Stirred Suspension Cultures Research Articles

The Proliferation of Pre-Pubertal Porcine Spermatogonia in Stirred Suspension Bioreactors Is Partially Mediated by the Wnt/β-Catenin Pathway.

Male survivors of childhood cancer are at risk of suffering from infertility in adulthood because of gonadotoxic chemotherapies. For adult men, sperm collection and preservation are routine procedures prior to treatment; however, this is not an option for pre-pubertal children. From young boys, a small biopsy may be taken before chemotherapy, and spermatogonia may be propagated in vitro for future transplantation to restore fertility. A robust system that allows for scalable expansion of spermatogonia within a controlled environment is therefore required. Stirred suspension culture has been applied to different types of stem cells but has so far not been explored for spermatogonia. Here, we report that pre-pubertal porcine spermatogonia proliferate more in bioreactor suspension culture, compared with static culture. Interestingly, oxygen tension provides an avenue to modulate spermatogonia status, with culture under 10% oxygen retaining a more undifferentiated state and reducing proliferation in comparison with the conventional approach of culturing under ambient oxygen levels. Spermatogonia grown in bioreactors upregulate the Wnt/ β-catenin pathway, which, along with enhanced gas and nutrient exchange observed in bioreactor culture, may synergistically account for higher spermatogonia proliferation. Therefore, stirred suspension bioreactors provide novel platforms to culture spermatogonia in a scalable manner and with minimal handling.

Proteomic Analysis of Exosomes during Cardiogenic Differentiation of Human Pluripotent Stem Cells.

Efforts to direct the specification of human pluripotent stem cells (hPSCs) to therapeutically important somatic cell types have focused on identifying proper combinations of soluble cues. Yet, whether exosomes, which mediate intercellular communication, play a role in the differentiation remains unexplored. We took a first step toward addressing this question by subjecting hPSCs to stage-wise specification toward cardiomyocytes (CMs) in scalable stirred-suspension cultures and collecting exosomes. Samples underwent liquid chromatography (LC)/mass spectrometry (MS) and subsequent proteomic analysis revealed over 300 unique proteins from four differentiation stages including proteins such as PPP2CA, AFM, MYH9, MYH10, TRA2B, CTNNA1, EHD1, ACTC1, LDHB, and GPC4, which are linked to cardiogenic commitment. There was a significant correlation of the protein composition of exosomes with the hPSC line and stage of commitment. Differentiating hPSCs treated with exosomes from hPSC-derived CMs displayed improved efficiency of CM formation compared to cells without exogenously added vesicles. Collectively, these results demonstrate that exosomes from hPSCs induced along the CM lineage contain proteins linked to the specification process with modulating effects and open avenues for enhancing the biomanufacturing of stem cell products for cardiac diseases.

Xenogeneic-Free System for Biomanufacturing of Cardiomyocyte Progeny From Human Pluripotent Stem Cells.

Functional heart cells and tissues sourced from human pluripotent stem cells (hPSCs) have great potential for substantially advancing treatments of cardiovascular maladies. Realization of this potential will require the development of cost-effective and tunable bioprocesses for manufacturing hPSC-based cell therapeutics. Here, we report the development of a xeno-free platform for guiding the cardiogenic commitment of hPSCs. The system is based on a fully defined, open-source formulation without complex supplements, which have varied and often undetermined effects on stem cell physiology. The formulation was used to systematically investigate factors inducing the efficient commitment to cardiac mesoderm of three hPSC lines. Contractile clusters of cells appeared within a week of differentiation in planar cultures and by day 13 over 80% of the cells expressed cardiac progeny markers such as TNNT2. In conjunction with expansion, this differentiation strategy was employed in stirred-suspension cultures of hPSCs. Scalable differentiation resulted in 0.4–2 million CMs/ml or ∼5–20 TNNT2-positive cells per seeded hPSC without further enrichment. Our findings will contribute to the engineering of bioprocesses advancing the manufacturing of stem cell-based therapeutics for heart diseases.

