.97% in flasks (Fig. S6). In 2D cultures, we measured a fraction of dead cells of the order of 12% at harvest, even though this value underestimates the cumulated mortality which is drastically impacted by cell passaging and confluency [START_REF] Li | Scalable and physiologically relevant microenvironments for human pluripotent stem cell expansion and differentiation[END_REF]. Further expansion improvement is theoretically within reach by vanishing the cell death rate. Taking again t + ~14 h for the cell cycle length of hiPSC, the glass ceiling is calculated to be AF max (t=6.5 d)~2200. However, the value of t + ~14 h cannot be taken as granted. For instance, a cell density-dependence on the proliferation rate has been reported.

Indeed, hPSC exhibit a decelerated proliferation due to a prolonged G1 phase as cell density increases [START_REF] Wu | Increased Culture Density Is Linked to Decelerated Proliferation, Prolonged G1 Phase, and Enhanced Propensity for Differentiation of Self-Renewing Human Pluripotent Stem Cells[END_REF].

Similarly, smaller expansion folds were observed when the inoculation density either as single cells or preclusters in a synthetic hydrogel exceeds 10 6 cells/mL [START_REF] Lei | A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation[END_REF]. We may then anticipate that the actual average cell cycle duration could be longer than the one reported above upon single-cell lineage tracking in small colonies of hPSCs. As a consequence, even though future work should aim at a rigorous in situ measurement of the division rate within the encapsulated hiPSC cysts, we cannot exclude that the unprecedented hiPSC expansion rates reported in this work are approaching the glass ceiling.

Among other specificities of the C-STEM technology, we mentioned the protective role of the alginate shell and the scale-independence of the culture conditions. By contrast with other suspension cultures that need to design specific low-shear impellers (e.g. the vertical-wheel bioreactor, [START_REF] Borys | Overcoming bioprocess bottlenecks in the largescale expansion of high-quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors[END_REF][START_REF] Nogueira | Strategies for the expansion of human induced pluripotent stem cells as aggregates in singleuse Vertical-Wheel TM bioreactors[END_REF], or to add shear-dampener polymers (e.g. pluronics, [START_REF] Manstein | High density bioprocessing of human pluripotent stem cells by metabolic control and in silico modeling[END_REF]) in order to avoid stirring-induced cell damages, our capsules permit the use of standard industrial scale bioreactors.

However, all the benefits cannot be assigned to this sole shielding effect. Indeed, previous works had already proposed to embed either hESC aggregates or microcarriers within hydrogel beads (which are referred to as capsules in these original works) [START_REF] Serra | Microencapsulation technology: A powerful tool for integrating expansion and cryopreservation of human embryonic stem cells[END_REF] to improve cell viability. Nonetheless, expansion rates were not reported to be larger than 10 in 19 days. Similarly, two recent works describe stem cells encapsulation in hollow capsules [START_REF] Fattahi | Core-shell hydrogel microcapsules enable formation of human pluripotent stem cell spheroids and their cultivation in a stirred bioreactor[END_REF][START_REF] Horiguchi | Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle[END_REF]. However, the absence of ECM leads to the formation of aggregates and a modest amplification (estimated to 70-fold in 8 days from the size of the encapsulated spheroids). We thus propose that the significant amplification increase obtained with the C-STEM technology mostly originates from the stem cell niche-like environment that is engineered within each capsule and that drives hiPSC multicellular organization into cysts.

