Microcarrier-based platforms for in vitro expansion and differentiation of human pluripotent stem cells in bioreactor culture systems

Accepted Manuscript Title: Microcarrier-based platforms for in vitro expansion and differentiation of human pluripotent stem cells in bioreactor culture systems Author: Sara M. Badenes Tiago G. Fernandes Carlos A.V. Rodrigues Maria Margarida Diogo Joaquim M.S. Cabral PII: DOI: Reference:

S0168-1656(16)31433-X http://dx.doi.org/doi:10.1016/j.jbiotec.2016.07.023 BIOTEC 7635

To appear in:

Journal of Biotechnology

Received date: Revised date: Accepted date:

17-5-2016 26-7-2016 28-7-2016

Please cite this article as: Badenes, Sara M., Fernandes, Tiago G., Rodrigues, Carlos A.V., Diogo, Maria Margarida, Cabral, Joaquim M.S., Microcarrierbased platforms for in vitro expansion and differentiation of human pluripotent stem cells in bioreactor culture systems.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2016.07.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Microcarrier-based platforms for in vitro expansion and differentiation of human pluripotent stem cells in bioreactor culture systems

Sara M. Badenes, Tiago G. Fernandes, Carlos A.V. Rodrigues, Maria Margarida Diogo, Joaquim M. S. Cabral* Department of Bioengineering, and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Portugal

*Corresponding author: Joaquim M. S. Cabral Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; Tel: +351218419063; Fax: +351218419062; E-mail: [email protected]

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Highlights

Microcarrier-based culture systems achievements for hPSC manufacturing are reviewed. Crucial aspects that influence the performance of these systems are discussed. Integrated platforms for hPSC expansion and directed differentiation are evaluated. Recent progress includes addressing GMP-compliant microcarriers-based systems.

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Abstract Human pluripotent stem cells (hPSC) have attracted a great attention as an unlimited source of cells for cell therapies and other in vitro biomedical applications such as drug screening, toxicology assays and disease modeling. The implementation of scalable culture platforms for the large-scale production of hPSC and their derivatives is mandatory to fulfill the requirement of obtaining large numbers of cells for these applications. Microcarrier technology has been emerging as an effective approach for the large scale ex vivo hPSC expansion and differentiation. This review presents recent achievements in hPSC microcarrier-based culture systems and discusses the crucial aspects that influence the performance of these culture platforms.

Recent

progress

includes

addressing

chemically-defined

culture

conditions for manufacturing of hPSC and their derivatives, with the development of xeno-free media and microcarrier coatings to meet good manufacturing practice (GMP) quality requirements. Finally, examples of integrated platforms including hPSC expansion and directed differentiation to specific lineages are also presented in this review.

Keywords: human pluripotent stem cells (hPSC), hPSC derivatives, microcarriers, bioreactors, large scale production, integrated expansion and differentiation

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1. Introduction Human pluripotent stem cells (hPSC) are in vitro cell lines derived from the late epiblast stage of embryonic development (Thomson et al., 1998). These cells have the capacity to differentiate into virtually all cell types in the human body (Davidson et al., 2015) which makes them attractive for a wide rage of applications (Irion et al., 2008). Firstly, they can be used for in vitro studies of human development and organogenesis that would otherwise be difficult to perform (Yin et al., 2016). These include recent advances in the study of central nervous system development (Lancaster et al., 2013), gut formation (Forster et al., 2014), hepatic modeling (Huch et al., 2015), functional kidney studies (Takasato et al., 2015), among others (Ader and Tanaka, 2014). Also, given their apparent unlimited self-renewal capacity, hPSC may be used as renewable and unlimited source of cells and tissues for drug development (Engle and Puppala, 2013), toxicity screening (Scott et al., 2013), and regenerative medicine applications (Angelos and Kaufman, 2015). Furthermore, the seminal work in somatic cell reprogramming to generate human induced pluripotent stem cells (hiPSC) by Yamanaka and coworkers (Takahashi et al., 2007) also opened up the possibility of personalized treatments and the development of human in vitro models of disease (Inoue et al., 2014). Nevertheless, it is anticipated that standard cell culture technologies will not meet the foreseen high demand for hPSC and their derivatives (Fernandes et al., 2014). One important issue in translating hPSC into possible applications is establishing defined, robust and reproducible culture systems to derive, maintain and expand these cells in vitro without compromising their differentiation potential, and to perform their controlled differentiation without exposing them to xenogeneic agents that could compromise their clinical use (McDevitt and Palecek, 2008). This will involve recreating the necessary molecular signals that regulate hPSC self-renewal and differentiation. Furthermore, given the high cell numbers necessary for cellular

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therapies, scale up and scale out approaches using suspension culture systems may be an important option to consider (Rodrigues et al., 2011). For example, it is estimated that over 108 beta cells per transplant, with multiple transplants required per patient, may be needed for the treatment of type 1 diabetes (Shapiro et al., 2000). Therefore, stirred suspension bioreactor systems, which provide densities of 106–107 cells/ml, are appealing for generating stem cell therapeutics, especially given the limitations for scale-up of traditional dish cultures (Fan et al., 2015). Microcarriers in particular, are a valuable option for the scale up of adherent cultures to stirred suspension systems. When compared to tissue culture plates, the three-dimensional (3D) shape of the microcarriers significantly increases the available surface area for cell adhesion, providing a higher surface-to-volume ratio. Thus, microcarriers have been successfully used to expand and differentiate hPSC in stirred culture systems with high efficiency in terms of cell yields (Oh et al., 2009; Storm et al., 2010). In this review, we provide a review of the stirred suspension systems developed for hPSC expansion and differentiation, with a special emphasis on microcarrier-based cultures. We critically assess the importance of environmental factors that influence these cultures (Fig. 1), particularly (I) cell inoculation improvements, (II) cell shear sensitivity evaluations, (III) medium composition progresses, and (IV) novel microcarriers, including the development of novel surface coatings for hPSC culture; and we provide examples of microcarrier-based systems for hPSC expansion and differentiation. Finally, we discuss future developments and provide an updated and integrated perspective on the challenges for developing clinically relevant scalable systems for culturing hPSC on microcarriers.

2. In vitro culture of human pluripotent stem cells

2.1. Pluripotent stem cell cultivation: reconstructing the niche

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Historically, hPSC were first isolated and cultured using undefined medium containing Fetal Bovine Serum (FBS), or serum extracts, high concentrations of basic fibroblast growth factor (bFGF), and feeder layers of supporting cells (Thomson et al., 1998). While both mouse and human feeders (Amit et al., 2003) have been replaced by the inclusion of extracellular matrix coatings (i.e. Matrigel™) (Xu et al., 2001), the typical system for routine culture of hPSC still includes undefined components, usually isolated from animal sources. This undefined environment makes fundamental research studies difficult to design and interpret, and translation of hPSC-derived products to the clinic fundamentally unattainable. Nevertheless, in the last few years, significant progress has been made towards the development of chemically defined culture systems for the production of increased cell quantities, under good manufacturing practices (GMP), free of contaminants and with low batchto-batch variability. The generation of robust and scalable culture systems for adherent hPSC expansion, under clinically compliant GMP conditions, thus involves the combination of both xeno-free and chemically defined adhesion substrates and culture medium. FBS and serum extracts as well as animal feeder layers and xenogeneic adhesion substrates have now been removed from most media compositions designed to sustain selfrenewal and pluripotency of hPSC. Furthermore, and although the nutritional requirements may vary according to cell type, there is a set of essential components that can be defined when culturing animal cells, particularly carbon/energy source (e.g. glucose and glutamine), nitrogen source (e.g. glutamine), vitamins and growth factors. In the case of hPSC, signaling components that need to be fed to the system include stimulators of different canonical signaling pathways, namely TGFβ/Activin/Nodal and bFGF. TGF-β/Activin/Nodal signaling plays a major role in maintaining hPSCs self-renewal and pluripotency through the activation of SMAD2/3 via ALK4/5/7 (James et al., 2005). FGF signaling is also involved in pluripotency

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maintenance of hPSC via intracellular activation of PI3K/AKT and/or Ras-RefMEK/ERK downstream pathways. Activated AKT promotes cell proliferation, survival, growth and motility (Manning and Cantley, 2007). Ras-Ref-MEK/ERK signaling, also known as MAPK/ERK, is believed to cooperate with PI13/AKT pathway in maintaining hPSC pluripotency, although its mechanism of action remains unclear (Li et al., 2007). Accordingly, sustained activation of these signaling pathways in hPSC converge towards the activation of a core transcriptional network involving OCT4, SOX2 and NANOG, maintaining human pluripotency in culture. Based on this knowledge, a xeno- and feeder-free culture medium specifically formulated for the growth and expansion of hPSC has been developed by Guokai Chen and co-workers at James Thomson’s laboratory (Chen et al., 2011b). The so-called E8™ medium, which merely comprises eight components (L-ascorbic acid, selenium, transferrin, NaHCO3, insulin, FGF2, TGFβ1 and DMEM/F12), has the potential to reduce both manufacturing costs and lot-to-lot variability, while simplifying quality control. This and other highly defined culture media compositions should significantly help facilitating the translation of basic research to the clinic. Chemically defined medium can be integrated with adhesive molecules to create clinically compliant GMP conditions for adherent culture of hPSC. Matrigel™ was first used to replace feeder cells, and although derived from animal sources, some of its components have shown to support hPSC proliferation and survival. For example, different laminins have been reported to effectively support hPSC growth (Xu et al., 2001). Additionally, it has also been stated that the specific laminin isoforms -111, 332 and -511 support the adhesion and proliferation of hPSC due to their affinity to integrin α6β1 (Miyazaki et al., 2008). Since integrins are responsible to mediate cell adhesion, the identification of integrins in hPSC led to the development and use of other supporting extracellular matrix (ECM) proteins for hPSC maintenance in culture, in particular vitronectin and laminin-511 (Braam et al., 2008; Domogatskaya et al.,