Hydrodynamic characterization within a spinner flask and a rotary wall vessel for stem cell culture

Stirred suspension culture is becoming a popular method for expanding human pluripotent stem cells (hPSCs). While stirring generates adequate fluid motions to lift the cells and facilitates mass transfers (of nutrients, dissolved gases, and metabolic wastes), excessive stirring could impose hydrodynamic forces deleterious for the growth of the cells. In this study, computational fluid dynamics (CFD) simulations were performed to first investigate hydrodynamic characteristics of fluid flows in a spinner flask, a common stirred suspension culture vessel used in laboratories. Flow patterns and distributions of shear stresses and the Kolmogorov length scales at varying impeller speeds were obtained. Comparison of the Kolmogorov length scales and sizes of hPSC aggregates, measured in the authors’ previous experimental study, showed a strong correlation between the two. In addition to the spinner flask which generated complex and transient turbulent flows, this study investigated a newly developed rotary wall vessel that had been designed to produce laminar, circular Couette flows in order to control shear stress. CFD simulations revealed significantly more uniform and homogeneous flows compared to those in the spinner flask, suggesting that the rotary wall vessel is a suitable culture vessel to investigate roles of shear stress on hPSCs in suspension.

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.

Optimizing adipose tissue extract isolation with stirred suspension culture

ABSTRACTObjectives: Adherent culture which is used to collect adipose tissue extract (ATE) previously brings the problem of inhomogeneity and non-repeatability. Here we aim to extract ATE with stirred suspension culture to speed up the extraction process, stabilize the yield, and improve consistent potency metrics of ATE. Materials and methods: ATE was collected with adherent culture (ATE-A) and stirred suspension culture (ATE-S) separately. Protein yield and composition were detected by SDS-PAGE, while cytokines in ATE were determined with ELISA. The adipogenic and angiogenic potential of ATE were compared by western blot and qPCR. In addition, haematoxylin and eosin staining and lactate dehydrogenase (LDH) activity assays were used to analyze the cell viability of adipose tissue cultured with different methods. Results: The yield of ATE-S was consistent while ATE-A varied notably. Characterization of the protein composition and exosome-like vesicles (ELVs) indicated no significant difference between ATE-S and ATE-A. The concentrations of cytokines (VEGF, bFGF, and IL-6) showed no significant difference, while IGF in ATE-S was higher than that in ATE-A. ATE-S showed upregulated adipogenic and angiogenic potential compared to ATE-A. Morever, stirred suspension culture decreased the LDH activity of ATE while increased the number of viable adipocytes and reduced adipose tissue necrosis. Conclusion: Compared with adherent culture, stirred suspension culture is a reliable, time- and labor-saving method to collect ATE, which might be used to improve the downstream applications of ATE.

Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred-Suspension Culture.

Human induced pluripotent stem cells (hiPSCs) hold great hopes for application in regenerative medicine due to their inherent capacity to self-renew and differentiate into cells from the three embryonic germ layers. For clinical applications, a large quantity of hiPSCs produced in standardized and scalable culture processes is required. Several groups, including ours, have developed methodologies for scaled-up hiPSC production in stirred bioreactors in chemically defined medium. In this study, we optimized the critical steps and factors that affect hiPSC expansion and yield in stirred-suspension cultures, including inoculation conditions, seeding density, aggregate size, agitation rate, and cell passaging method. After multiple passages in stirred-suspension bioreactors, hiPSCs remained pluripotent, karyotypically normal, and capable of differentiating into all three germ layers.

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single-use bioreactors.

The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell-based therapies and cell-loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400ml. The simple and robust two-step process reported here first generates hiPSC aggregates of 324±71μm diameter in 7days in 125ml spinner flasks (100ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000ml bioreactors (1000ml volume), finally yielding hiPSC aggregates of 198±58μm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16-fold increase in hiPSC quantity at the 100ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000ml scale, an additional 10-fold increase was achieved. Taken together, 16×106 hiPSCs were expanded into 2×109 hiPSCs in 14days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell-based therapies and brings their clinical potential closer to fruition.

Impact of fluidic agitation on human pluripotent stem cells in stirred suspension culture.