Interestingly, numerous studies have recently proposed biomimetic controllable environments that can be used to develop hPSC-based embryo models and more specifically epiblast models [START_REF] Shahbazi | Mechanisms of human embryo development: from cell fate to tissue shape and back[END_REF][START_REF] Vianello | Understanding the Mechanobiology of Early Mammalian Development through Bioengineered Models[END_REF][START_REF] Irizarry | Bioengineered pluripotent stem cell models: new approaches to explore early human embryo development[END_REF]. In all cases, these 3D stem cell niche mimics drive PSCs self-organization, luminogenesis, and polarization into pseudo-stratified epithelia [START_REF] Taniguchi | Lumen Formation Is an Intrinsic Property of Isolated Human Pluripotent Stem Cells[END_REF][START_REF] Shahbazi | Pluripotent state transitions coordinate morphogenesis in mouse and human embryos[END_REF][START_REF] Zheng | Controlled modelling of human epiblast and amnion development using stem cells[END_REF]. This cyst configuration, which seems to be key in developmental processes, may be regarded as an optimized configuration for hPSC expansion with minimal loss of viability [START_REF] Zhu | Amnion-on-a-chip: modeling human amniotic development in mid-gestation from pluripotent stem cells[END_REF]. First, it is well accepted that 2D hiPSC cultures exhibit intra-colony heterogeneities in pluripotency marker genes [START_REF] Hamidi | Mesenchymal-epithelial transition regulates initiation of pluripotency exit before gastrulation[END_REF], viability (69) and cell morphology [START_REF] Wakao | Morphologic and Gene Expression Criteria for Identifying Human Induced Pluripotent Stem Cells[END_REF], which are very striking between the center and the edges of the colonies. In this respect, the closed spherical symmetry of a cyst intrinsically suppresses the "center-edge phenotype" and may result in more homogeneous cell population [START_REF] Hashimoto | Epiblast Formation by TEAD-YAP-Dependent Expression of Pluripotency Factors and Competitive Elimination of Unspecified Cells[END_REF]. Additionally, whereas cellular crowding or compaction are known to inhibit proliferation or even trigger apoptosis via caspase-dependent mechanisms (72) a cyst configuration is less prone to stress building in bulk due to the presence of a lumen. Similarly, fast proliferation rate may contribute to stress relaxation and reduce cell extrusion occurrence as observed in epithelia under compression [START_REF] Fadul | The forces and fates of extruding cells[END_REF][START_REF] Hannezo | Mechanochemical Feedback Loops in Development and Disease[END_REF]. Besides mechanical stress, chemical stresses are known to increase the mutation rate [START_REF] Thompson | Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions[END_REF][START_REF] Halliwell | Nucleosides Rescue Replication-Mediated Genome Instability of Human Pluripotent Stem Cells[END_REF]. The use of bioreactors with precise adjustment of physioxia, pH, lactate, glucose and nutriments supply is thus instrumental and could be optimized beyond the present achievement.

Finally, relying on the observation that chromosome segregation fidelity is unambiguously higher in native contexts of epithelia of primary cells [START_REF] Knouse | Chromosome Segregation Fidelity in Epithelia Requires Tissue Architecture[END_REF][START_REF] Mckinley | Cellular aspect ratio and cell division mechanics underlie the patterning of cell progeny in diverse mammalian epithelia[END_REF], it also becomes tempting to speculate that, beyond the gain in amplification it provides, the preserved histology of our in-vitro epiblast-like colonies could also be beneficial to the maintenance of the genetic integrity [START_REF] Singla | Autophagy-mediated apoptosis eliminates aneuploid cells in a mouse model of chromosome mosaicism[END_REF].

In summary, our work has shown that hollow alginate capsules with reconstituted niche-like microenvironement can promote the formation and growth of 3D hPSC colonies and provide the necessary protection for scaling up the production in stirred tank bioreactors. Self-organized encapsulated epiblast-like colonies seem to be instrumental for optimal expansion by preserving stem cells physiological properties. We have demonstrated that our biomimetic stem cell platform C-STEM can deliver unprecedented scalability and we anticipate that cell quality is preserved on the basis of extremely high viability, which is taken as a primary signature of cell fitness.

Future works should focus on assessing the hPSCs quality in in vivo-like culture systems, since the emergence of mutations during culture may be the last limitation to overcome for cell therapy bioproduction.

MATERIALS AND METHODS

Ethics statements

The generation, use and storage of hiPSCs were performed with approval from the "Comité de Protection des

Personnes" (CPP) Ile de France (DC 2015-2595 and 2016-A00773-48).

Human pluripotent stem cell lines

Throughout the present work, we used 4 hiPSC lines. Among these, 3 hiPS cells, namely IMAGINi004-A (referred to as iPS004), IMAGINi005-A (iPS005) and IMAGINi013-A (iPS013) were derived from peripheral blood mononuclear cells (PBMC) according to the protocol described in [START_REF] Quelennec | Generation of two induced pluripotent stem cell lines IMAGINi004-A and IMAGINi005-A from heathy donors[END_REF]. Briefly, PBMCs were transduced using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific) following the manufacturer's instructions. After 2-3 weeks, colonies were manually picked and expanded at least 10 passages. The 4 th hiPSC line is a commercial line from ThermoFisher: Gibco TM episomal hiPSC line (A18945) generated using cord blood derived CD34+ progenitors with 7 episomally expressed factors (Oct4, Sox2, Klf4, Myc, Nanog, Lin28, and SV40 T). This commercial cell line is referred to as iPS C. For the sake of availability , in order to allow other groups to reproduce our findings, all experiments reported here were performed with iPS C, except for those that are described in Figures 2F, 3A,B,D 4E and Figures S4 S5, which were carried out to demonstrate that the findings were not cell line-dependent.

