Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow.

Human mesenchymal stem cells (hMSCs) are currently being explored as a promising cell-based therapeutic modality for various diseases, with more market approvals for clinical use expected over the next few years. To facilitate this transition, addressing the bottlenecks of scale, lot-to-lot reproducibility, cost, regulatory compliance, and quality control is critical. These challenges can be addressed by closing the process and adopting automated manufacturing platforms. In this study, we developed a closed and semi-automated process for passaging and harvesting Wharton's jelly (WJ)-derived hMSCs (WJ-hMSCs) from multi-layered flasks using counterflow centrifugation. The WJ-hMSCs were expanded using regulatory compliant serum-free xeno-free (SFM XF) medium, and they showed comparable cell proliferation (population doubling) and morphology to WJ-hMSCs expanded in classic serum-containing media. Our closed semi-automated harvesting protocol demonstrated high cell recovery (~98%) and viability (~99%). The cells washed and concentrated using counterflow centrifugation maintained WJ-hMSC surface marker expression, colony-forming units (CFU-F), trilineage differentiation potential, and cytokine secretion profiles. The semi-automated cell harvesting protocol developed in the study can be easily applied for the small- to medium-scale processing of various adherent and suspension cells by directly connecting to different cell expansion platforms to perform volume reduction, washing, and harvesting with a low output volume.

Emerging Trends in Biodegradable Microcarriers for Therapeutic Applications.

Microcarriers (MCs) are adaptable therapeutic instruments that may be adjusted to specific therapeutic uses, making them an appealing alternative for regenerative medicine and drug delivery. MCs can be employed to expand therapeutic cells. MCs can be used as scaffolds for tissue engineering, as well as providing a 3D milieu that replicates the original extracellular matrix, facilitating cell proliferation and differentiation. Drugs, peptides, and other therapeutic compounds can be carried by MCs. The surface of the MCs can be altered, to improve medication loading and release, and to target specific tissues or cells. Allogeneic cell therapies in clinical trials require enormous volumes of stem cells, to assure adequate coverage for several recruitment locations, eliminate batch to batch variability, and reduce production costs. Commercially available microcarriers necessitate additional harvesting steps to extract cells and dissociation reagents, which reduces cell yield and quality. To circumvent such production challenges, biodegradable microcarriers have been developed. In this review, we have compiled key information relating to biodegradable MC platforms, for generating clinical-grade cells, that permit cell delivery at the target site without compromising quality or cell yields. Biodegradable MCs could also be employed as injectable scaffolds for defect filling, supplying biochemical signals for tissue repair and regeneration. Bioinks, coupled with biodegradable microcarriers with controlled rheological properties, might improve bioactive profiles, while also providing mechanical stability to 3D bioprinted tissue structures. Biodegradable materials used for microcarriers have the ability to solve in vitro disease modeling, and are advantageous to the biopharmaceutical drug industries, because they widen the spectrum of controllable biodegradation and may be employed in a variety of applications.

An allied reprogramming, selection, expansion and differentiation platform for creating hiPSC on microcarriers.

OBJECTIVES: Induced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges. MATERIALS AND METHODS: Five sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures. RESULTS: Improvement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50-fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (ß-catenin, E-cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ-layers, cardiomyocytes and haematopoietic stem cells were further demonstrated. CONCLUSIONS: Our method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.

Selection of O-negative induced pluripotent stem cell clones for high-density red blood cell production in a scalable perfusion bioreactor system.

OBJECTIVES: Large-scale generation of universal red blood cells (RBCs) from O-negative (O-ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year-round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high-density culture platform for large-scale generation of RBCs in vitro. MATERIALS AND METHODS: We generated 10 O-ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high-density cultures of erythroblasts in vitro. RESULTS: Based on their tri-lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small-scale generation of high-density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation. CONCLUSIONS: This study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume-controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

Multiomics analyses of cytokines, genes, miRNA, and regulatory networks in human mesenchymal stem cells expanded in stirred microcarrier-spinner cultures.