The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges, in part, on the availability of a robust and scalable culture system that can readily produce a clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one such promising platform due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development within the past several years, and little is known beyond empirically optimized culture parameters. In particular, detailed characterizations of different agitation rates and their influence on the propagation of hPSCs are often not reported in the literature. In the current study, we systematically investigated various agitation rates to characterize their impact on cell yield, viability, and the maintenance of pluripotency. Additionally, we closely examined the distribution of cell aggregates and how the observed culture outcomes are attributed to their size distribution. Overall, our results showed that moderate agitation maximized the propagation of hPSCs to approximately 38-fold over 7 days by keeping the cell aggregates below the critical size, beyond which the cells are impacted by the diffusion limit, while limiting cell death caused by excessive fluidic forces. Furthermore, we observed that fluidic agitation could regulate not only cell aggregation, but also expression of some key signaling proteins in hPSCs. This indicates a new possibility to guide stem cell fate determination by fluidic agitation in stirred suspension cultures. Biotechnol. Bioeng. 2017;114: 2109-2120. © 2017 Wiley Periodicals, Inc.

Inter-microcarrier transfer and phenotypic stability of stem cell-derived Schwann cells in stirred suspension bioreactor culture.

Emerging bioreactor technologies offer an effective way for scaled-up production of large numbers of cells for cell therapy applications. One of the clinical paradigms where cell therapy can be an asset is restorative neurosciences. Nerve repair can benefit from the injections of stem cells and/or Schwann cells, acting as a source for axon myelination, myelin debris clearance, and trophic support. We have adapted microcarrier-based suspension bioreactor culture for Schwann cells (SCs) differentiated from a new stem cell source - skin-derived precursors (SKPs). SKP-derived SCs attach and grow on different types of microcarriers in both static and stirred culture, with Cytodex 3 and CultiSpher-S found most effective. Inter-microcarrier migration of SKP-SCs represents a key mechanism for rapid expansion and colonization in stirred suspension culture. We have shown that microcarrier-expanded SKP-SCs cells express Schwann cell markers p75-NTR, GFAP and S100 and retain their key ability to myelinate axons both in vitro and in vivo. Scaled-up microcarrier-based production of SKP-SCs in suspension bioreactors appears feasible for timely generation of sufficient cell numbers for nerve repair strategies.

Inverse problem analysis of pluripotent stem cell aggregation dynamics in stirred-suspension cultures

The cultivation of stem cells as aggregates in scalable bioreactor cultures is an appealing modality for the large-scale manufacturing of stem cell products. Aggregation phenomena are central to such bioprocesses affecting the viability, proliferation and differentiation trajectory of stem cells but a quantitative framework is currently lacking. A population balance equation (PBE) model was used to describe the temporal evolution of the embryonic stem cell (ESC) cluster size distribution by considering collision-induced aggregation and cell proliferation in a stirred-suspension vessel. For ESC cultures at different agitation rates, the aggregation kernel representing the aggregation dynamics was successfully recovered as a solution of the inverse problem. The rate of change of the average aggregate size was greater at the intermediate rate tested suggesting a trade-off between increased collisions and agitation-induced shear. Results from forward simulation with obtained aggregation kernels were in agreement with transient aggregate size data from experiments. We conclude that the framework presented here can complement mechanistic studies offering insights into relevant stem cell clustering processes. More importantly from a process development standpoint, this strategy can be employed in the design and control of bioreactors for the generation of stem cell derivatives for drug screening, tissue engineering and regenerative medicine.

Comparative study of in vitro expansion of bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BMD-MSCs) are of great interest for tissue engineering, but require expansion before they can be used for therapeutic applications. We compared three different culture techniques for their potential for large scale expansion of rat BMD-MSCs, i.e. monolayer cultures, stirred suspension cultures and pour-off cultures, and found that pour-off cultures supported the biggest expansion in BMD-MSCs as measured by the fibroblastic-colony forming unit assay (CFU-f). BMD-MSCs expanded in stirred suspension cultures stopped proliferating altogether and, although monolayer cultures allowed for expansion of BMD-MSCs, they favoured a differentiated phenotype over uncommitted MSCs. Only BMD-MSCs expanded in pour-off cultures were able to differentiate into both osteoblastic and adipocytic lineages and maintain CFU-f numbers. These data suggest that pour-off cultures are a viable method of BMD-MSC expansion.