2D hiPSC culture

All hiPSC lines were maintained on Matrigel (Corning Ref. 354234) and cultured in mTeSR1 medium (StemCell Technologies 85875). Cultures were fed daily, passaged with an enzyme-free reagent, ReleSR (StemCell Technologies 05873) every 3-6 days (around 80% confluency) and replated as small clusters (between 100 and 200 µm) at a density of about 5000-10000 cells/cm 2 . Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

3D hiPSC Encapsulation

Prior to encapsulation, 2D stem cell colonies were detached using ReLeSR for 1 minute and dissociated into a near single cell solution using Accutase (Stem Cell Thechnologies 07920). HiPSCs were then mixed in a 50/50 volume ratio with Matrigel at 4°C to keep the suspension in a liquid state. The final concentration of cells in the cell/matrix solution was thus between 0.4-1.0×10 6 viable cells/mL, referred to as the encapsulation density. The encapsulation system is similar to the one described in [START_REF] Alessandri | A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC)[END_REF]. In brief, ethylene tetrafluoroethylene (ETFE,) tubings are connected to the three inlets of a 3D printed (using the DLP Micro Plus Hi-Res printer from EnvisionTEC) microfluidic co-laminar flow device. An extruded and polished glass microcapillary tip (of diameter ~100 µm for most experiments reported in this work, at the exception of those shown in Fig. 5A-B that were carried out with a nozzle diameter of 150 µm) is glued to the outlet of the nozzle for a better control of the flow. The cell/matrix suspension is loaded into the inner channel of the 3-way device, which is kept refrigerated thanks to an in-line cooling system in order to prevent premature gelation of Matrigel. A solution of sodium alginate (Novamatrix Pronova SLG100, 0,25 g #4202101 at 2% in distilled water) is injected into the outer channel. To prevent alginate gelation within the microfluidic device due to calcium release from the suspended cells, a calcium-free solution (Sorbitol 300mM, Sigma-Aldrich 85529) is used in the intermediate channel of the co-extrusion chip and serves as a barrier against calcium diffusion. Typical flow rates for the 3 solutions were on the order of 120 ml/h for all three channels: (alginate solution, the sorbitol solution and the cell+matrix suspension). At these rates, the composite solution forms a liquid jet that fragments into droplets (of about twice the size of the nozzle) due to the spontaneous Rayleigh-Plateau instability. To avoid subsequent coalescence of the train of droplet, an alginate charging part and a copper ring are connected to a high voltage (2000V) generator are introduced. A high-speed camera (PHANTOM VEO 1310L) was used to visualize droplet formation and splay (Fig. S1and Movie S1).

When the composite droplets contact the collecting calcium bath (at 100mM), the outer layer of alginate readily gelates. As a consequence, the inner cell/matrix solution remains entrapped inside a closed, spherical and permeable micro-compartment. Within 1 min following encapsulation, capsules are rinsed with medium (DMEM)

to reduce the basal calcium concentration. Finally, ther are transferred to suspension culture medium.

Re-encapsulation was performed by dissolution of alginate shells using short rReleSR rinse, followed by cell dissociation using TrypLE (Trypsin-based, dissociation enzyme, ThermoFischer) for 20 minutes at 37° . Then the obtained cells were processed following the classic encapsulation protocol.

3D stem cell suspension culture in static T-Flasks or bioreactors

Static suspension cultures of encapsulated hiPSC were carried out using T-Flasks (from 5 to 30 ml) maintained in a cell culture incubator at 37°C and 5% CO 2 . The medium (mTeSR1) was supplemented with 10µM Y-27632 for ROCK inhibition only during the first 24 hours of culture. From culture day 3, the medium was exchanged every day as described hereafter. The contents of the T-Flasks were transferred into Falcon Tubes. After capsules sedimentation (within a few minutes), the supernatant was removed and replenished as the capsules were transferred back into a T-Flasks. The volume of culture medium was kept constant for the first 4 days of culture (~ 4× the capsules volume). Then, the volume was steadily increased every day in order to maintain a cell concentration bellow 10 6 cells/mL.