Mesenchymal stem cells (MSCs) are of great clinical interest as a form of allogenic therapy due to their excellent regenerative and immunomodulatory effects for various therapeutic indications. Stirred suspension bioreactors using microcarriers (MC) have been used for large-scale production of MSCs compared to planar cultivation systems. Previously, we have demonstrated that expansion of MSCs in MC-spinner cultures improved chondrogenic, osteogenic, and cell migration potentials as compared to monolayer-static cultures. In this study, we sought to address this by analyzing global gene expression patterns, miRNA profiles and secretome under both monolayer-static and MC-spinner cultures in serum-free medium at different growth phases. The datasets revealed differential expression patterns that correlated with potentially improved MSC properties in cells from MC-spinner cultures compared to those of monolayer-static cultures. Transcriptome analysis identified a unique expression signature for cells from MC-spinner cultures, which correlated well with miRNA expression, and cytokine secretion involved in key MSC functions. Importantly, MC-spinner cultures and conditioned medium showed increased expression of factors that possibly enhance pathways of extracellular matrix dynamics, cellular metabolism, differentiation potential, immunoregulatory function, and wound healing. This systematic analysis provides insights for the efficient optimization of stem cell bioprocessing and infers that MC-based bioprocess manufacturing could improve post-expansion cellular properties for stem cell therapies.

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor.

BACKGROUND: The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor. METHODS: Five hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions. RESULTS: Three cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 106 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 106 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process. CONCLUSION: A streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional-type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry.

Sub-confluent culture of human mesenchymal stromal cells on biodegradable polycaprolactone microcarriers enhances bone healing of rat calvarial defect.

In the current emerging trend of using human mesenchymal stromal cell (MSCs) for cell therapy, large quantities of cells are needed for clinical testing. Current methods of culturing cells, using tissue culture flasks or cell multilayer vessels, are proving to be ineffective in terms of cost, space and manpower. Therefore, alternatives such as large-scale industrialized production of MSCs in stirred tank bioreactors using microcarriers (MCs) are needed. Moreover, the development of biodegradable MCs for MSC expansion can streamline the bioprocess by eliminating the need for enzymatic cell harvesting and scaffold seeding for bone-healing therapies. Our previous studies described a process of making regulated density (1.06 g/cm3) porous polycaprolactone biodegradable MCs Light Polycarprolactone (LPCL) (MCs), which were used for expanding MSCs from various sources in stirred suspension culture. Here, we use human early MSCs (heMSCs) expanded on LPCL MCs for evaluation of their osteogenic differentiation potential in vitro as well as their use in vivo calvarial defect treatment in a rat model. In summary, (i) in vitro data show that LPCL MCs can be used to efficiently expand heMSCs in stirred cultures while maintaining surface marker expression; (ii) LPCL MCs can be used as scaffolds for cell transfer for transplantation in vivo; (iii) 50% sub-confluency, mid-logarithmic phase, on LPCL MCs (50% confluent) exhibited higher secretion levels of six cytokines (interleukin [IL]-6, IL-8, Vascular endothelial growth factor (VEGF), Monocyte Chemoattractant Protein-1 (MCP-1), growth-regulated oncogene-α (GRO-α) and stromal cell-derived factor-1α (SDF-1α)) as compared with 100% confluent, stationary phase cultures (100% confluent); (iv) these 50% confluent cultures demonstrated better in vitro osteogenic differentiation capacity as compared with 100% confluent cultures (higher levels of calcium deposition and at earlier stage); the improved bone differentiation capacity of these 50% confluent cultures was also demonstrated at the molecular level by higher expression of early osteoblast genes Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP), collagen type I, osterix and osteocalcin); and (v) in vivo implantation of biodegradable LPCL MCs covered with 50% heMSCs into rats with calvarial defect demonstrated significantly better bone formation as compared with heMSCs obtained from monolayer cultures (5.1 ± 1.6 mm3 versus 1.3 ± 0.7 mm3). Moreover, the LPCL MCs covered with 50% heMSCs supported better in vivo bone formation compared with 100% confluent culture (2.1 ± 1.3 mm3). Taken together, our study highlights the potential of implanting 50% confluent MSCs propagated on LPCL MCs as optimal for bone regeneration. This methodology allows for the production of large numbers of MSCs in a three-dimensional (3D) stirred reactor, while supporting improved bone healing and eliminating the need for a 3D matrix support scaffold, as traditionally used in bone-healing treatments.