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2008). Vitronectin has been identified to support hPSC by binding to integrin αVβ5. Also, E-cadherin, which mediates cell-cell interactions, thus being involved in colony formation and self-renewal, has been used as well as substrate for long-term culture of hPSC (Nagaoka et al., 2010). Therefore, the use of recombinant human (rh) proteins, such as (rh) laminin-511, vitronectin and E-cadherin, represents a significant breakthrough in the culture of hPSC. However, such biological substrates are expensive to manufacture, which makes their use difficult to implement in largescale expansion of hPSC under chemically defined conditions (Villa-Diaz et al., 2013). One interesting alternative is the use of synthetic peptide-acrylate surfaces (PAS), which have been recently shown to support hPSC in culture (Melkoumian et al., 2010). These surfaces are made of acrylate conjugated to biologically active peptides, such as vitronectin and fibronectin. Such material is compatible to common sterilization procedures, which is significantly important when considering their use in biomedical applications (Ross et al., 2012). In conclusion, defined substrates and medium components have been designed to support hPSC proliferation and maintenance in vitro. Several materials and soluble components have been combined to recreate the signaling landscape required by hPSC to maintain their undifferentiated phenotype. Such efforts were focused on the introduction of defined and clinically compliant components, which could be used to produce hPSC under GMP conditions. In the following sections, we focus on different microcarrier-based systems that have been applied for scaling up the expansion and differentiation of hPSC.

2.2. Microcarriers supporting human pluripotent stem cell culture Several materials have been used as the backbone for the commercially available microcarriers

(e.g.

polystyrene,

dextran).

The

surface

of

the

commercial

microcarriers is chemically derivatized with functional groups, such as positively

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charged groups (e.g. DEAE ) or non-ionic materials from biological origin (e.g. collagen). Some commercial microcarriers are also functionalized with defined ECM proteins (e.g. fibronectin). However, in order for the microcarriers to support hPSC expansion and differentiation, reports have shown that an additional surface coating by immersion of the microcarriers in ECM substrates may be necessary. Coating the microcarriers with Matrigel™ led to successful hESC expansion and differentiation on microcarriers (Fernandes et al., 2009; Oh et al., 2009; Kehoe et al., 2010; Bardy et al., 2013; Ting et al., 2014). Nevertheless, the use of Matrigel™ limits the applicability of these systems in the manufacturing for cell-based therapies that requires the use of GMP-compliant microcarriers, which means xeno-free materials and xeno-free additional surface coatings. More recently, it has been reported the use of animal or human-derived ECM glycoproteins (mouse laminin, and human plasma purified vitronectin and fibronectin (Chen et al., 2011a; Heng et al., 2012)) as surface coatings for microcarriers. However, some of these proteins may suffer from batch to batch variation and some of them are obtained from animal sources. Most ECM proteins may also be obtained by recombinant DNA technology, and for example, recombinant vitronectin was used to coat polystyrene microcarriers for a chemically defined hiPSC expansion system (Badenes et al., 2016a). On the other hand, synthetic polymer- or peptide-conjugated substrates, which circumvent the consistency issues and the high costs associated with the production of recombinant substrates, have been developed to support hPSC expansion in planar conditions (Klim et al., 2010; Melkoumian et al., 2010; Villa-Diaz et al., 2010). One of these synthetic surfaces (designated by Synthemax) was used to functionalize novel commercial microcarriers that were already successfully translated to stirred culture systems, with no need for an additional surface coating, as described in a few recent reports (Badenes et al., 2015; Silva et al., 2015).

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The selection of the microcarrier core and the additional surface coating for a specific hPSC culture will depend on the specific biological system to be developed (expansion/differentiation), on the specific intrinsic characteristics of the cell line and the stirred culture system to be used. Overall, the microcarriers should comply with the following criteria: be able to promote an efficient cell adhesion and a robust cell proliferation and/or differentiation, to allow an efficient and technically simple cell harvesting, and at the same time to exhibit adequate physical and mechanical properties for application in dynamic systems. The possibility of sterilizing and reusing the microcarriers, as well as their price, are also important factors that affect the cost-effectiveness of the production process (Badenes et al., 2016b).

2.3. Microcarrier-based bioreactor systems for human pluripotent stem cell culture Microcarrier technology combined with stirred culture systems offers a promising platform for the large-scale production of hPSC and their derivatives, providing high cell yields due to the high surface-to-volume ratio. Moreover, stirred culture systems are highly scalable, well-characterized systems from the hydrodynamics standpoint (Nienow, 2006), provide a good homogeneity and they promote an easy sampling. Cell attachment and proliferation on microcarriers can be highly influenced by the agitation speed and regime and impeller design, among other factors. Therefore, careful consideration should be taken when selecting the bioreactor system for scaling-up stem cell culture. Spinner flasks, laboratory scale stirred culture systems, have been widely used for expansion and differentiation of hPSC. However, spinner flask culture must be performed inside the CO2 incubator since these culture flasks are not equipped with control systems for culture parameters, such as pH and dissolved oxygen (pO2). Using controlled stirred tank reactors, process monitoring and control are facilitated. The control and monitoring of the production process for stem cell manufacturing is crucial to guarantee product quality, to ensure process

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robustness and reproducibility and to satisfy GMP requirements (Kirouac and Zandstra, 2008; Placzek et al., 2009). Real-time on-line monitoring tools are recommended to improve the understanding and control of the manufacturing processes (Hu and Oberg, 1990; Lim et al., 2007). The key process variables that are generally monitored and controlled in a microcarrier-based stirred bioreactor culture system are the physicochemical environment parameters (pH, pO2, dissolved carbon dioxide (pCO2), temperature), the medium components (nutrients and metabolites), and cell density and viability (Rodrigues et al., 2011). These process variables can strongly affect stem cell self-renewal and directed differentiation. hESC cultures in computer-controlled stirred tank bioreactors (small working volumes) were already performed using Matrigel™-coated Cytodex 3 microcarriers in 300 mL conditioned medium (Serra et al., 2010) and Synthemax II microcarriers in 180 mL of Cellartis Defined Culture System (DEF-CS™ basal medium) (Silva et al., 2015). Nevertheless, the implementation of efficient fully-controlled large-scale microcarrierbased hPSC culture systems is still a challenge. The critical issues are the development of reasonably priced chemically defined and xeno-free microcarrier coatings and media, and the development of large-scale systems to the culture of these shear stress-sensitive cells.

2.3.1. Operational parameters In order to establish a microcarrier-based stirred culture system for hPSC culture, several operational parameters have to be considered since they directly affect cell growth, pluripotency and lineage commitment.

Seeding The seeding of the cells onto the microcarriers is a critical step of the hPSC expansion process. The agitation protocol during the first 24h of culture has to be

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optimized in order to maximize microcarrier-cell interaction and homogeneity in cell distribution and to avoid the formation of cell-microcarriers clumps. However, nonagitated periods are needed to guarantee the anchorage of the cells to the microcarriers. Reducing the volume of the culture medium during cell seeding, like to one-half or one-third of the final operating volume, favor cell-to-cell and cell-tomicrocarriers interactions, which potentially increase cell adhesion. The importance of carefully optimizing the cell seeding protocol was analyzed in a murine ESC culture system. The results indicated that a higher efficiency may be obtained when using an intermittent stirring for the first 24 h than when using continuous stirring (Storm et al., 2010). When seeding hPSC onto microcarriers, most of the seeding protocols reported include a 24 h-step under static conditions in half of the final working volume, in order to enhance cell-microcarrier contact. Since the initial cell attachment efficiency of cells to the microcarrier surface impacts the kinetics of cell proliferation, higher attachment efficiency subsequently results in higher cell yields at the end of the culture and/or more rapid cell growth. Attachment efficiencies are affected by the initial cell and microcarrier concentrations, the microcarrier coating, the agitation regime, the impeller configuration, and the microenvironment conditions (pH, temperature, medium composition). Moreover, in the seeding protocol it is also important to generate an even distribution of cells on the microcarriers. Most of the reports describe the inoculation of microcarriers using hESC as clumps, generated by collagenase treatment. However, this seeding method results in a significant loss of viable cells (seeding efficiencies of only ~30%) and a low reproducibility (Lock and Tzanakakis, 2009; Kehoe et al., 2010). Latest studies reported the seeding of hPSC on microcarriers as single cells obtained by incubation with a proteolytic enzyme and the use of rho-associated kinase (ROCK) inhibitor to limit dissociation-induced apoptosis. Single-cell seeding generates homogeneous cell-microcarriers aggregates and increases seeding efficiencies to