Stirred suspension cultures were performed in different bioreactors. For all experiments reported in Fig. 4 and Fig.

5A-B, we used benchtop STBRs, including a 30 mL (Minibio, ABLE® Bioreactor Systems) or 500 mL bioreactors (Applikon Biotechnology & Global Process Concept). For the experiments reported in Fig. 5C-D, we used a 10 liter -scale bioreactor (Global Process Concept).

In all cases, the bioreactors were inoculated with 15% capsule-to-medium volume. The bioreactor culture starts at a volume representing 30% of the final working volume. At Day1, the medium was replaced with fresh medium without ROCK inhibitor. From day 2 to 5, the culture is performed in a fed-batch mode up to the final working volume [START_REF] Kropp | Progress and challenges in large-scale expansion of human pluripotent stem cells[END_REF]. Then, we switched to repeated-batch mode, where 90% of the media was daily renewed to maintain sufficient nutriment supply. The final capsule-to-medium volume was 4.2 ± 0.3% and the pH was maintained at 7.2 ±0.2.

Dissolved Oxygen (DO) level is calibrated at 100% in starting conditions by injecting air into the bioreactor headspace. During the run, the oxygen level is monitored and controlled. In normoxic conditions the oxygen is controlled at 100% while in hypoxic conditions the set point is at 20% DO. Oxygen level is regulated by sparging nitrogen and/or air to maintain the set point. 10L scale bioreactors were regulated in hypoxic conditions. During one week of culture, the stirring speed is set at 150 rpm that is sufficient to keep capsules resuspension and bioreactor homogeneity along the run.

Time-lapse microscopy of encapsulated cyst growth and image analysis

Time-lapse microscopy was performed using a Nikon Biostation IM microscope with a 10x objective. Capsules containing hiPSCs were transferred to a 35 mm Petri dish 24 hours after encapsulation. Approximately 10 to 20 capsules were placed in the petri dish containing 5 mL of fresh Y-27632-free mTeSR1 medium. Cyst growth was monitored over 7 days. Practically, images were taken every 6 to 10 minutes at preselected Z-focal planes to ensure acquisition at proper focus in case of undesired drift Image analysis was performed using ImageJ. The external and internal effective radii of the cysts, R out and R in , were measured from the equatorial corresponding cross sections S according to R out,in =(S out,in /π) 1/2 after applying appropriate bandpass filters and thresholds. The volume V of the cells was calculated as V=4π/3(R out 3 -R in 3 ). Capsule circularity was defined as C=a 2 /b 2 , where a and b are the short and long axes of the approximated ellipse.

In vitro trilineage differentiation

Small cell clusters were collected from hiPSC cultures (2D or decapsulated-dissociated 3D hiPSCs colonies) and transferred into low attachment dishes (Corning, Ultra-low attachment 6 well plate). Three-dimensional aggregates of cells that are an amalgam of the three developmental germ layers (Embryoid bodies) are obtained and cultured in suspension for 7 to 9 days with DMEM/F-12 medium containing 20% pluriQ Serum Replacement (GlobalStem), 1% non-essential amino acids, 1% penicillin-streptomycin and 0.2% β-mercaptoethanol (ThermoFisher Scientific) in a humidified atmosphere containing 5% CO2. Culture medium was refreshed every two days. EBs were then collected for RNA analyses or transferred onto gelatin-coated dishes for 1 week. For immunocytochemistry analysis, cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT).

After washing in PBS/1%BSA blocking solution for 1 hour, cells were incubated overnight at 4°C with primary antibodies, washed 3 times in PBS, and incubated with secondary antibodies for 2 hours at RT. Antibodies were diluted in PBS/1%BSA/0.1%triton solution. The list of antibodies used in this work and their origin are listed in Table S1. Nuclei were stained with a DAPI solution. Immuno-fluorescence staining was analyzed using the Celena S™ Digital Imaging System (Logos Biosystems).

RNA extraction and RT-PCR analyses

Total RNA was extracted using the RNeasy Mini Kit (Qiagen). cDNA was synthesized using a high capacity cDNA RT kit (ThermoFisher Scientific) from 1μg of total RNA. The expression of pluripotency markers as well as the trilineage differentiation potential of the cells were evaluated by TaqMan® hiPSC Scorecard™ assay according to the manufacturer's protocol. This scorecard compares the gene expression pattern of key pluripotency and germ lineage markers relative to a reference standard that consists of 9 different human ES and iPS lines. Data analysis was performed using the cloud based TaqMan® hiPSC Scorecard TM analysis software.