Scaled-Up Inertial Microfluidics: Retention System for Microcarrier-Based Suspension Cultures.

Recently, particle concentration and filtration using inertial microfluidics have drawn attention as an alternative to membrane and centrifugal technologies for industrial applications, where the target particle size varies between 1 µm and 500 µm. Inevitably, the bigger particle size (>50 µm) mandates scaling up the channel cross-section or hydraulic diameter (DH > 0.5 mm). The Dean-coupled inertial focusing dynamics in spiral microchannels is studied broadly; however, the impacts of secondary flow on particle migration in a scaled-up spiral channel is not fully elucidated. The mechanism of particle focusing inside scaled-up rectangular and trapezoidal spiral channels (i.e., 5-10× bigger than conventional microchannels) with an aim to develop a continuous and clog-free microfiltration system for bioprocessing is studied in detail. Herein, a unique focusing based on inflection point without the aid of sheath flow is reported. This new focusing mechanism, observed in the scaled-up channels, out-performs the conventional focusing scenarios in the previously reported trapezoidal and rectangular channels. Finally, as a proof-of-concept, the utility of this device is showcased for the first time as a retention system for a cell-microcarrier (MC) suspension culture.

Inertial-Based Filtration Method for Removal of Microcarriers from Mesenchymal Stem Cell Suspensions.

Rapidly evolving cell-based therapies towards clinical trials demand alternative approaches for efficient expansion of adherent cell types such as human mesenchymal stem cells (hMSCs). Using microcarriers (100-300 µm) in a stirred tank bioreactor offers considerably enhanced surface to volume ratio of culture environment. However, downstream purification of the harvested cell product needs to be addressed carefully due to distinctive features and fragility of these cell products. This work demonstrates a novel alternative approach which utilizes inertial focusing to separate microcarriers (MCs) from the final cell suspension. First, we systematically investigated MC focusing dynamics inside scaled-up curved channels with trapezoidal and rectangular cross-sections. A trapezoidal spiral channel with ultra-low-slope (Tan(α) = 0.0375) was found to contribute to strong MC focusing (~300 < Re < ~400) while managing high MC volume fractions up to ~1.68%. Accordingly, the high-throughput trapezoidal spiral channel successfully separated MCs from hMSC suspension with total cell yield~94% (after two passes) at a high volumetric flow rate of ~30 mL/min (Re~326.5).

Defined Serum-Free Medium for Bioreactor Culture of an Immortalized Human Erythroblast Cell Line.

Anticipated shortages in donated blood supply have prompted investigation of alternative approaches for in vitro production of red blood cells (RBCs), such as expansion of conditional immortalization erythroid progenitors. However, there is a bioprocessing challenge wherein factors promoting maximal cell expansion and growth-limiting inhibitory factors are yet to be investigated. The authors use an erythroblast cell line (ImEry) derived from immortalizing CD71+CD235a+ erythroblast from adult peripheral blood for optimization of expansion culture conditions. Design of experiments (DOE) is used in media formulation to explore relationships and interactive effects between factors which affect cell expansion. Our in-house optimized medium formulation produced significantly higher cell densities (3.62 ± 0.055) × 106 cells mL-1 , n = 3) compared to commercial formulations (2.07 ± 0.055) × 106 cells mL-1 , n = 3; at 209 h culture). Culture media costs per unit of blood is shown to have a 2.96-3.09 times cost reduction. As a proof of principle for scale up, ImEry are expanded in a half-liter stirred-bioreactor under controlled settings. Growth characteristics, metabolic, and molecular profile of the cells are evaluated. ImEry has identical O2 binding capacity to adult erythroblasts. Amino acid supplementation results in further yield improvements. The study serves as a first step for scaling up erythroblast expansion in controlled bioreactors.