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over 50-80% (Fan et al., 2014; Badenes et al., 2015). Nevertheless, enzymatic treatment with TryPLE or Accutase can induce cell damage by digesting cell-surface proteins. More recently, EDTA-based protocol to dissociate hPSC was successfully implemented to inoculate vitronectin-coated polystyrene microcarriers (Badenes et al., 2016a), achieving higher cell yields when compared to cells inoculated after Accutase-treatment. In this protocol, ROCK inhibitor was only used for the first 24h of culture. EDTA chelates divalent cations and promotes cell dissociation and attachment by sequestering calcium and magnesium. This method can minimize cell death and allows for rapid cell attachment upon inoculation and resumption of the cell cycle. After the expansion culture, the size of the cell-microcarrier aggregates to start the differentiation protocol will affect the efficiency of the process due to the diffusional gradients of nutrients, oxygen, growth factors and small molecules within the aggregates. Some studies have shown the influence of the cell-microcarrier aggregates size in the differentiation of hESC to cardiomyocytes on microcarriers (Lecina et al., 2010; Lam et al., 2014; Lam et al., 2015), which is discussed later in section 4.

Agitation Stirring in a microcarrier-based culture system should be high enough to guarantee that a) the microcarriers-containing cells are in a homogenous suspension, b) the mass transfer rates of oxygen and other nutrients to the cells are not limiting and c) the microcarriers do not form large clumps. However, agitation has to be controlled in order to avoid cell damage and cell detachment from the microcarriers (Jing et al., 2011; Gupta et al., 2014). The rate of stirring has a significant influence on cell yield and therefore on the expansion and differentiation processes efficiency. The optimal

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rate depends on the type of cells and microcarriers and on the design of the impeller and culture vessel. It was demonstrated that the shear stress caused by stirring hPSC attached to microcarriers is cell line-specific. The HES-3 hESC line showed to be more shear sensitive when compared to the HES-2 cell line, displaying a decrease in growth yield and expression of pluripotency markers at the agitation rate of 30 rpm (0.08 m/s) (Leung et al., 2011), in 100mL-Bell-FloTM spinner flask (radius of impeller = 25mm). Moreover, an agitation rate of 80 rpm (0.18 m/s) resulted in a lower cell yield for the H9 hESC line when 60 or 45 rpm (0.14 or 0.11 m/s) were employed (Lock and Tzanakakis, 2009) in a 125mL-ProCulture® spinner flask (radius of impeller = 21.5mm). A multifactorial approach and a response surface methodology were applied recently for Gibco™ hiPSC line culture to evaluate the influence of agitation rate (Badenes et al., 2016a), in a 50-mL StemSpanTM spinner flask (radius of impeller = 13.5mm). In a range of agitation speeds, from 30 to 70 rpm (0.04 to 0.10 m/s), the optimal condition predicted by the model to reach the maximum cell yield was 44 rpm (0.06 m/s). It was also demonstrated that the extent of the shear forces applied during the hESC expansion phase has an effect on the further differentiation process (Ting et al., 2014). The effect of the agitation during the directed commitment of hPSC on microcarriers was investigated for the generation of cardiomyocytes (Ting et al., 2014) and hepatocyte-like cells (Park et al., 2014), where the hydrodynamic forces evolved in stirred culture systems is defined as a critical parameter for the success of cell differentiation. The conclusions of these studies are discussed later in section 4.

Oxygen

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It is well known that the dissolved oxygen concentration in the culture medium can affect stem cell growth and stem cell fate. Therefore, the monitoring and control of the optimal pO2 in the culture medium are essential for the in vitro hPSC cultures. In planar culture systems it has been shown that low levels of oxygen (2-6%) are beneficial for the in vitro maintenance of hESC, in contrast to atmospheric conditions (Want et al., 2012). Also, hypoxic environments have also been shown to improve differentiation of hPSC towards neurons, cardiomyocytes, hematopoietic progenitors, endothelial cell and chondrocytes (Millman et al., 2009). The impact of pO2 upon hESC growth on microcarriers was evaluated in pO2controlled bioreactors, suggesting that using 30% of air saturation (which corresponds to 6% of oxygen) improved cell expansion (Serra et al., 2010). When compared with cultures in uncontrolled spinner vessels, placed inside incubators (where 20% oxygen is available), the fold increase in cell concentration in the bioreactor was 2.5 times higher. Nevertheless, there are few reports on the effect of oxygen in hPSC microcarrier-based cultures either for cell expansion or differentiation due to the fact that the implemented systems are mainly non-controlled spinner flasks.

Feeding The optimization of the feeding regime during hPSC expansion and differentiation using microcarriers is also an essential point to increase cell production and minimize the volume of culture medium necessary and the costs associated. The ideal feeding regime should allow a physiological environment with the minimum variation of nutrients concentration and pH during culture (GE Healthcare, 2005). The impact of feeding regime towards hPSC metabolism has been studied (Chen et al., 2010), suggesting that a controlled glucose concentration at 1-1.5 g/L improve cell expansion and reduce lactate accumulation. Nevertheless, most of the implemented

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hESC expansion cultures on microcarriers include a daily medium exchange of 80% of the culture total medium volume (Oh et al., 2009; Chen et al., 2011a; Badenes et al., 2016a), mainly due to the fast degradation of medium-containing growth factors. More recently, a higher cell yield was attained in a stirred spinner flask culture by changing the feeding regime from once to twice a day (1.3x106 cells/ml to 6.1x106 cells/ml) (Bardy et al., 2013). These feeding strategies increase the cost and associated labor. Moreover, current hPSC differentiation methods rely on laborious feeding strategies, using small molecule inhibitors or growth factors to induce the signals for specific lineage commitment. In order to provide greater efficiencies, optimization of medium feeding strategies during expansion and differentiation are still needed. One published work evaluates feeding strategies in a controlled stirred bioreactor system for hPSC culture on microcarriers (Serra et al., 2010). By using a continuous perfusion operation mode, it was possible to reduce fluctuations in the concentration of medium components, which contributed to enhance cell metabolism and growth, when compared to semi-continuous operation mode. Nevertheless, this study was performed using a mouse embryonic fibroblast conditioned medium.

Cell harvesting hPSC

harvesting

protocols

after

the

expansion/differentiation

process

on

microcarriers have to guarantee an efficient cell-microcarrier separation and cell recovery, without compromising cell quality in terms of viability, potency and functionality. Although this an essential step for a successful manufacturing process, little research has been made in the optimization of harvesting methods to collect hPSC and their derivatives from microcarrier-based culture. Typically, detachment of the cells from coated microcarriers requires incubation with an enzyme (TryPLE or Accutase) and further filtration with a cell strainer to separate the cells from the

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microcarriers. The parameters that can affect cell viability and recovery efficiency during this operation are enzyme concentration and incubation time, mode of mixing and filter loading capacity. This method can result in cell surface protein degradation, cannot easily ensure 100% detachment of cells and is impractical for large quantities of cells. The present challenge is the adaptation of existing technologies to develop a robust GMP-grade large-scale harvesting process for hPSC and their derivatives recovery and gentle removal of microcarriers. On the other hand, other alternatives were studied for human mesenchymal stem cell (hMSC) harvesting in order to avoid the proteolytic enzyme treatment, such as the use of microcarriers coated with thermosensitive polymers that allow cell detachment with a small temperature variation (Yang et al., 2010) and the use of synthetic dissolvable microcarriers that are easily digested by pectinase incubation (Henry et al., 2014).