DNA isolation, genomic stability and authenticity analysis

DNA isolation was performed using the PureLink™ Genomic DNA Mini Kit (Invitrogen). Molecular karyotype was performed using an Infinium Core-24 v1.2 Kit (Illumina) containing 300000 SNPs. Data were analyzed with BeadStudio/GenomeStudio software (Illumina). The percentage of SNP concordance between iPSC samples before and 7 days after encapsulation was assessed for the 3 derived iPS cell lines. SNP files of all samples were extracted from genome studio software. The percentage of concordance between two paired samples (before and after encapsulation) was evaluated by comparing the genotype of each informative SNP (Fig. S5)

Flow cytometry analysis

The hiPSCs colonies were dissociated with Accutase for 10 minutes at 37°C for 2D cultures or with TrypLE Select (ThermoFisher Scientific 11598846) for 30 minutes at 37°C for 3D cultures after capsule removal. Then, cells were fixed and permeabilized using the Transcription Factor Staining Buffer Set (ThermoFisher 11500597).

Cells were suspended in the permeabilization buffer at a density of 0.5-1×10 6 cells in 100 μl and incubated with the specific antibodies or isotype controls (Table S1) for 45 minutes at room temperature in the dark. Cells were washed twice with the staining buffer and analyzed using either BD Canto II (at the TBMCore CNRS UMS 3427 -INSERM US 005) or a BD Accuri TM C6 plus and data was post-processed with FlowJo software.

Cell growth and viability analysis

Cell counting was performed using the Nucleo counter NC3000 or NC200 (Chemometec). Live/dead analysis was performed using CalceinAM/Ethidium homodimer-1 (ThermoFisher L3224) according to the manufacturer recommendations, and samples were imaged using either the EVOS FL or EVOS M5000 auto Imaging system (ThermoFischer).

Immunostaining, Microscopy, and Image Analysis

For daily brightfield imaging of 2D cultures and encapsulated hiPSC cysts, a widefield EVOS FL or EVOS M5000 automated microscope was used. Encapsulated 3D hiPSC colonies were harvested for confocal microscopy at several timepoints. The alginate capsule was removed prior to fixation by incubating the samples in PBS without divalent cations for at least 5 minutes with agitation at RT. Both 2D and 3D stem cell colonies were fixed with 4% PFA for 30-60 minutes at RT in the dark. Following fixation, the samples were washed 3x with 0.1% Tween20 in PBS. A permeabilization step was done in parallel with excess PFA quenching in a PBS solution containing 0.3% Triton X-100 and 100 mM Glycine for 30 minutes, followed by 3x washing with 0.1% Tween20 in PBS. The samples were incubated in primary and secondary antibodies (Table S1) in 1% BSA + 0.1% Tween20 in PBS overnight at 4° with gentle orbital agitation, including a 3x rinsing with 1% BSA + 0.1% Tween20 in PBS after each incubation. To maintain alginate capsules during fixation and staining, the decapsulation step was skipped and all solutions (including 4% PFA) were supplemented with calcium and magnesium. All samples were imaged on either a Leica SP5 or SP8 confocal microscope (Bordeaux Imaging Center, BIC).

Statistical Analysis

All statistical analyses were performed using GraphPad Prism8. To test significant differences between bioreactor and flask, 2wayANOVA with sidak's multiple comparaison test was used. the SNP concordance analysis. Decapsulated and dissociated cells were fixed and stained for OCT4, SOX2 and NANOG and analyzed by flowcytometry, BD Accuri C6 plus (Table S1).

Fig. 1 .

 1 Fig. 1. Encapsulation of human pluripotent stem cells (hPSCs) and suspension culture of 3D lumenized

Fig. 2 .

 2 Fig. 2. Morphology, growth and stemness of in capsulo self-assembled 3D hPSC colonies

Fig. 3 .

 3 Fig. 3. Maintenance of pluripotency and genomic integrity in 3D hPSC colonies

Fig. 4 .Fig. 5 .

 45 Fig. 4. Amplification of 3D hiPSC colonies at the scale of a single capsule, in a static suspension and in

Fig. 6 .