High density continuous production of murine pluripotent cells in an acoustic perfused bioreactor at different oxygen concentrations

Strategies for the production of pluripotent stem cells (PSCs) rely on serially dissociated adherent or aggregate-based culture, consequently limiting robust scale-up of cell production, on-line control and optimization of culture conditions. We recently developed a method that enables continuous (non-serially dissociated) suspension culture-mediated reprogramming to pluripotency. Herein, we use this method to demonstrate the scalable production of PSCs and early derivatives using acoustic filter technology to enable continuous oxygen-controlled perfusion culture. Cell densities of greater than 1 × 10⁷ cells/mL were achieved after 7 days of expansion at a specific growth rate (µ) of 0.61 ± 0.1 day⁻¹ with a perfusion rate (D) of 5.0 day⁻¹. A twofold increase in maximum cell density (to greater than 2.5 × 10⁷ cells/mL) was achieved when the medium dissolved oxygen was reduced (5% DO). Cell densities and viabilities >80% were maintained for extended production periods during which maintenance of pluripotency was confirmed by stable expression of pluripotency factors (SSEA-1 and Nanog), as well as the capacity to differentiate into all three germ layers. This work establishes a versatile biotechnological platform for the production of pluripotent cells and derivatives in an integrated, scalable and intensified stirred suspension culture.

Effects of agitation speed on the ex vivo expansion of cord blood hematopoietic stem/progenitor cells in stirred suspension culture

The mononuclear cells were cultivated in stirred flasks at different agitation speeds of 30 rpm, 45 rpm, 60 rpm and 80 rpm. At the agitation speed of 30 rpm, total cells achieved higher expansion folds and the CFC density increased. When at higher agitation speed of 60 rpm or 80 rpm, the number of cells dropped rapidly and characteristics of hematopoietic stem/progenitor cells (HSPCs) were not maintained. Moreover, the culture duration of 6–9 days was better for HSPCs ex vivo expansion. These data indicated that HSPCs should be cultured at relatively low agitation speed and for a short-term period when cultured in stirred suspension system.

Reprogramming in suspension

Current practice for the generation and maintenance of induced pluripotent stem cells (iPSCs) involves static culture in dishes. Two groups now report that mouse iPSCs can be generated efficiently in stirred suspension culture.

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology.

Temperature-responsive poly(N-isopropylacrylamide)-grafted microcarriers for large-scale non-invasive harvest of anchorage-dependent cells

Cell cultivation on the surface of microcarriers in stirred suspension is an essential method for the large-scale culture of anchorage-dependent cells. For applying this method to the field of cell therapy and for obtaining a large number of intact cells, non-invasive cell harvest without proteolytic enzyme treatment is an advantageous method. In this regard, temperature-responsive microcarriers that bearing poly(N-isopropylacrylamide) (PIPAAm)-grafted chains on the outermost surface were developed for harvesting cultured cells by temperature alteration. PIPAAm-grafted beads with the various grafted amount of PIPAAm and various bead diameters were synthesized for optimizing cell proliferation and thermally-induced detachment on the surface. The chinese hamster ovary (CHO-K1) cells adhered on the surface of all PIPAAm-grafted beads at 37 °C, while the adhering cells were found to detach themselves from the surfaces at 20 °C. The efficiency of thermally-induced cell detachment increased with increasing the grafted amount of PIPAAm and the diameter of bead. An efficient cell proliferation on bead surfaces in stirred suspension culture and subsequent thermally-induced cell detachment were achieved by the precise regulation of both the grafted amount of PIPAAm and diameter of bead. The temperature-responsive microcarriers exhibiting temperature-dependent cell adhesion and detachment will be an attractive candidate for the large-scale cell culture of therapeutic cells.