3. Human pluripotent stem cell expansion on microcarriers Expansion of hPSC is currently performed in planar tissue culture plates. However, these conditions are characterized by low cell yields, due to the limited surface area available for cell proliferation, and are characterized by the lack of control of important culture parameters. There is a need for a scalable and robust platform to produce hPSC to meet the required large number of cells for drug testing applications and future cell therapies. Different strategies for expansion and/or differentiation of hPSC on scalable suspension culture systems have been explored, including the culture of the cells encapsulated within hydrogels, culturing the cells as aggregates or adhered to microcarriers. Initially, microcarrier-based culture systems were implemented for mouse ESC expansion as model system (Abranches et al., 2007; Fernandes et al., 2009; Fernandes-Platzgummer et al., 2014). This extensive work has provided important proof-of-concept data to the establishment of hESC

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expansion processes based on microcarriers, and more recently these systems are been applied towards the development of microcarrier-based hiPSC cultures (Bardy et al., 2013; Fan et al., 2014; Badenes et al., 2016a). The cell yields obtained in suspension hESC cultures based on microcarriers are higher than the ones obtained on planar platforms (3.5x106 cells/ml compared to 0.8-1.5x106 cells/ml) (Oh et al., 2009), due to the larger surface area. The first reports on microcarrier-based cultures for hPSC expansion were focused on the screening of different microcarriers, and were established in low-attachment plates under static conditions, using non-defined ECM extracts, such as Matrigel™, as surface for cell adherence on microcarriers, and conditioned medium with serum replacers, such as Knockout™ Serum Replacement, that are undefined and may contain animal-derived products. The established microcarriers for hPSC cultures, under static conditions, are the trimethyl-ammonium coated polystyrene-based Hillex II microcarriers (Phillips et al., 2008), the denatured collagen coated dextranbased Cytodex 3 microcarriers (Nie et al., 2009), positively charged cellulosebased DE-53 (Chen et al., 2010) and dextran-based Cytodex 1 microcarriers (Chen et al., 2011a)); all of them were used with an additional Matrigel™ coating (Table 1). Then, these systems have started to be implemented in spinner flasks under dynamic conditions. A 3 to 6 fold expansion was obtained using Matrigel™-coated Cytodex 3 microcarriers (Serra et al., 2010), DE-53 (Oh et al., 2009; Leung et al., 2011), and 7 to 10 fold expansion was achieved by coating collagen (Lock and Tzanakakis, 2009), polystyrene (Kehoe et al., 2010), DE-53 (Chen et al., 2011a; Bardy et al., 2013) and Cytodex 1 microcarriers with Matrigel™ (Ting et al., 2014). The same range of fold expansion was observed when using Cytodex 3 (Fernandes et al., 2009) and gelatin-based CultiSpher S (Storm et al., 2010) microcarriers with no Matrigel™ coating, in mouse embryonic fibroblast (MEF)-

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conditioned media (Table 1). These studies focus the optimization of initial cell density per surface area available and the agitation rate during spinner flask culture. Recent approaches have been made towards the development of an hPSC culture system under chemically-defined and xeno-free conditions, in terms of microcarrier materials, additional surface coatings and media composition. Under static conditions, in low-attachment plates, a 7 days-culture was performed using laminin- or vitronectin-coated plastic microcarriers and StemPro medium which led to an 8-fold increase in the number of hESC (Heng et al., 2012). Moving to dynamic conditions, the three established chemically-defined hPSC cultures are (Table 1): (1) polystyrene microcarriers coated with a combination of cation poly-L-lysine (PLL) and ECM proteins (laminin or vitronectin) and mTeSR™1 or TeSR™2 media (Fan et al., 2014; Lam et al., 2014); (2) Synthemax II microcarriers and mTeSR™1 or Cellartis DEFCS™ media (Badenes et al., 2015; Silva et al., 2015); and (3) vitronectin coatedpolystyrene microcarriers and E8™ medium (Badenes et al., 2016a). Importantly, at the end of the expansion process, the quality of the produced hPSC has to be analysed namely concerning their viability, pluripotency state and their differentiation potential. In the implemented microcarrier-based cultures, cells expressed high levels of the pluripotency markers such as OCT4, NANOG, SOX2 and

SSEA4,

assessed

by

different

techniques

(e.g.

quantitative

PCR,

immunocytochemistry and flow cytometry). Also, the stability of cell karyotype at the end of the dynamic culture was evaluated. Formation of embryoid bodies (EB) is routinely employed to assess the hPSC ability to differentiate spontaneously into progeny of the three embryonic germ layers. After 4 weeks of culture, EB’s included cells displaying relevant markers for endoderm (SOX17, FOXA2, AFP), mesoderm (ISL1, T, αSMA) and ectoderm (PAX6, TUBB3, TUJ1) lineages. In the following section, microcarrier-based systems implemented for subsequent controlled and

19

directed differentiation to specific lineages (e.g. neural and cardiac fates) will be discussed.

4. Controlled Differentiation of human pluripotent stem cells on microcarriers and integrated culture systems hPSC have the potential to differentiate into all the cell types of the human body, which makes them attractive for many applications. It can be predicted that most of these applications will require hPSC differentiation into specialized cell types. In particular, in the case of regenerative medicine applications, hPSC cannot be used in their undifferentiated state as they can generate teratomas upon transplantation. Thus, in this scenario, hPSC have to be differentiated into more mature progenitor cells – without tumorigenic potential – or into terminally differentiated cells before their use. Methods for hPSC differentiation are performed as two-dimensional (2D) monolayer cultures or as 3D cell aggregates and most of the times consist of multistep and complex protocols where hPSC are manipulated (e.g. passaging, cell selection) and sequentially cultured in different culture media, containing different combinations of growth factors, until they acquire the desired phenotype. Differentiation systems where cells are cultured as a 2D monolayer are difficult to perform on a large scale, mostly if they are performed in culture platforms (e.g. culture flasks, multi-tray flasks) which have to be extensively scaled-out to provide a sufficient surface area for large-scale production. hPSC differentiation as 3D aggregates, where the aggregates are suspended in the culture medium, is in principle more easily scaled-up if the methodologies used in culture dishes are adapted to bioreactors with large volumes. These methods, however, rely on cell aggregation, which may not be straightforward, in particular under stirred conditions (Kempf et al., 2016). Nevertheless, the successful hPSC culture as suspension aggregates for expansion and differentiation into cardiomyocytes (Kempf et al., 2014)

20

or hepatocyte-like cells (Vosough et al., 2013), in stirred bioreactors, has been reported. Microcarriers can also be used to culture and differentiate hPSC in suspension and facilitate the transition to larger scale bioprocesses. Most of the existing reports describe that hPSC cultured on microcarriers do not form single layers of cells but, instead, attach predominantly to each other, forming cell-microcarrier (hPSC/MC) aggregates. In this context, the application of microcarrier technology to hPSC differentiation can be seen as a way to assist cell aggregation and to stabilize and minimize agglomeration of the aggregates (Lecina et al., 2010). Alternatively, microcarriers can be used in integrated bioprocesses where hiPSC are expanded and the hPSC/MC aggregates formed in the process are subsequently induced to differentiate (Lam et al., 2015). In this case, the proliferative capacity of hPSC may be exploited during an initial phase, allowing the use of a smaller cell inoculum. When the necessary cell density is achieved, differentiation can be induced, usually by switching the culture medium formulation used. Examples of microcarrier processes for hPSC differentiation The generation of large quantities of neural progenitor cells (NPC) from hiPSC is required for many applications, including the development of novel cell therapies for neurodegenerative diseases. Neural differentiation of hiPSC has been achieved using inhibitors of BMP (e.g. Noggin) and/or Lefty/Activin/TGFβ pathways (e.g. SB431542) (Chambers et al., 2009) and microcarrier-based systems have been developed to adapt the already established NPC generation protocols for bioreactor culture. Bardy et al. (Bardy et al., 2013) used a 2-stage method for NPC generation using spinner flask bioreactors. An initial expansion phase was performed, where hiPSC were cultured on cellulose DE-53 microcarriers coated with Matrigel™, using mTeSR™1 medium, in an optimized feeding scheme which consisted of two 80%

21

medium exchanges/day. After the expansion step, neural differentiation was induced by replacing the culture medium with N2B27 medium supplemented with Noggin. The integrated process (7 days of expansion plus 18 days of differentiation) led to the generation of 333 NPC/hiPSC seeded (Table 2), with a purity of ≈80% PSANCAM positive cells (Bardy et al., 2013). The analysis of the transcripts from the hPSC/MC aggregates obtained also showed upregulation of NPC markers such as PAX6, SOX1 and NESTIN as well as the neuronal marker MAP2 when compared to undifferentiated cells. The process could also be used with hESC, with comparable results (Table 2). The NPC were shown to have the potential to further differentiate into neurons, astrocytes and oligodendrocytes after replating onto laminin-coated culture plates. A different process was described where Noggin was replaced by two small molecule inhibitors of SMAD signaling (dorsomorphin and SB431542), leading to a more cost-effective process (Chambers et al., 2009). hESC were expanded and differentiated on Geltrex-coated Cytodex 1 microcarriers (Qiu et al., 2015). 9 days after induction of neural differentiation, the cells obtained were 96.6% positive for the NCAM and 97.5% positive for PSA-NCAM markers. The generated NPC were also transplanted as single cells or as aggregates into the striatum of NOD-SCID IL2Rgc null mice and it was observed that the cells were able to survive and to differentiate into neurons in vivo, one month post-transplantation. Dual inhibition of SMAD signaling protocol was recently applied using defined and xeno-free conditions to generate NPC from hiPSC on vitronectin-coated polystyrene microcarriers (Badenes et al., 2016a). The differentiation protocol was accomplished in low-attachment static plates inoculated with hPSC expanded on these microcarriers in a 50 mL-spinner flask. Replated cells after differentiation protocol presented neuroepithelial cells arranged in neural rosette structures, which expressed PAX6 and NESTIN markers. Quantitative RT-PCR demonstrated an