 6 Fig. 6. C-STEM pipeline: encapsulation and scale-independent culture of encapsulated 3D hPSC colonies in

Fig. S2 .

 S2 Fig. S2. Phenotype of encapsulated 3D hPSC colonies (A) Graph of hPSC cyst radius as a function of time. Radii were measured by monitoring n=25, 47, 52, 61 capsules at days 4, 5, 6 and 7 respectively. (B) 3D hPSC colony co-stained for stemness markers NANOG (cyan) and OCT4 (green) and with Hoechst (blue). Projection of maxima (upper panel) and equatorial plane (lower panel) of confocal images. Scale bar is 50µm. (C) Confocal equatorial plane of a fully confluent hPSC capsule (t=10 days post-encapsulation) co-stained for OCT4 (green), SOX2 (red), and nuclei (Hoechst, blue) and showing that the lumen has collapsed. Scale bar is 100µm. (D) Sequence of phase-contrast microscopy images showing luminogenesis, growth and collapse of an encapsulated epiblast-like colony. The time interval between successive images is 18h. Scale bar=100µm. (E) Confocal equatorial plane of a collapsed 3D hPSC colony co-stained for F-actin (Phalloidin, magenta) and nuclei (Hoechst, blue), Scale bar=100µm.

Fig. S3 .

 S3 Fig. S3. Stemness staining of encapsulated 3D hPSC colonies Four different representative encapsulated hPSC colonies co-stained for OCT4 (green) SOX2 (red) and nuclei (Hoechst, blue). The 2 colonies in the upper panels (A) and (B) are collected at day 5. The 2 colonies in the lower panels (C) and (D) are collected at day7. White arrows and dots illustrate the quantification method used to determine OCT4 (green) and SOX2 (red) co-staining and the percentage of positive cells within the encapsulated cysts shown in Figure 2D of the main text. Scale bars are 50 µm.

Fig. S4 .

 S4 Fig. S4. Scorecard quantitative comparison of gene expression profiles from trilineage differentiation assays of 2D and 3D hPSC colonies 2D cultures and 3D in capsulo cultures of hPSC were assessed with trilineage differentiation assay and scorecard qPCR panel. Three iPSC lines were differentiated and analyzed. Algorithm score generated by the manufacturer confirm a consistent decreased of the self-renewal genes compared to the undifferentiated reference. Up-regulation of the 3 germs layers means that both 2D and 3D stem cell colonies can engage in differentiation. The grey box plot indicates the undifferentiated reference.

Fig. S5 .

 S5 Fig. S5. Analysis of high-resolution SNP arrays before and after C-STEM amplification(A) Qualitative analysis of high-resolution SNP arrays before and after one-week of encapsulation: preencapsulation (left) and post-encapsulation (right). Absence of duplications and deletions for the 3 distinct iPS cell lines (iPS004 top row, iPS005 middle row, iPS013 lower row). (B)Tablesummarizing

Fig. S6 .

 S6 Fig. S6. Cell viability in 2D and encapsulated 3D hPSC cultures (A) Fluorescence microscopy image of a 2D stem cell colony stained for live (Calcein, green) and dead (ethidium, red). Pictures were taken before daily media changes to avoid removal of free-floating cells. Scale bar is 500 µm. (B) Fluorescence microscopy image of live (green) & dead (red) staining of 3D hPSC colonies at day 7 post encapsulation. Scale bar is 500 µm. (C) Percentage of cell mortality at harvest for 2D culture and. encapsulated 3D hPSC colonies. Stem cells cultivated in 2D were harvested at day 4, 5 or 6 before reaching confluency. Stem cells cultivated for 6 to 7 days in capsules were harvested and dissociated after capsule dissolution. Viability was assessed with Nucleo counter NC3000. The 'count and viability assay' use the cell stain Acridine Orange for cell detection, and the nucleic acid stain DAPI for detecting non-viable cells.

Fig. S7 .

 S7 Fig. S7. Encapsulated epiblast-like colonies resilience to hydrodynamic damagesFrom one encapsulation batch, 2 suspension cultures were seeded in parallel: capsules were cultivated either in static suspension (flasks) or in constantly agitated suspension (500ml stirred tank bioreactor). after 7 dyas of suspension culture, capsules were collected, dissolved; 3D stem cell colonies were dissociated, fixed and stained for stem cell markers OCT4, SOX2 and NANOG. Fig.S7shows flow cytometry dot-plots of the two different culture conditions (static flask versus stirred suspension in a bioreactor)

Fig. S9 .