Stirred Suspension Culture Improves Embryoid Body Formation and Cardiogenic Differentiation of Genetically Modified Embryonic Stem Cells

Embryonic stem cells (ESCs) can propagate unlimitedly in vitro and differentiate into cardiomyocytes, which have been proposed as unlimited cell sources for cardiac cell therapy. This was limited by difficulties in large-scale generation of pure cardiomyocytes. In this study, we used stirred bioreactors to optimize the differentiation condition for mass production of embryoid bodies (EBs) derived from genetically modified mouse ESCs. Stirred suspension culture could more efficiently produce EBs and have a more uniform EB population without large necrotic centers, compared with the conventional static culture. Importantly, the cardiac-specific gene expressions (GATA binding protein 4, α-cardiac myosin heavy chain and myosin light chain-2v) were increased within EBs cultured in stirred bioreactor. Stirred suspension culture significantly increased the proportion of spontaneously contracting EBs, yielded a greater percentage of α-sarcomeric actinin-positive cells detected via flow cytometry, and harvested relatively more cardiomyocytes after G418 selection. Stirred suspension culture provided a more ideal culture condition facilitating the growth of EBs and enhancing the cardiogenic differentiation of genetically modified ESCs, which may be valuable in large-scale generation of pure cardiomyocytes.

Creation of mouse embryonic stem cell-derived cardiac cell sheets

Research on heart tissue engineering is an exciting and promising area. Although we previously developed bioengineered myocardium using cell sheet-based tissue engineering technologies, the issue of appropriate cell sources remained unresolved. In the present study, we created cell sheets of mouse embryonic stem (ES) cell-derived cardiomyocytes after expansion in three-dimensional stirred suspension cultures. Serial treatment of the suspension cultures with noggin and granulocyte colony-stimulating factor significantly increased the number of cardiomyocytes by more than fourfold compared with untreated cultures. After drug selection for ES cells expressing the neomycin-resistance gene under the control of the α-myosin heavy chain promoter, almost all of the cells showed spontaneous beating and expressed several cardiac contractive proteins in a fine striated pattern. When ES-derived cardiomyocytes alone were seeded onto temperature-responsive culture dishes, cell sheets were not created, whereas cocultures with cardiac fibroblasts promoted cell sheet formation. The cardiomyocytes in the cell sheets beat spontaneously and synchronously, and expressed connexin 43 at the edge of adjacent cardiomyocytes. Furthermore, when the extracellular action potential was recorded, unidirectional action potential propagation was observed. The present findings suggest that stirred suspension cultures with appropriate growth factors are capable of producing cardiomyocytes effectively and easily, and that ES-derived cardiac cell sheets may be a promising tool for the development of bioengineered myocardium.

Pseudoislets in stirred-suspension culture exhibit enhanced cell survival, propagation and insulin secretion

Cells from primary islets and beta-cell lines form pseudoislets (PIs) in static cultures. Interestingly, MIN6 beta-cells with aberrant regulation of proliferation form PIs which cease to grow after a week in culture. This growth arrest is attributed to a pro-apoptotic and anti-proliferative PI environment. We hypothesized that cell necrosis due to poor nutrient transport in dishes rather than apoptosis effects the observed PI size restriction. Formation of beta-cell PIs was explored in stirred-suspension bioreactors with enhanced mass transfer. Cells in stirred-suspension proliferated continuously and the PI size increased for two weeks. Bioreactor PIs displayed regulated basal insulin secretion and enhanced responsivity to glucose and incretins. Compared to dishes, cell viability in the bioreactor was higher with lower released lactate dehydrogenase activity. Similar expression of p21 and p27 in monolayers and PIs did not suggest an anti-proliferative PI milieu. Caspase-2, -8 and -9 activities were comparable in dish and bioreactor PIs, and the latter continued to grow after one week of culture. Thus, apoptosis is not sufficient to explain the differences in PI size between dishes and bioreactor. Moreover, the bioreactor method described here may be used to generate PIs with increased cell viability and function for research and clinical applications.