22

increase of transcription levels of representative genes of NPC (PAX6, SOX1), and decrease in pluripotency marker gene expression (OCT4 and NANOG). The integrated expansion and differentiation of hESC into definitive endoderm (DE) was also described using microcarriers in spinner flasks (Lock and Tzanakakis, 2009). Cells were cultured with MEF-conditioned medium using collagen-coated microcarriers with an additional Matrigel™ coating. Cells were initially expanded as undifferentiated hESC and, when cell concentration reached a peak, DE differentiation was induced by changing the culture medium to a formulation containing Activin A, Wnt3a and a low concentration of serum. After this medium change, a decline in cell number was noted but afterwards a stable total cell concentration of ≈4x105 cells/mL was maintained. Approximately 84% of the cells differentiated with this method co-expressed the DE markers SOX17 and FOXA2. The authors compared spinner-flask microcarrier cultures with the standard 2D culture dishes and found that comparable concentrations of cells expressing SOX17 and FOXA2 were obtained with both systems: 9.4x104 DE cells/cm2 using microcarriers versus 10 x104 DE cells/cm2 in dishes. Moreover, to demonstrate that the microcarrier system provides a way to reduce the differentiation culture medium requirements, the authors demonstrated that, with the use of the microcarriers, 2.14 x105 cells could be generated per mL of medium consumed while cell culture in 2D dishes in static conditions generated 1.25 x105 cells/mL of culture medium consumed. The development of cell therapies for liver failure would greatly benefit with the development of large-scale bioprocesses. A microcarrier-based system for expansion of hESC and directed differentiation to hepatocyte-like cells has been reported by Park et al. (Park et al., 2014). The authors cultured the cells in spinner flasks, using Cytodex 3 microcarriers coated with Matrigel™. An initial 2 days expansion phase (using mTeSR™1 medium) was followed by a hepatic differentiation step, following a previously reported protocol (Roelandt et al., 2010).

23

The authors observed that stirring rates above 30 rpm, during the differentiation phase, led to extensive cell detachment and consequently many empty microcarriers, whereas stirring rates of 20-25 rpm could be successfully applied, minimizing the microcarrier agglomeration observed in static culture. The hepatocyte-like cells generated with this process showed differentiation levels similar to those observed in cells cultured on 2D dishes, as shown by RT-PCR and functional assays. However, when compared to primary hepatocytes, the hESC-derived hepatocyte-like cells have a lower albumin and urea secretion rates as well as lower CYP3A4 activity, suggesting a more immature phenotype. Hemangioblasts are progenitor cells capable of undergoing differentiation into vascular

and

hematopoietic

lineages.

The

generation

of

hPSC-derived

hemangioblasts on microcarriers, described by Lu et al. (Lu et al., 2013), constitutes a scalable platform capable of generating large numbers of cells that can be used for the treatment of human blood and vascular diseases. DE-53 cellulose microcarriers coated with Matrigel™ were used for hPSC expansion, using mTeSR™1, which led to the formation of hPSC/MC aggregates. Afterwards, a 2-step hemangioblast differentiation process was performed. The cells developed into hemangioblasts, which were identical to those formed by control cells cultured under 2D conditions and could be differentiated into hematopoietic and endothelial cells. A higher number of blast cells was obtained with the microcarrier system (4.41 blast cells/hESC seeded) than with the standard 2D system (0.21 blast cells/hESC seeded). This system was also described to work either with hESC or hiPSC. However, this study was performed only under static conditions and the application of the microcarrierbased culture in bioreactors is not described. Cardiovascular diseases are the leading cause of death worldwide. Since the human adult heart has a limited regenerative capacity, in cases of myocardial infarction, the generation of hPSC-derived cardiomyocytes could constitute a promising alternative

24

to heart transplantation which, currently, is the only definitive treatment. Estimates suggest that 1-10 x 109 cells may be required in these cases (Laflamme and Murry, 2005; Kempf et al., 2016) and thus methods for large-scale cardiomyocyte (CM) production will certainly be required.

Lecina et al. (Lecina et al., 2010) used

microcarriers to assist hESC aggregate formation and studied the effect of microcarrier shape, size and surface coating on CM differentiation, using a chemically defined, serum-free medium and a differentiation protocol based on p38 mitogen-activated protein kinase (MAPK) inhibition with SB203580. The best results were obtained with laminin-coated TOSOH-10 microcarriers, which are 10 μm protamine derivatized microcarriers. The TOSOH-10 microcarriers attach to clumps of hESC, forming hPSC/MC aggregates which are able to support CM differentiation. This microcarrier-based system was shown to generate 0.62 CM/hESC seeded in the system, under static conditions and 0.33 CM/hESC in spinner flasks. The lower CM/hESC ratio in the spinner flasks was attributed to reduced cell growth in stirred conditions, caused by the shear stress in serum-free conditions. After 16 days of differentiation in the spinner flask, 17% of the population was positive for Myosin Heavy Chain (MHC) and 13.6% for sarcomeric α-actinin, which gives an average of 15.7% of CM. Moreover, the CM generated were also positive, as demonstrated by immunostaining, for other cardiac-specific markers, such as myosin light chain, desmin and troponin-I. Cell functionality was also assessed by drug induced QT interval prolongation with Astemizole. The same group describes an alternative version of this system (Lam et al., 2014) using a method for CM differentiation in spinner flasks based on modulation of the Wnt signaling pathway. In this work, hESC were cultured on polystyrene microcarriers (≈100 μm diameter) coated with PLL and murine laminin. After 7 days of expansion with mTeSR™1, spherical hPSC/MC aggregates were formed. Cells were then differentiated into CM using the method described by Lian et al (Lian et al.,

25

2012; Lian et al., 2013). Four distinct differentiation methodologies were compared: i) replating the hPSC/MC aggregates on laminin-coated plates; ii) culturing the hPSC/MC aggregates in static suspension or iii) in agitated suspension; iv) culturing the hPSC/MC aggregates in spinner flasks. The hydrodynamic shear introduced in condition iii), when hPSC/MC aggregates were agitated by a shaking platform, resulted in extensive cell detachment from the microcarriers. This effect could be successfully minimized with the implementation of an optimized agitation regimen, including two ≈16h periods without agitation. The authors were then able to develop a conjoint expansion and differentiation process in a spinner flask, by using the optimized agitation scheme previously developed. The CM yield from hESC seeded was compared among the differentiation methods, being the best results obtained with the integrated process in the spinner flask (condition iv, Table 2). In the end of the culture ≈48% of the cells were MHC positive, ≈56% were Cardiac troponin T (cTNT) positive and 9.6 CM/hESC seeded were generated (versus 3.8 CM/hESC in monolayer cultures). The generated cells had the expected molecular, structural and functional properties. The effect of shear on CM differentiation on microcarriers was further demonstrated in a different study (Ting et al., 2014) where systems with different levels of shear stress were compared: static culture conditions, a “wave bioreactor”-like rocker platform (shear rates ≈0.1 dyn/cm2 (Oncul et al., 2010)) and the more conventional spinner flask (≈1.1-2.4 dyn/cm2 (Sucosky et al., 2004; Santos et al., 2011)). In this work, hPSC were cultured on Cytodex 1 microcarriers, coated with Matrigel™. The cell yield was higher in the spinner flask and the rocker systems, with smaller hPSC/MC aggregates generated during the expansion phase, with mTeSR™1. Also, a more defined spherical shape was observed in these aggregates when compared with the static culture. When cell-laden microcarriers were harvested from the 3 different systems and differentiated (in static culture plates) using the Wnt modulation

26

protocol, the highest differentiation efficiencies were attained with the rocking platform, being this system selected for the development of an integrated hPSC expansion/CM differentiation bioprocess. The study refers that, under the conditions tested, if the hPSC/MC aggregates formed during the expansion step were continuously agitated during the 12-days differentiation period, CM were not generated in comparable levels to static culture. This result again suggests that the shear stress caused by agitation has a significant impact on the CM differentiation process. The authors found that intermittent agitation during the first 3 days, followed by continuous agitation for the rest of the differentiation period, significantly improved the CM generation efficiency. With this optimized process a cell population with cTNT and MHC expression levels of ≈66% and ≈60% respectively was obtained as well as a 31.75 CM/hESC seeded ratio (Table 2). The process was also successfully tested with hiPSC (Table 2). Recently, directed differentiation of hiPSC into CM was performed under defined and xeno-free conditions using vitronectin-coated polystyrene microcarriers and ready-touse media (from Thermo Fisher Scientific) (Badenes et al., 2016a). The cardiac commitment was accomplished in low-attachment static plates inoculated with hPSC expanded on these microcarriers in a 50 mL-spinner flask. Beating cell-microcarrier aggregates were observed at day 10 of differentiation and replated cells presented contracting colonies that stained positively for the cardiac marker cTNT. Quantitative RT-PCR demonstrated an increase of transcription levels of representative genes of cardiac markers (early markers ISL1 and GATA4 and late markers TNNT2 and NKX2.5), and a decrease in pluripotency marker gene expression (OCT4 and NANOG).