 S9 Fig. S9. Stemness maintenance assessment of hiPSCs through 2 consecutive encapsulationsAfter a first encapsulation of hPSCs and 7 days of suspension culture, capsules were collected and dissolved, and 3D colonies were dissociated, resuspended and used for another round of encapsulation/suspension culture. At day 7 and 14 cells were dissociated, fixed, and stained for the stemness markers OCT4, SOX2, and NANOG. Individual histograms from flow cytometry show stable phenotypes.

  ).

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2021.07.05.451086	this version posted July 5, 2021.	The copyright holder for this preprint (which
	was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
			. CC-BY-NC-ND 4.0 International license available under a

  To compare between 2 groups, a student T-test was used. All statistical significance is reported in terms of p-values <0.05. helped analyze the growth of 3D colonies and write the manuscript. KA CR JH conceived and produced the microfluidic chips and performed high speed camera recordings. All data are available in the main text or the supplementary materials.

	Competing interests : MF and KA are the founders of TFT; MF, KA, PC and PN are shareholders of Treefrog
	therapeutics. MF KA and PN have a patent pertaining to discoveries presented in this manuscript. Patent no:
	WO2018096277A1.
	Data and materials availability :

ACKNOWLEDGMENTS

Funding : This work was supported in part by funding grants from European commission H2020-EIC-SMEInst (grant agreement number: 881113 C-stemGMP), iLAB2018 Bpi France, Region Nouvelle Aquitaine and Agence Nationale pour la Recherche (ANR-17-C18-0026-02). We acknowledge the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging supported by the French Research Agency (ANR-10-INBS-04). We also acknowledge the TBMCore facility.

(CNRS UMS 3427 -INSERM US 005). We also thank Nicolas Doulet and Marion Pilorge for helping build the collaboration between Treefrog Therapeutics and the Imagine Institute.

Author contributions : KA NL MF designed the project and supervised experiments and analysis; PC performed the experiments, analyzed the data and wrote the article; Experiments were performed both at the Imagine Institute and at Treefrog Therapeutics (TFT). AL helped performing experiments, analysing the data and writing the article; EL FM ML designed bioreactor cultures and scale-up, EL JP HW EJ MD performed bioreactor cultures; JC EW EQ CB EP contributed to 2D cultures, encapsulations, trilineage assay and SNP analysis; BG contributed to data analysis and writing.; PVL proposed a mathematical formulation for PDT and viability. PN Each crossing event corresponds to a black spot in the image. (D) Intensity threshold of (c). Elementary binary operations are performed to ensure that each crossing event corresponds to a single white spot.

(E) Particle analysis (using ImageJ plugin). Each white spot is counted independently, and its coordinates are stored by the algorithm, providing details on the time and location of the crossing event.

(F) Example of typical production curves for three different spraying nozzles (chips A, B and C). The corresponding production rates are included in the legend. The values are usually estimated with longer datasets (recordings >500 ms).

MOVIE S1. Formation and collection of capsules in calcium bath.

Video taken with a high speed camera showing how the train of droplets splays during the encapsulation process under electric field. Acquisition was performed at a frame rate of 10.000 fps. Scale bar is 1mm.

MOVIE S2. 3D rendering of an equatorially sectioned 3D hPSC colony.

The image shows nuclear localization of OCT4 (green) and phalloidin (gray). The diameter of the cyst is 110 um.

MOVIE S3. Time-lapse of an encapsulated 3D hPSC colony : between encapsulation and harvest

Phase contrast sequence (using Biostation Nikon IM) of a growing hPSC colony inside a capsule. Video starts at day 1 after encapsulation. Scale bar is 100 µm.

MOVIE S4. Time-lapse of an encapsulated 3D hPSC colony : between encapsulation and lumen collapse

Phase contrast sequence (using Biostation Nikon IM) of a growing hPSC colony inside a capsule. Video starts at day 1 after encapsulation. prolonged until full collapse of the lumen. Scale bar is 100 µm.

MOVIE S5. Encapsulated 3D hPSCs colonies cultured in a 10 liter stirred-tank bioreactor.

Video taken with a Digital Single-Lens Reflex camera shows the flow of hPSC-laden capsules induced by the motion of the bioreactor's impeller.