Tunable Volumetric Density and Porous Structure of Spherical Poly-ε-caprolactone Microcarriers, as Applied in Human Mesenchymal Stem Cell Expansion.

Polymeric microspheres may serve as microcarrier (MC) matrices, for the expansion of anchorage-dependent stem cells. They require surface properties that promote both initial cell adhesion and the subsequent spreading of cells, which is a prerequisite for successful expansion. When implemented in a three-dimensional culture environment, under agitation, their suspension under low shear rates depends on the MCs having a modest negative buoyancy, with a density of 1.02-1.05 g/cm3. Bioresorbable poly-ε-caprolactone (PCL), with a density of 1.14 g/cm3, requires a reduction in volumetric density, for the microspheres to achieve high cell viability and yields. Uniform-sized droplets, from solutions of PCL dissolved in dichloromethane (DCM), were generated by coaxial microfluidic geometry. Subsequent exposure to ethanol rapidly extracted the DCM solvent, solidifying the droplets and yielding monodisperse microspheres with a porous structure, which was demonstrated to have tunable porosity and a hollow inner core. The variation in process parameters, including the molecular weight of PCL, its concentration in DCM, and the ethanol concentration, served to effectively alter the diffusion flux between ethanol and DCM, resulting in a broad spectrum of volumetric densities of 1.04-1.11 g/cm3. The solidified microspheres are generally covered by a smooth thin skin, which provides a uniform cell culture surface and masks their internal porous structure. When coated with a cationic polyelectrolyte and extracellular matrix protein, monodisperse microspheres with a diameter of approximately 150 µm and densities ranging from 1.05-1.11 g/cm3 are capable of supporting the expansion of human mesenchymal stem cells (hMSCs). Validation of hMSC expansion was carried out with a positive control of commercial Cytodex 3 MCs and a negative control of uncoated low-density PCL MCs. Static culture conditions generated more than 70% cell attachment and similar yields of sixfold cell expansion on all coated MCs, with poor cell attachment and growth on the negative control. Under agitation, coated porous microspheres, with a low density of 1.05 g/cm3, achieved robust cell attachment and resulted in high cell yields of ninefold cell expansion, comparable with those generated by commercial Cytodex 3 MCs.

Biodegradable poly-ε-caprolactone microcarriers for efficient production of human mesenchymal stromal cells and secreted cytokines in batch and fed-batch bioreactors.

Large numbers of human mesenchymal stromal cells (MSCs) used for a variety of applications in tissue engineering and cell therapy can be generated by scalable expansion in a bioreactor using microcarriers (MCs) systems. However, the enzymatic digestion process needed to detach cells from the growth surface can affect cell viability and potentially the potency and differentiation efficiency. Thus, the main aim of our study was to develop biocompatible and biodegradable MCs that can support high MSC yields while maintaining their differentiation capability and potency. After cell expansion, the cells that covered MCs can be directly implanted in vivo without the need for cell harvesting or use of scaffold. Poly-ε-caprolactone (PCL) is known as a biocompatible and biodegradable material. However, it cannot be used for generation of MCs because its high density (1.14 g/cm3) would exclude its applicability for suspension MCs in stirred reactors. In this article, we describe expansion and potency of MSCs propagated on low-density (1.06 g/cm3) porous PCL MCs coated with extracellular matrices (LPCLs) in suspended stirred reactors. Using these LPCLs, cell yields of about 4 × 104 cells/cm2 and 7- to 10-fold increases were obtained using four different MSC lines (bone marrow, cord blood, fetal and Wharton's jelly). These yields were comparable with those obtained using non-degradable MCs (Cytodex 3) and higher than two-dimensional monolayer (MNL) cultures. A fed-batch process, which demonstrated faster cell expansion (4.5 × 104 cells/cm2 in 5 days as compared with 7 days in batch culture) and about 70% reduction in growth media usage, was developed and scaled up from 100-mL spinner flask to 1-L controlled bioreactor. Surface marker expression, trilineage differentiation and clonogenic potential of the MSCs expanded on LPCL were not affected. Cytokine secretion kinetics, which occurred mostly during late logarithmic phase, was usually comparable with that obtained in Cytodex 3 cultures and higher than MNL cultures. In conclusion, biodegradable LPCL can be used to efficiently expand a variety of MSC lines in stirred scalable reactors in a cost-effective manner while maintaining surface markers expression, differentiation capability and high levels of cytokine secretion. This study is the first step in testing these cell-biodegradable porous MC aggregates for tissue engineering and cell therapy, such as bone and cartilage regeneration, or wound healing.