5. Conclusion and future perspectives

27

In the nearby future, the expected number of products derived from hPSC for clinical applications emphasize the need to establish scalable hPSC manufacturing processes. Although the existing work clearly shows that microcarrier-based integrated bioprocesses for cell expansion and differentiation are promising as scalable manufacturing platforms for hPSC-derived products, there are still points that may require further investigation in the near future. A huge variety of microcarrier particles, with different properties in terms of size, shape and biomaterial composition are used and, most often, selected empirically based on a screening of the different products available. It can be envisioned that for each specific application (expansion, differentiation into specific mature cells) the microcarriers have to be carefully designed in order to constitute not only a support for cell attachment but also to provide physical, mechanical and/or biochemical cues for the desired differentiation fate. Possible strategies for the development of these improved

microcarriers

would

include

testing

biomaterials

with

different

characteristics (e.g. stiffness, surface properties) as well as the conjugation of biomolecules to the material (Conway and Schaffer, 2012). Although most studies described the use Matrigel™ as a coating for the microcarriers, different ECM molecules or combinations of molecules could be tested, to better understand the exact and optimal ECM requirements for the different processes. Also, medium development is critical for the progress of hPSC-derived therapeutics production and the ultimate goal is to establish defined and xeno-free culture conditions for each stage of the derived hPSC manufacturing, the expansion and the differentiation processes. Regarding large-scale manufacturing, although there are several studies on hPSC expansion and differentiation on stirred microcarriers-based culture systems, they are implemented in small bench-scale vessels with no control and no monitoring of culture environmental parameters. Only two reported works used computer-

28

controlled stirred tank bioreactors, however culture volumes were only 180 and 300 mL (Serra et al., 2010; Silva et al., 2015). While the microcarrier-based systems are being developed with scalability in mind, the real scale-up for example to 1L-stirred tank bioreactor has not yet been demonstrated for hPSC cultures. The high cost of media for expansion and differentiation processes has been one of the bottlenecks for large-scale process development. The design and optimization of feeding strategies for medium nutrients, growth factors and small molecules are facilitated when using computer-controlled stirred tank bioreactors, which will be useful to provide higher efficiencies in expansion and differentiation processes, and also in the associated costs. Furthermore, an important question when designing a microcarrier-based bioprocess is cell recovery. Since hPSC have the tendency to create multilayers of cells on the microcarriers, often forming very large cell-microcarrier aggregates, the cell recovery can be challenging and can be time and resource consuming. There are several factors that can affect cell viability and functionality, and also the recovery process efficiency. The challenge to dissociate and separate large quantities of cells from microcarriers is crucial for the overall manufacturing process. Several reports have been published with different types of microcarriers and hPSC lines, however there have been no systematic studies comparing the methods and conditions for harvesting cells from microcarriers. It will be crucial to develop efficient methods for cell retrieval and complete removal of the microcarriers. One future tendency is the implementation of hPSC culture on synthetic dissolvable microcarriers, which can be quickly dissolved by a non-proteolytic enzyme. This allows easy collection of the cells without the need of the microcarrier separation step, facilitating the downstream processing. Another aspect of downstream processing is the purification of hPSC derivatives from mixed cell populations. If the final product are hPSC-derived differentiated cells,

29

it will be important to know the “final” yield of functional cells after the harvesting procedures as well as the downstream processing required (e.g. removal of residual hPSC) in order to fully be able to develop a process capable of delivering the number of high quality cells required for the different applications. Also, it is important to understand the effects of unwanted cells in cell replacement therapy (e.g. tumorigenicity related to undifferentiated hPSC remains one of the concerns) in order to provide future directions to address these difficulties. Meeting GMP requirements is the ultimate challenge as most of the current hPSC processes are being developed for research purposes. The goal is the development of a defined xeno-free integrated manufacturing process, including the expansion, the differentiation and the downstream purification, within a closed system with suitable monitoring and control systems. Moreover, quality control has also to be integrated in the production of hPSC-derivatives, with the implementation of standardized assays (e.g. gene and protein marker expression analysis and functionality characterization).

Acknowledgements The authors thank the financial support from Fundação para a Ciência e a Tecnologia (FCT), Portugal, through iBB - Institute for Bioengineering and Biosciences (UID/BIO/04565/2013) and Programa Operacional Regional de Lisboa 2020 (Project N. 007317). S.M. Badenes, T.G. Fernandes and C.A.V. Rodrigues acknowledge

FCT

SFRH/BPD/86316/2012,

for

financial

support

SFRH/BD/78758/2011

respectively).

References

30

and

(SFRH/BPD/74449/2010, SFRH/BPD/82056/2011,

Abranches, E., Bekman, E., Henrique, D., et al., 2007. Expansion of mouse embryonic stem cells on microcarriers. Biotechnol Bioeng. 96, 1211-21. Ader, M.,Tanaka, E.M., 2014. Modeling human development in 3D culture. Curr Opin Cell Biol. 31, 23-8. Amit, M., Margulets, V., Segev, H., et al., 2003. Human feeder layers for human embryonic stem cells. Biol Reprod. 68, 2150-6. Angelos, M.G.,Kaufman, D.S., 2015. Pluripotent stem cell applications for regenerative medicine. Curr Opin Organ Transplant. 20, 663-70. Badenes, S.M., Fernandes, T.G., Rodrigues, C.A., et al., 2015. Scalable expansion of human-induced pluripotent stem cells in xeno-free microcarriers. Methods Mol Biol. 1283, 23-9. Badenes, S.M., Fernandes, T.G., Cordeiro, C.S., et al., 2016a. Defined Essential 8 Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems. PLoS One. 11, e0151264. Badenes, S.M., Fernandes-Platzgummer, A.M., Rodrigues, C.A.V., et al., 2016b. Microcarrier Culture Systems for Stem Cell Manufacturing, in: Cabral, J.M.S., da Silva, C.L., Chase, L.G. and Diogo, M.M. (Eds.), Stem Cell Manufacturing. (in press), pp. Bardy, J., Chen, A.K., Lim, Y.M., et al., 2013. Microcarrier suspension cultures for high-density expansion and differentiation of human pluripotent stem cells to neural progenitor cells. Tissue Eng Part C Methods. 19, 166-80. Braam, S.R., Zeinstra, L., Litjens, S., et al., 2008. Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin. Stem Cells. 26, 2257-65. Chambers, S.M., Fasano, C.A., Papapetrou, E.P., et al., 2009. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 27, 275-80.

31

Chen, A.K.L., Chen, X.L., Choo, A.B.H., et al., 2011a. Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells. Stem Cell Research. 7, 97-111. Chen, G., Gulbranson, D.R., Hou, Z., et al., 2011b. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 8, 424-9. Chen, X., Chen, A., Woo, T.L., et al., 2010. Investigations into the metabolism of two-dimensional colony and suspended microcarrier cultures of human embryonic stem cells in serum-free media. Stem Cells Dev. 19, 1781-92. Conway, A.,Schaffer, D.V., 2012. Biophysical regulation of stem cell behavior within the niche. Stem Cell Res Ther. 3, 50. Davidson, K.C., Mason, E.A.,Pera, M.F., 2015. The pluripotent state in mouse and human. Development. 142, 3090-9. Domogatskaya, A., Rodin, S., Boutaud, A., et al., 2008. Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro. Stem Cells. 26, 2800-9. Engle, S.J.,Puppala, D., 2013. Integrating human pluripotent stem cells into drug development. Cell Stem Cell. 12, 669-77. Fan, Y., Hsiung, M., Cheng, C., et al., 2014. Facile engineering of xeno-free microcarriers for the scalable cultivation of human pluripotent stem cells in stirred suspension. Tissue Eng Part A. 20, 588-99. Fan, Y., Wu, J., Ashok, P., et al., 2015. Production of human pluripotent stem cell therapeutics under defined xeno-free conditions: progress and challenges. Stem Cell Rev. 11, 96-109. Fernandes, A.M., Marinho, P.A., Sartore, R.C., et al., 2009. Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system. Braz J Med Biol Res. 42, 515-22. Fernandes, T.G., Rodrigues, C.A.V., Diogo, M.M., et al., 2014. Stem Cell Bioprocessing for Regenerative Medicine. J Chem Technol Biotechnol. 89, 34-47.

32

Fernandes-Platzgummer, A., Diogo, M.M., da Silva, C.L., et al., 2014. Maximizing mouse embryonic stem cell production in a stirred tank reactor by controlling dissolved oxygen concentration and continuous perfusion operation. Biochem Eng J. 82, 81-90. Forster, R., Chiba, K., Schaeffer, L., et al., 2014. Human intestinal tissue with adult stem cell properties derived from pluripotent stem cells. Stem Cell Reports. 2, 838-52. Gupta, P., Ismadi, M.Z., Verma, P.J., et al., 2014. Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells. Cytotechnology. Heng, B.C., Li, J., Chen, A.K., et al., 2012. Translating human embryonic stem cells from 2-dimensional to 3-dimensional cultures in a defined medium on lamininand vitronectin-coated surfaces. Stem Cells Dev. 21, 1701-15. Henry, D., Hervy, M.,Walerack, C., 2014. Cell Culture Article and Methods Thereof, Incorporated, C. WO 2014/209865 A1. Hu, W.S.,Oberg, M.G., 1990. Monitoring and control of animal cell bioreactors: biochemical engineering considerations. Bioprocess Technol. 10, 451-81. Huch, M., Gehart, H., van Boxtel, R., et al., 2015. Long-term culture of genomestable bipotent stem cells from adult human liver. Cell. 160, 299-312. Inoue, H., Nagata, N., Kurokawa, H., et al., 2014. iPS cells: a game changer for future medicine. EMBO J. 33, 409-17. Irion, S., Nostro, M.C., Kattman, S.J., et al., 2008. Directed differentiation of pluripotent stem cells: from developmental biology to therapeutic applications. Cold Spring Harb Symp Quant Biol. 73, 101-10. James, D., Levine, A.J., Besser, D., et al., 2005. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development. 132, 1273-82.

33

Jing, D., Parikh, A.,Tzanakakis, E.S., 2011. Stem Cell Bioprocessing for Regenerative Medicine, in: Stachowiak, M.K. and Tzanakakis, E.S. (Eds.), Stem Cells: From Mechanisms to Tecnologies. World Scientific, pp. Kehoe, D.E., Jing, D., Lock, L.T., et al., 2010. Scalable stirred-suspension bioreactor culture of human pluripotent stem cells. Tissue Eng Part A. 16, 405-21. Kempf, H., Olmer, R., Kropp, C., et al., 2014. Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells in scalable suspension culture. Stem Cell Reports. 3, 1132-46. Kempf, H., Andree, B.,Zweigerdt, R., 2016. Large-scale production of human pluripotent stem cell derived cardiomyocytes. Adv Drug Deliv Rev. 96, 18-30. Kirouac, D.C.,Zandstra, P.W., 2008. The systematic production of cells for cell therapies. Cell Stem Cell. 3, 369-81. Klim, J.R., Li, L., Wrighton, P.J., et al., 2010. A defined glycosaminoglycan-binding substratum for human pluripotent stem cells. Nat Methods. 7, 989-94. Laflamme, M.A.,Murry, C.E., 2005. Regenerating the heart. Nat Biotechnol. 23, 845-56. Lam, A.T., Chen, A.K., Li, J., et al., 2014. Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture. Stem Cell Res Ther. 5, 110. Lam, A.T., Chen, A.K., Ting, S.Q., et al., 2015. Integrated processes for expansion and differentiation of human pluripotent stem cells in suspended microcarriers cultures. Biochem Biophys Res Commun. Lancaster, M.A., Renner, M., Martin, C.A., et al., 2013. Cerebral organoids model human brain development and microcephaly. Nature. 501, 373-9. Lecina, M., Ting, S., Choo, A., et al., 2010. Scalable platform for human embryonic stem cell differentiation to cardiomyocytes in suspended microcarrier cultures. Tissue Eng Part C Methods. 16, 1609-19.

34

Leung, H.W., Chen, A., Choo, A.B., et al., 2011. Agitation can induce differentiation of human pluripotent stem cells in microcarrier cultures. Tissue Eng Part C Methods. 17, 165-72. Li, J., Wang, G., Wang, C., et al., 2007. MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal. Differentiation. 75, 299-307. Lian, X., Hsiao, C., Wilson, G., et al., 2012. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A. 109, E1848-57. Lian, X., Zhang, J., Azarin, S.M., et al., 2013. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. Nat Protoc. 8, 162-75. Lim, M., Ye, H., Panoskaltsis, N., et al., 2007. Intelligent bioprocessing for haemotopoietic cell cultures using monitoring and design of experiments. Biotechnol Adv. 25, 353-68. Lock, L.T.,Tzanakakis, E.S., 2009. Expansion and differentiation of human embryonic stem cells to endoderm progeny in a microcarrier stirred-suspension culture. Tissue Eng Part A. 15, 2051-63. Lu, S.J., Kelley, T., Feng, Q., et al., 2013. 3D microcarrier system for efficient differentiation of human pluripotent stem cells into hematopoietic cells without feeders and serum Regen Med. 8, 413-24. Manning, B.D.,Cantley, L.C., 2007. AKT/PKB signaling: navigating downstream. Cell. 129, 1261-74. McDevitt, T.C.,Palecek, S.P., 2008. Innovation in the culture and derivation of pluripotent human stem cells. Curr Opin Biotechnol. 19, 527-33. Melkoumian, Z., Weber, J.L., Weber, D.M., et al., 2010. Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells. Nat Biotechnol. 28, 606-10.

35

Millman, J.R., Tan, J.H.,Colton, C.K., 2009. The effects of low oxygen on selfrenewal and differentiation of embryonic stem cells. Curr Opin Organ Transplant. 14, 694-700. Miyazaki, T., Futaki, S., Hasegawa, K., et al., 2008. Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem Biophys Res Commun. 375, 27-32. Nagaoka, M., Si-Tayeb, K., Akaike, T., et al., 2010. Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum. BMC Dev Biol. 10, 60. Nie, Y., Bergendahl, V., Hei, D.J., et al., 2009. Scalable Culture and Cryopreservation of Human Embryonic Stem Cells on Microcarriers. Biotechnology Progress. 25, 20-31. Nienow, A.W., 2006. Reactor engineering in large scale animal cell culture. Cytotechnology. 50, 9-33. Oh, S.K., Chen, A.K., Mok, Y., et al., 2009. Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res. 2, 219-30. Oncul, A.A., Kalmbach, A., Genzel, Y., et al., 2010. Characterization of flow conditions in 2 L and 20 L wave bioreactors using computational fluid dynamics. Biotechnol Prog. 26, 101-10. Park, Y., Chen, Y., Ordovas, L., et al., 2014. Hepatic differentiation of human embryonic stem cells on microcarriers. J Biotechnol. 174, 39-48. Phillips, B.W., Horne, R., Lay, T.S., et al., 2008. Attachment and growth of human embryonic stem cells on microcarriers. J Biotechnol. 138, 24-32. Placzek, M.R., Chung, I.M., Macedo, H.M., et al., 2009. Stem cell bioprocessing: fundamentals and principles. J R Soc Interface. 6, 209-32. Qiu, L., Lim, Y.M., Chen, A.K., et al., 2015. Microcarrier-expanded Neural Progenitor Cells can Survive, Differentiate, and Innervate Host Neurons Better when

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Transplanted

as

Aggregates.

Cell

Transplant.

in

press,

DOI:

10.3727/096368915X690378. Rodrigues, C.A., Fernandes, T.G., Diogo, M.M., et al., 2011. Stem cell cultivation in bioreactors. Biotechnol Adv. 29, 815-29. Roelandt, P., Pauwelyn, K.A., Sancho-Bru, P., et al., 2010. Human embryonic and rat adult stem cells with primitive endoderm-like phenotype can be fated to definitive endoderm, and finally hepatocyte-like cells. PLoS One. 5, e12101. Ross, A.M., Himabindu, N., Ryan, A.L., et al., 2012. Synthetic substrates for longterm stem cell culture. Polymer. 53, 2533-2539. Santos, F., Andrade, P.Z., Abecasis, M.M., et al., 2011. Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions. Tissue Eng Part C Methods. 17, 1201-10. Scott, C.W., Peters, M.F.,Dragan, Y.P., 2013. Human induced pluripotent stem cells and their use in drug discovery for toxicity testing. Toxicol Lett. 219, 49-58. Serra, M., Brito, C., Sousa, M.F., et al., 2010. Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control. J Biotechnol. 148, 208-15. Shapiro, A.M., Lakey, J.R., Ryan, E.A., et al., 2000. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med. 343, 230-8. Silva, M.M., Rodrigues, A.F., Correia, C., et al., 2015. Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization. Stem Cells Transl Med. 4, 731-42. Storm, M.P., Orchard, C.B., Bone, H.K., et al., 2010. Three-dimensional culture systems for the expansion of pluripotent embryonic stem cells. Biotechnol Bioeng. 107, 683-95. Sucosky, P., Osorio, D.F., Brown, J.B., et al., 2004. Fluid mechanics of a spinnerflask bioreactor. Biotechnol Bioeng. 85, 34-46.

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Takahashi, K., Tanabe, K., Ohnuki, M., et al., 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131, 861-72. Takasato, M., Er, P.X., Chiu, H.S., et al., 2015. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature. 526, 564-8. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., et al., 1998. Embryonic stem cell lines derived from human blastocysts. Science. 282, 1145-7. Ting, S., Chen, A., Reuveny, S., et al., 2014. An intermittent rocking platform for integrated expansion and differentiation of human pluripotent stem cells to cardiomyocytes in suspended microcarrier cultures. Stem Cell Res. 13, 202-13. Villa-Diaz, L.G., Nandivada, H., Ding, J., et al., 2010. Synthetic polymer coatings for long-term growth of human embryonic stem cells. Nat Biotechnol. 28, 581-3. Villa-Diaz, L.G., Ross, A.M., Lahann, J., et al., 2013. Concise review: The evolution of human pluripotent stem cell culture: from feeder cells to synthetic coatings. Stem Cells. 31, 1-7. Vosough, M., Omidinia, E., Kadivar, M., et al., 2013. Generation of functional hepatocyte-like cells from human pluripotent stem cells in a scalable suspension culture. Stem Cells Dev. 22, 2693-705. Want, A.J., Nienow, A.W., Hewitt, C.J., et al., 2012. Large-scale expansion and exploitation of pluripotent stem cells for regenerative medicine purposes: beyond the T flask. Regen Med. 7, 71-84. Xu, C., Inokuma, M.S., Denham, J., et al., 2001. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 19, 971-4. Yang, H.S., Jeon, O., Bhang, S.H., et al., 2010. Suspension culture of mammalian cells using thermosensitive microcarrier that allows cell detachment without proteolytic enzyme treatment. Cell Transplant. 19, 1123-32. Yin, X., Mead, B.E., Safaee, H., et al., 2016. Engineering Stem Cell Organoids. Cell Stem Cell. 18, 25-38.

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Table 1 Examples of microcarrier-based processes for hPSC expansion. Cell type

Microcarrier/ additional coating

Medium

hESC

Hillex II

Conditioned medium

hESC

Cytodex 3/Feeder cells or Matrigel™

Conditioned medium

hESC

DE-53/Matrigel™

Conditioned medium

hESC

DE-53/Matrigel™

Conditioned medium

hESC

Cytodex 3

Conditioned medium

hESC

collagen-coated microcarriers/Matrigel™

Conditioned medium

hESC

DE-53/Matrigel™

hESC

CultiSpher S

hESC

Cytodex 3/Matrigel™

hESC

Cytodex 3/Matrigel™

hESC and hiPSC

polystyrene microcarriers/Matrigel™

Conditioned medium

hESC

DE-53/Matrigel™

Conditioned medium

hESC

Cytodex 1/Matrigel™ or mouse laminin

Conditioned medium

hESC

DE-53/Matrigel™ or mouse laminin

Conditioned medium

hESC

DE-53/ Matrigel™ or mouse laminin

Conditioned medium

Inoculation method TrypLE-treated single-cells Dispase-treated cell clumps Collagenase-treated cell clumps Collagenase-treated cell clumps TrypLE-treated single-cells

mTeSR™ 1/ StemPro Conditioned medium + ROCK inhibitor Conditioned medium + ROCK inhibitor Conditioned medium + ROCK inhibitor

Culture system

Maximum cell expansion

Culture plates (static) Culture plates (static) Culture plates (static)

0.7x10 cells/mL 3 fold (day 5) 6 2.7/1.7x10 cells/mL 5/3 fold (day 2.5) 6 1.5x10 cells/mL 2 fold (day 7)

6

6

Spinner flask Spinner flask

3.5x10 cells/mL 6 fold (day 5) 6 1.5x10 cells/mL 7 fold (day 14) 6

Collagenase-treated cell clumps

Spinner flask

2.0x10 cells/mL 10 fold (day 8)

N/A

Culture plates (static)

1.5/2.0x10 cells/mL 15/20 fold (day 9)

N/A

Spinner flask

N/A

Spinner flask

N/A

Stirred tank reactor

6

6

Collagenase-treated cell clumps Collagenase-treated cell clumps Collagenase-treated cell clumps Collagenase-treated cell clumps Collagenase-treated cell clumps

40

Spinner flask Spinner flask Culture plates (static) Culture plates (static)

1.0x10 cells/mL 10 fold (day 7) 6 1.2x10 cells/mL 3 fold (day 11) 6 2.3x10 cells/mL 5 fold (day 11, 30% pO2) 6 0.4x10 cells/mL 7 fold (day 8) 6 2.4x10 cells/mL 4 fold (day 6)

(Phillips et al., 2008) (Nie et al., 2009) (Oh et al., 2009) (Fernandes et al., 2009) (Lock and Tzanakakis, 2009) (Chen et al., 2010) (Storm et al., 2010) (Serra et al., 2010) (Kehoe et al., 2010) (Leung et al., 2011)

6

1.4/0.9x10 cells/mL 17/11 fold (day 7) 6

1.1/1.0x10 cells/mL 14/12 fold (day 7) 6

Spinner flask

Reference

3.5/2.0x10 cells/mL 9/5 fold (day 7)

(Chen et al., 2011a)

hESC

polystyrene microcarriers/mouse laminin or human vitronectin

StemPro

hiPSC

DE-53/Matrigel™

mTeSR™ 1

hiPSC

DE-53/Matrigel™

hiPSC

DE-53/Matrigel™

hESC

Cytodex 1/Matrigel™

mTeSR™ 1

hESC

polystyrene microcarriers/PLL+murine laminin

hiPSC

polystyrene microcarriers/PLL+vitronectin

hiPSC

Synthemax II microcarriers

hESC

Synthemax II microcarriers

hESC

Synthemax II microcarriers polystyrene microcarriers/human vitronectin

hiPSC

Accutase-treated single-cells Collagenase-treated cell clumps Collagenase-treated cell clumps Collagenase-treated cell clumps Dispase-treated cell clumps

Culture plates (static) Culture plates (static)

mTeSR™ 1

As small clumps

Spinner flask

TeSR™ 2 + ROCK inhibitor mTeSR™ 1 + ROCK inhibitor mTeSR™ 1 + ROCK inhibitor

Accutase-treated single-cells Accutase-treated single-cells

mTeSR™ 1 (once daily medium change) mTeSR™ 1 (twice daily medium change)

Spinner flask Spinner flask Spinner flask

Spinner flask

6

1.5/1.4 x10 cells/mL 8 fold (day 7) 6 1.3x10 cells/mL 8 fold (day 7) 6 3.0x10 cells/mL 10 fold (day 7) 6 6.1x10 cells/mL 20 fold (day 7) 6 2.9x10 cells/mL 7 fold (day 7) 6 3x10 cells/mL 15 fold (day 7) 6 1.6x10 cells/mL 19 fold (day 6)

Spinner flask

N/A

N/A

Spinner flask

2.1x10 cells/mL 7 fold (day 10)

Cellartis DEF-CS™

N/A

Stirred tank reactor

N/A

E8™ medium + ROCK inhibitor

EDTA-treated small cell clumps

Spinner flask

1.4x10 cells/mL 3.5 fold (day 10)

(Heng et al., 2012)

(Bardy et al., 2013)

(Ting et al., 2014) (Lam et al., 2014) (Fan et al., 2014) (Badenes et al., 2015)

6

N/A, not available.

41

6

(Silva et al., 2015) (Badenes et al., 2016a)

Table 2 Examples of microcarrier-based processes for hPSC differentiation. Cell type

Microcarrier/ additional coating

Culture system

Differentiation

Final cell concentration

Purity

Yield

78% PSA-NCAM positive cells

333 NPC/hiPSC

83% PSA-NCAM positive cells

371 NPC/hiPSC

Reference

6

hiPSC

DE-53/Matrigel™

Spinner flask

NPC

10.6x10 total cells/mL (7d expansion + 18d differentiation) 6 10.0x10 total cells/mL (7d expansion + 16d differentiation)

(Bardy et al., 2013)

hESC

DE-53/Matrigel™

Spinner flask

NPC

hESC

Cyotdex 1/Geltrex

Spinner flask

NPC

N/A

97% NCAM positive and 98% PSANCAM

N/A

(Qiu et al., 2015)

hiPSC

polystyrene microcarriers/vitronectin

Culture plates (static)

NPC

N/A

N/A

N/A

(Badenes et al., 2016a)

hESC

collagen-coated microcarriers/Matrigel™

84% co-expression of SOX17 and FOXA2

3.4 DE cells/hESC

(Lock and Tzanakakis, 2009)

N/A

N/A

(Park et al., 2014)

4.4 blast cells/hESC

(Lu et al., 2013)

0.33 CM/hESC

(Lecina et al., 2010)

9.6 CM/hESC

(Lam et al., 2014)

5

Spinner flask

4.0x10 cells/mL (8d expansion + 3d differentiation)

DE

5

hESC

Cytodex 3/Matrigel™

Spinner flask

Hepatocyte-like cells

5.9x10 total cells/mL (2d expansion + 18d differentiation)

hESC

DE-53/Matrigel™

Culture plates (static)

Hemangioblast

N/A

N/A

hESC

TOSOH-10/laminin

Spinner flask

CM

≈1.3x 0 total cells /mL (16d differentiation)

hESC

polystyrene microcarriers/PLL+laminin

Spinner flask

CM

hESC

Cytodex 1/Matrigel™

Rocker platform

CM

≈3.4x10 total cells/mL (7d expansion + 20d differentiation) 6 2.4x10 total cells/mL (7d expansion + 12d differentiation) 6 2.3x10 total cells/mL (7d expansion + 12d differentiation)

17% MHC positive and 13.6% SA positive ≈48% MHC positive and ≈56% cTNT positive ≈66% cTNT positive and ≈60% MHC positive ≈47% cTNT positive and ≈47% MHC positive

N/A

N/A

6

6

hiPSC

Cytodex 1/Matrigel™

Rocker platform

CM

hiPSC

polystyrene microcarriers/vitronectin

Culture plates (static)

CM

N/A, not available.

42

31.8 CM/hESC 19.6 CM/hiPSC N/A

(Ting et al., 2014)

(Badenes et al., 2016a)

Figure captions Fig. 1. Schematic of the critical parameters for the development of a culture system for scalable expansion and/or controlled differentiation of hPSC using microcarriers for cell attachment.

43