Superior Red Blood Cell Generation from Human Pluripotent Stem Cells Through a Novel Microcarrier-Based Embryoid Body Platform.

In vitro generation of red blood cells (RBCs) from human embryonic stem cells and human induced pluripotent stem cells appears to be a promising alternate approach to circumvent shortages in donor-derived blood supplies for clinical applications. Conventional methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) rely on embryoid body (EB) formation and/or coculture with xenogeneic cell lines. However, most current methods for hPSC expansion and EB formation are not amenable for scale-up to levels required for large-scale RBC generation. Moreover, differentiation methods that rely on xenogenic cell lines would face obstacles for future clinical translation. In this study, we report the development of a serum-free and chemically defined microcarrier-based suspension culture platform for scalable hPSC expansion and EB formation. Improved survival and better quality EBs generated with the microcarrier-based method resulted in significantly improved mesoderm induction and, when combined with hematopoietic differentiation, resulted in at least a 6-fold improvement in hematopoietic precursor expansion, potentially culminating in a 80-fold improvement in the yield of RBC generation compared to a conventional EB-based differentiation method. In addition, we report efficient terminal maturation and generation of mature enucleated RBCs using a coculture system that comprised primary human mesenchymal stromal cells. The microcarrier-based platform could prove to be an appealing strategy for future scale-up of hPSC culture, EB generation, and large-scale generation of RBCs under defined and xeno-free conditions.

Integrated processes for expansion and differentiation of human pluripotent stem cells in suspended microcarriers cultures.

Current methods for human pluripotent stem cells (hPSC) expansion and differentiation can be limited in scalability and costly (due to their labor intensive nature). This can limit their use in cell therapy, drug screening and toxicity assays. One of the approaches that can overcome these limitations is microcarrier (MC) based cultures in which cells are expanded as cell/MC aggregates and then directly differentiated as embryoid bodies (EBs) in the same agitated reactor. This integrated process can be scaled up and eliminate the need for some culture manipulation used in common monolayer and EBs cultures. This review describes the principles of such microcarriers based integrated hPSC expansion and differentiation process, and parameters that can affect its efficiency (such as MC type and extracellular matrix proteins coatings, cell/MC aggregates size, and agitation). Finally examples of integrated process for generation cardiomyocytes (CM) and neural progenitor cells (NPC) as well as challenges to be solved are described.

Fabrication of uniform-sized poly-ɛ-caprolactone microspheres and their applications in human embryonic stem cell culture.

The generation of liquefied poly-ɛ-caprolactone (PCL) droplets by means of a microfluidic device results in uniform-sized microspheres, which are validated as microcarriers for human embryonic stem cell culture. Formed droplet size and size distribution, as well as the resulting PCL microsphere size, are correlated with the viscosity and flow rate ratio of the dispersed (Q d) and continuous (Q c) phases. PCL in dichloromethane increases its viscosity with concentration and molecular weight. Higher viscosity and Q d/Q c lead to the formation of larger droplets, within two observed formation modes: dripping and jetting. At low viscosity of dispersed phase and Q d/Q c, the microfluidic device is operated in dripping mode, which generates droplets and microspheres with greater size uniformity. Solutions with lower molecular weight PCL have lower viscosity, resulting in a wider concentration range for the dripping mode. When coated with extracellular matrix (ECM) proteins, the fabricated PCL microspheres are demonstrated capable of supporting the expansion of human embryonic stem cells.

Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures.

Human pluripotent stem cells (hPSC) are self-renewing cells having the potential of differentiation into the three lineages of somatic cells and thus can be medically used in diverse cellular therapies. One of the requirements for achieving these clinical applications is development of completely defined xeno-free systems for large-scale cell expansion and differentiation. Previously, we demonstrated that microcarriers (MCs) coated with mouse laminin-111 (LN111) and positively charged poly-l-lysine (PLL) critically enable the formation and evolution of cells/MC aggregates with high cell yields obtained under agitated conditions. In this article, we further improved the MC system into a defined xeno-free MC one in which the MCs are coated with recombinant human laminin-521 (LN521) alone without additional positive charge. The high binding affinity of the LN521 to cell integrins enables efficient initial HES-3 cell attachment (87%) and spreading (85%), which leads to generation of cells/MC aggregates (400 µm in size) and high cell yields (2.4-3.5×10(6) cells/mL) within 7 days in agitated plate and scalable spinner cultures. The universality of the system was demonstrated by propagation of an induced pluripotent cells line in this defined MC system. Long-term pluripotent (>90% expression Tra-1-60) cell expansion and maintenance of normal karyotype was demonstrated after 10 cell passages. Moreover, tri-lineage differentiation as well as directed differentiation into cardiomyocytes was achieved. The new LN521-based MC system offers a defined, xeno-free, GMP-compatible, and scalable bioprocessing platform for the production of hPSC with the quantity and quality compliant for clinical applications. Use of LN521 on MCs enabled a 34% savings in matrix and media costs over monolayer cultures to produce 10(8) cells.

Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture.

INTRODUCTION: Myocardial infarction is accompanied by a significant loss of cardiomyocytes (CMs). Functional CMs, differentiated from human embryonic stem cells (hESCs), offer a potentially unlimited cell source for cardiac disease therapies and regenerative cardiovascular medicine. However, conventional production methods on monolayer culture surfaces cannot adequately supply the large numbers of cells required for such treatments. To this end, an integrated microcarrier (MC) bioprocessing system for hESC propagation and subsequent CM differentiation was developed. METHODS: Production of hESC-derived CMs was initially established in monolayer cultures. This control condition was compared against hESC expansion on laminin-coated MC with cationic surface charge, in a stirred serum-free defined culture. Following expansion, the hESC/MC aggregates were placed in a CM differentiation medium, using Wnt signalling modulators in four different culture conditions. This process eliminated the need for manual colony cutting. The final optimized protocol was tested in stirred spinner flasks, combining expansion and differentiation on the same MC, with only media changes during the culture process. RESULTS: In the propagation phase, a 15-fold expansion of viable pluripotent HES-3 was achieved, with homogeneous sized aggregates of 316 ± 11 µm. Of the four differentiation conditions, stirred spinner flask cultures (MC-Sp) provided the best controlled aggregate sizes and yielded 1.9 × 106 CM/ml, as compared to 0.5 × 106 CM/ml using the monolayer cultures method: a four-fold increase in CM/ml. Similar results (1.3 × 106 CM/ml) were obtained with an alternative hESC H7 line. The hESC/MC-derived CM expressed cardiac-specific transcription factors, structural, ion channel genes, and exhibited cross-striations of sarcomeric proteins, thus confirming their cardiac ontogeny. Moreover, E-4031 (0.3 µM) prolonged the QT-interval duration by 40% and verapamil (3 µM) reduced it by 45%, illustrating the suitability of these CM for pharmacological assays. CONCLUSIONS: We have demonstrated a robust and scalable microcarrier system for generating hESC-derived CM. This platform is enabled by defined microcarrier matrices and it integrates cell propagation and differentiation within a continuous process, in serum-free culture media. It can generate significant numbers of CM, which are potentially suitable for future clinical therapies.

Cationic surface charge combined with either vitronectin or laminin dictates the evolution of human embryonic stem cells/microcarrier aggregates and cell growth in agitated cultures.

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 µm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 µm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment.