High-titer manufacturing of SARS-CoV-2 Spike-pseudotyped VSV in stirred-tank bioreactors.

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms have been rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus (S-VSV)-vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 ± 0.42 ×106 cells/mL in 1-L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 ± 0.58 ×108 plaque-forming units (PFUs)/mL at 3 days postinfection. When compared to growth in plate-based cultures, this was a 29-fold increase in virus production, meaning a 1-L bioreactor produces the same amount of virus as 1,284 plates of 15 cm. In addition, the omicron BA.1 S-VSV reached a peak titer of 5.58 ± 0.35 × 106 PFU/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-PFU ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics.

Adipose-derived stem cells (ASCs) culture in spinner flask: improving the parameters of culture in a microcarrier-based system.

Prior to clinical use, extensive in vitro proliferation of human adipose-derived stem cells (ASCs) is required. Among the current options, spinner-type stirred flasks, which use microcarriers to increase the yield of adherent cells, are recommended. Here, we propose a methodology for ASCs proliferation through cell suspension culture using Cultispher-S® microcarriers (MC) under agitation in a spinner flask, with the aim of establishing a system that reconciles the efficiency of cell yield with high viability of the culture during two distinct phases: seeding and proliferation. The results showed that cell adhesion was potentiated under intermittent stirring at 70 rpm in the presence of 10% FBS for an initial cell concentration of 2.4 × 104 cells/mL in the initial 24 h of cultivation. In the proliferation phase, kinetic analysis showed that cell growth was higher under continuous agitation at 50 rpm with a culture medium renewal regime of 50% every 72 h, which was sufficient to maintain the culture at optimal levels of nutrients and metabolites for up to nine days of cultivation, representing an 11.1-fold increase and a maximum cell productivity of 422 cells/mL/h (1.0 × 105 viable cells/mL). ASCs maintained the immunophenotypic characteristics and mesodermal differentiation potential of both cell lines from different donors. The established protocol represents a more efficient and cost-effective method to obtain a high proliferation rate of ASCs in a microcarrier-based system, which is necessary for large-scale use in cell therapy, highlighting that the manipulation of critical parameters optimizes the ASCs production process.

Osteogenic Activity on NaOH-Etched Three-Dimensional-Printed Poly-ɛ-Caprolactone Scaffolds in Perfusion or Spinner Flask Bioreactor.

Bioreactor systems, for example, spinner flask and perfusion bioreactors, and cell-seeded three-dimensional (3D)-printed scaffolds are used in bone tissue engineering strategies to stimulate cells and produce bone tissue suitable for implantation into the patient. The construction of functional and clinically relevant bone graft using cell-seeded 3D-printed scaffolds within bioreactor systems is still a challenge. Bioreactor parameters, for example, fluid shear stress and nutrient transport, will crucially affect cell function on 3D-printed scaffolds. Therefore, fluid shear stress induced by spinner flask and perfusion bioreactors might differentially affect osteogenic responsiveness of pre-osteoblasts inside 3D-printed scaffolds. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine fluid shear stress and osteogenic responsiveness of MC3T3-E1 pre-osteoblasts seeded on the scaffolds in the bioreactors using finite element (FE)-modeling and experiments. FE-modeling was used to quantify wall shear stress (WSS) distribution and magnitude inside 3D-printed PCL scaffolds within spinner flask and perfusion bioreactors. MC3T3-E1 pre-osteoblasts were seeded on NaOH surface-modified 3D-printed PCL scaffolds, and cultured in customized static, spinner flask, and perfusion bioreactors up to 7 days. The scaffolds' physicochemical properties and pre-osteoblast function were assessed experimentally. FE-modeling showed that spinner flask and perfusion bioreactors locally affected WSS distribution and magnitude inside the scaffolds. The WSS distribution was more homogeneous inside scaffolds in perfusion than in spinner flask bioreactors. The average WSS on scaffold-strand surfaces ranged from 0 to 6.5 mPa for spinner flask bioreactors, and from 0 to 4.1 mPa for perfusion bioreactors. Surface modification of scaffolds by NaOH resulted in a surface with a honeycomb-like pattern and increased surface roughness (1.6-fold), but decreased water contact angle (0.3-fold). Both spinner flask and perfusion bioreactors increased cell spreading, proliferation, and distribution throughout the scaffolds. Perfusion, but not spinner flask bioreactors more strongly enhanced collagen (2.2-fold) and calcium deposition (2.1-fold) throughout the scaffolds after 7 days compared with static bioreactors, likely due to uniform WSS-induced mechanical stimulation of the cells revealed by FE-modeling. In conclusion, our findings indicate the importance of using accurate FE models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems. Impact Statement The success of cell-seeded three-dimensional (3D)-printed scaffolds depends on cell stimulation by biomechanical/biochemical factors to produce bone tissue suitable for implantation into the patient. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine wall shear stress (WSS) and osteogenic responsiveness of pre-osteoblasts seeded on the scaffolds using finite element (FE)-modeling and experiments. We found that cell-seeded 3D-printed PCL scaffolds within perfusion bioreactors more strongly enhanced osteogenic activity than within spinner flask bioreactors. Our results indicate the importance of using accurate FE-models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems.

Enhancing mesenchymal stem cells cultivated on microcarriers in spinner flasks via impeller design optimization for aggregated suspensions.

During the ex vivo expansion of umbilical cord-derived mesenchymal stem cells (hUCMSCs) in a stirred tank bioreactor, the formation of cell-microcarrier aggregates significantly affects cell proliferation and physiological activity, making it difficult to meet the quantity and quality requirements for in vitro research and clinical applications. In this study, computational fluid dynamic (CFD) simulations were used to investigate the effect of an impeller structure in a commercial spinner flask on flow field structure, aggregate formation, and cellular physiological activity. By designing a modified impeller, the aggregate size was reduced, which promoted cell proliferation and stemness maintenance. This study showed that increasing the stirring speed reduced the size of hUCMSC-microcarrier aggregates with the original impeller. However, it also inhibited cell proliferation, decreased activity, and led to spontaneous differentiation. Compared to low stirring speeds, high stirring speeds did not alter the radial flow characteristics and vortex distribution of the flow field, but did generate higher shear rates. The new impeller's design changed the flow field from radial to axial. The use of the novel impeller with an increased axial pumping rate (Qz) at a similar shear rate compared to the original impeller resulted in a 43.7% reduction in aggregate size, a 37.4% increase in cell density, and a better preservation of the expression of stemness markers (SOX2, OCT4 and NANOG). Increasing the Qz was a key factor in promoting aggregate suspension and size reduction. The results of this study have significant implications for the design of reactors, the optimisation of operating parameters, and the regulation of cellular physiological activity during MSC expansion.

Systematic review on spheroids from adipose-derived stem cells: Spontaneous or artefact state?

Three-dimensional (3D) cell cultures represent the spontaneous state of stem cells with specific gene and protein molecular expression that are more alike the in vivo condition. In vitro two-dimensional (2D) cell adhesion cultures are still commonly employed for various cellular studies such as movement, proliferation and differentiation phenomena; this procedure is standardized and amply used in laboratories, however their representing the original tissue has recently been subject to questioning. Cell cultures in 2D require a support/substrate (flasks, multiwells, etc.) and use of fetal bovine serum as an adjuvant that stimulates adhesion that most likely leads to cellular aging. A 3D environment stimulates cells to grow in suspended aggregates that are defined as "spheroids." In particular, adipose stem cells (ASCs) are traditionally observed in adhesion conditions, but a recent and vast literature offers many strategies that obtain 3D cell spheroids. These cells seem to possess a greater ability in maintaining their stemness and differentiate towards all mesenchymal lineages, as demonstrated in in vitro and in vivo studies compared to adhesion cultures. To date, standardized procedures that form ASC spheroids have not yet been established. This systematic review carries out an in-depth analysis of the 76 articles produced over the past 10 years and discusses the similarities and differences in materials, techniques, and purposes to standardize the methods aimed at obtaining ASC spheroids as already described for 2D cultures.

Physical optimization of cell proliferation and differentiation using spinner flask and microcarriers.

The traditional breeding industry has been increasingly saturated and caused environmental pollution, disease transmission, excessive resource use, and methane emission; however, it still cannot meet the needs of the growing population. To explore other alternatives, researchers focused on cell agriculture and cell-based meat, especially large-scale cell culture. As a prerequisite for production, large-scale culture technology has become an important bottleneck restricting cell-based meat industrialization. In this study, the single-factor variable method was adopted to examine the influence of Cytodex1 microcarrier pretreatment, spinner flask reaction vessel, cell culture medium, serum and cell incubation, and other influencing factors on large-scale cell cultures to identify the optimization parameters suitable for 3D culture environment. Collagen and 3D culture were also prospectively explored to promote myogenesis and cultivate tissue-like muscle fibers that contract spontaneously. This research lays a theoretical foundation and an exploratory practice for large-scale cell cultures and provides a study reference for the microenvironment of myoblast culture in vitro, a feasible direction for the cell therapy of muscular dystrophy, and prerequisites for the industrialized manufacturing of cell-based meat. Graphical summary: Research on large-scale myoblast culture using spinner flasks and microcarriers. For cell culture, the microcarriers were pretreated with UV and collagen. Cell seeding condition, spinner flask speed, resting time, and spinner flask culture microenvironment were then optimized. Finally, two culture systems were prepared: a culture system based on large-scale cell expansion and a culture system for myogenesis promotion and differentiation.

The combination of dextran sulphate and polyvinyl alcohol prevents excess aggregation and promotes proliferation of pluripotent stem cells in suspension culture.

OBJECTIVES: For clinical applications of cell-based therapies, a large quantity of human pluripotent stem cells (hPSCs) produced in standardized and scalable culture processes is required. Currently, microcarrier-free suspension culture shows potential for large-scale expansion of hPSCs; however, hPSCs tend to aggregate during culturing leading to a negative effect on cell yield. To overcome this problem, we developed a novel protocol to effectively control the sizes of cell aggregates and enhance the cell proliferation during the expansion of hPSCs in suspension. MATERIALS AND METHODS: hPSCs were expanded in suspension culture supplemented with polyvinyl alcohol (PVA) and dextran sulphate (DS), and 3D suspension culture of hPSCs formed cell aggregates under static or dynamic conditions. The sizes of cell aggregates and the cell proliferation as well as the pluripotency of hPSCs after expansion were assessed using cell counting, size analysis, real-time quantitative polymerase chain reaction, flow cytometry analysis, immunofluorescence staining, embryoid body formation, teratoma formation and transcriptome sequencing. RESULTS: Our results demonstrated that the addition of DS alone effectively prevented hPSC aggregation, while the addition of PVA significantly enhanced hPSC proliferation. The combination of PVA and DS not only promoted cell proliferation of hPSCs but also produced uniform and size-controlled cell aggregates. Moreover, hPSCs treated with PVA, or DS or a combination, maintained the pluripotency and were capable of differentiating into all three germ layers. mRNA-seq analysis demonstrated that the combination of PVA and DS significantly promoted hPSC proliferation and prevented cell aggregation through improving energy metabolism-related processes, regulating cell growth, cell proliferation and cell division, as well as reducing the adhesion among hPSC aggregates by affecting expression of genes related to cell adhesion. CONCLUSIONS: Our results represent a significant step towards developing a simple and robust approach for the expansion of hPSCs in large scale.

Microcarrier Screening and Evaluation for Dynamic Expansion of Human Periosteum-Derived Progenitor Cells in a Xenogeneic Free Medium.

An increasing need toward a more efficient expansion of adherent progenitor cell types arises with the advancements of cell therapy. The use of a dynamic expansion instead of a static planar expansion could be one way to tackle the challenges of expanding adherent cells at a large scale. Microcarriers are often reported as a biomaterial for culturing cells in suspension. However, the type of microcarrier has an effect on the cell expansion. In order to find an efficient expansion process for a specific adherent progenitor cell type, it is important to investigate the effect of the type of microcarrier on the cell expansion. Human periosteum-derived progenitor cells are extensively used in skeletal tissue engineering for the regeneration of bone defects. Therefore, we evaluated the use of different microcarriers on human periosteum-derived progenitor cells. In order to assess the potency, identity and viability of these cells after being cultured in the spinner flasks, this study performed several in vitro and in vivo analyses. The novelty of this work lies in the combination of screening different microcarriers for human periosteum-derived progenitor cells with in vivo assessments of the cells' potency using the microcarrier that was selected as the most promising one. The results showed that expanding human periosteum-derived progenitor cells in spinner flasks using xeno-free medium and Star-Plus microcarriers, does not affect the potency, identity or viability of the cells. The potency of the cells was assured with an in vivo evaluation, where bone formation was achieved. In summary, this expansion method has the potential to be used for large scale cell expansion with clinical relevance.

Scaffold Pore Geometry Guides Gene Regulation and Bone-like Tissue Formation in Dynamic Cultures.

Cells sense and respond to scaffold pore geometry and mechanical stimuli. Many fabrication methods used in bone tissue engineering render structures with poorly controlled pore geometries. Given that cell-scaffold interactions are complex, drawing a conclusion on how cells sense and respond to uncontrolled scaffold features under mechanical loading is difficult. In this study, monodisperse templated scaffolds (MTSC) were fabricated and used as well-defined porous scaffolds to study the effect of dynamic culture conditions on bone-like tissue formation. Human bone marrow-derived stromal cells were cultured on MTSC or conventional salt-leached scaffolds (SLSC) for up to 7 weeks, either under static or dynamic conditions (wall shear stress [WSS] using spinner flask bioreactors). The influence of controlled spherical pore geometry of MTSC subjected to static or dynamic conditions on osteoblast cell differentiation, bone-like tissue formation, structure, and distribution was investigated. WSS generated within the two idealized geometrical scaffold features was assessed. Distinct response to fluid flow in osteoblast cell differentiation were shown to be dependent on scaffold pore geometry. As revealed by collagen staining and microcomputed tomography images, dynamic conditions promoted a more regular extracellular matrix (ECM) formation and mineral distribution in both scaffold types compared with static conditions. The results showed that regulation of bone-related genes and the amount and the structure of mineralized ECM were dependent on scaffold pore geometry and the mechanical cues provided by the two different culture conditions. Under dynamic conditions, SLSC favored osteoblast cell differentiation and ECM formation, whereas MTSC enhanced ECM mineralization. The spherical pore shape in MTSC supported a more trabecular bone-like structure under dynamic conditions compared with MTSC statically cultured or to SLSC under either static or dynamic conditions. These results suggest that cell activity and bone-like tissue formation is driven not only by the pore geometry but also by the mechanical environment. This should be taken into account in the future design of complex scaffolds, which should favor cell differentiation while guiding the formation, structure, and distribution of the engineered bone tissue. This could help to mimic the anatomical complexity of the bone tissue structure and to adapt to each bone defect needs. Impact statement Aging of the human population leads to an increasing need for medical implants with high success rate. We provide evidence that cell activity and the amount and structure of bone-like tissue formation is dependent on the scaffold pore geometry and on the mechanical environment. Fabrication of complex scaffolds comprising concave and planar pore geometries might represent a promising direction toward the tunability and mimicry the structural complexity of the bone tissue. Moreover, the use of fabrication methods that allow a systematic fabrication of reproducible and geometrically controlled structures would simplify scaffold design optimization.

Enhanced chondrogenic differentiation of human mesenchymal stem cells in silk fibroin/chitosan/glycosaminoglycan scaffolds under dynamic culture condition.

Cartilage tissue damage and diseases are the most common clinical situation that occurs because of aging and injury, thereby causing pain and loss of mobility. The inability of cartilage tissue to self-repair is instrumental in developing tissue engineered substitutes. To this effect, the present study aims to engineer cartilage construct by culturing umbilical cord blood-derived human mesenchymal stem cells (hMSCs) on novel 3D porous scaffolds developed from natural biopolymers, silk fibroin (SF) and chitosan (CS), with addition of cartilage matrix components, glucosamine (Gl) and chondroitin sulfate (Ch). The presence of Gl and Ch is expected to enhance cartilage regeneration. The developed SF/CS-Gl-Ch scaffolds possess desired pore size in the range 56.55-168.15 µm, 88-92% porosity, 44.7-46.8̊ contact angle, controlled swelling and biodegradability. Upon culturing under dynamic condition in a spinner flask bioreactor, the scaffold supported hMSCs attachment, proliferation, and further promoted chondrogenic differentiation. Cartilage-specific matrix and gene (Collagen II, Sox9 and aggrecan) expression analyses by histology, immunophenotype, immunofluorescence and quantitative PCR studies showed superiority of cell-scaffold construct generated in dynamic culture towards cartilage tissue generation as compared to cell aggregates formed by pellet culture. This study demonstrates the potentiality of SF/CS-Gl-Ch porous scaffold for the development of tissue construct for cartilage regeneration under dynamic culture condition.

Dynamic formation of cellular aggregates of chondrocytes and mesenchymal stem cells in spinner flask.

OBJECTIVES: Cellular aggregates are readily applicable in cell-based therapy. The effects of agitation and inoculation density on the aggregation of cells in spinner flask and the molecular mechanism of aggregation were investigated. MATERIALS AND METHODS: The aggregation kinetics of cells in spinner flask was evaluated with bovine articular chondrocytes (bACs), rabbit bone marrow-derived mesenchymal stem cells (rMSCs) and their mixture. The morphology of cellular aggregates was studied with scanning electron microscopy and gene expression of cell adhesion-related molecules was analysed. RESULTS: It was shown that suspension culture in spinner flask induced the aggregation of bACs and rMSCs. Both cells exhibited increased aggregation rate and aggregate size with decreasing agitation rate and increasing cell inoculation density. Additionally, aggregate size increased with extended culture time. By analysing gene expression of integrin ß1 and cadherin, it was indicated that these molecules were potentially involved in the aggregation process of bACs and rMSCs, respectively. Aggregates composed of both bACs and rMSCs were also prepared, showing rMSCs in the core and bACs in the periphery. CONCLUSIONS: Cellular aggregates were prepared in dynamic suspension culture using spinner flask, the key parameters to the aggregation process were identified, and the molecular mechanism of aggregation was revealed. This would lay a solid foundation for the large-scale production of cellular aggregates for cell-based therapy, such as cartilage regeneration.

Chondrogenic differentiation of mesenchymal stem cells on silk fibroin:chitosan-glucosamine scaffold in dynamic culture.

AIM: Cartilage damage is a common age-related problem that leads to progressive proteoglycan loss. Glucosamine stimulates proteoglycan synthesis and, therefore, its effect on the cartilage extracellular matrix synthesis over silk fibroin:chitosan (SF:CS) tissue-engineered scaffold was investigated for cartilage construct generation. MATERIALS & METHODS: Human mesenchymal stem cells (hMSCs) were cultured and differentiated over SF:CS-glucosamine porous scaffold, under dynamic culture condition in spinner flask bioreactor. RESULTS: hMSCs-seeded scaffold in dynamic culture exhibited homogenous cell distribution, proliferation and higher cell density at the core than static culture. Glucosamine in scaffold promoted proteoglycan and collagenous matrix synthesis as revealed by histological and immunofluorescence studies. Quantitative-PCR analysis showed upregulation of cartilage-specific genes, thereby confirming the chondrogenic differentiation. CONCLUSION: The chondrogenic differentiation of hMSCs was enhanced by the synergistic effect of glucosamine incorporated in SF:CS scaffold and influence of 3D dynamic culture environment, thereby resulting in chondrogenic phenotype of the cells that promoted cartilage regeneration.

Aggregation of Culture Expanded Human Mesenchymal Stem Cells in Microcarrier-based Bioreactor.

Three-dimensional aggregation of human mesenchymal stem cells (hMSCs) has been used to enhance their therapeutic properties but current fabrication protocols depend on laboratory methods and are not scalable. In this study, we developed thermal responsive poly(N-isopropylacrylamide) grafted microcarriers (PNIPAM-MCs), which supported expansion and thermal detachment of hMSCs at reduced temperature (23.0 °C). hMSCs were cultured on the PNIPAM-MCs in both spinner flask (SF) and PBS Vertical-Wheel (PBS-VW) bioreactors for expansion. At room temperature, hMSCs were detached as small cell sheets, which subsequently self-assembled into 3D hMSC aggregates in PBS-VW bioreactor and remain as single cells in SF bioreactor owing to different hydrodynamic conditions. hMSC aggregates generated from the bioreactor maintained comparable immunomodulation and cytokine secretion properties compared to the ones made from the AggreWell®. The results of the current study demonstrate the feasibility of scale-up production of hMSC aggregates in the suspension bioreactor using thermal responsive microcarriers for integrated cell expansion and 3D aggregation in a close bioreactor system and highlight the critical role of hydrodynamics in self-assembly of detached hMSC in suspension.

Enhanced chondrogenesis of mesenchymal stem cells over silk fibroin/chitosan-chondroitin sulfate three dimensional scaffold in dynamic culture condition.

Chondroitin sulfate (Ch) is one of the main structural components of cartilage tissue, therefore, its presence in tissue engineered scaffold is expected to enhance cartilage regeneration. Previously, silk fibroin/chitosan (SF/CS) blend was proven to be a potential biomaterial for tissue development. In this study, the effect of Ch on physicochemical and biological properties of SF/CS blend was investigated and scaffolds with 0.8 wt% Ch was found to be favorable. The scaffolds possess pore size of 37-212 µm, contact angle 46.2-50.3°, showed controlled swelling and biodegradation. The biocompatibility of scaffold was confirmed by subcutaneous implantation in mouse. Human mesenchymal stem cells (hMSCs) seeded scaffolds cultured under spinner flask bioreactor promoted cell attachment, proliferation, distribution, and metabolic activity in vitro. The histology and immunofluorescence studies revealed that combined effect of Ch and dynamic condition resulted in higher glycosaminoglycan secretion and native cartilage type matrix synthesis in comparison to SF/CS scaffolds used as control. Higher expression of collagen-II, Sox9, aggrecan and decrease in collagen-I expression represented by quantitative polymerase chain reaction study confirmed the progression of chondrogenic differentiation. This study successfully demonstrates the potentiality of SF/CS-Ch scaffold for hMSCs recruitment and redirecting cartilage tissue regeneration with enhanced chondrogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2576-2587, 2018.

In vitro cartilage construct generation from silk fibroin- chitosan porous scaffold and umbilical cord blood derived human mesenchymal stem cells in dynamic culture condition.

Cartilage construct generation includes a scaffold with appropriate composition to mimic matrix of the damaged tissue on which the stem cells grow and differentiate. In this study, umbilical cord blood (UCB) derived human mesenchymal stem cells (hMSCs) were seeded on freeze dried porous silk-fibroin (SF)/chitosan (CS) scaffolds. Influence of static and dynamic (spinner flask bioreactor) culture conditions on the developing cartilage construct were studied by in-vitro characterization for viability, proliferation, distribution, and chondrogenic differentiation of hMSCs over the scaffold. Constructs developed in spinner flask consisted of 62% live cells, and exhibited 543% more cell density at the core than constructs cultured in static system. Quantification of DNA and glycosaminoglycans accumulation after 21 days showed the progression of chondrogenic differentiation of hMSCs was higher in dynamic culture compared to static one. In constructs generated under dynamic condition, histology staining for proteoglycan matrix, and fluorescence staining for collagen-II and aggrecan showed positive correlation between early and late stage chondrogenic markers, which was further confirmed by quantitative PCR analysis, showing low collagen-I expression and highly expressed Sox9, collagen-II and aggrecan. The present study demonstrated that construct generated by combining 3D SF/CS scaffold with UCB-hMSCs under dynamic condition using spinner flask bioreactor can be used for cartilage tissue regeneration for future medical treatments. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 397-407, 2018.

An Integrated Bioprocess for the Expansion and Chondrogenic Priming of Human Periosteum-Derived Progenitor Cells in Suspension Bioreactors.

The increasing use of microcarrier-based suspension bioreactors for scalable expansion of adult progenitor cells in recent years reveals the necessity of such approaches to address bio manufacturing challenges of advanced therapeutic medicinal products. However, the differentiation of progenitor cells within suspension bioreactors for the production of tissue modules is of equal importance but not well investigated. This study reports on the development of a bioreactor-based integrated process for expansion and chondrogenic priming of human periosteum-derived stem cells (hPDCs) using Cultispher S microcarriers. Spinner flask-based expansion and priming of hPDCs were carried out over 12 days for expansion and 14 days for priming. Characterization of the cells were carried out every 3rd day. Our study showed that hPDCs were able to expand till confluency with fold increase of 3.2±0.64 and to be subsequently primed toward a chondrogenic state within spinner flasks. During expansion, the cells maintained their phenotypic markers, trilineage differentiation capabilities and viability. Upon switching to TGF-ß containing media the cells were able to differentiate toward chondrogenic lineage by clustering into mm-sized macrotissues containing hundreds of microcarriers. Chondrogenic priming was further evidenced by the expression of relevant markers at the mRNA level while maintaining their viability. Ectopic implantation of macrotissues highlighted that they were able to sustain their chondrogenic properties for 8 weeks in vivo. The method indicated here, suggests that expansion and relevant priming of progenitor cells can be carried out in an integrated bioprocess using spinner flasks and as such could be potentially extrapolated to other stem and progenitor cell populations.

Clinical-Grade Manufacturing of Therapeutic Human Mesenchymal Stem/Stromal Cells in Microcarrier-Based Culture Systems.

The therapeutic potential of mesenchymal stem/stromal cells (MSC) has triggered the need for high cell doses in a vast number of clinical applications. This demand requires the development of good manufacturing practices (GMP)-compliant ex vivo expansion protocols that should be effective to deliver a robust and reproducible supply of clinical-grade cells in a safe and cost-effective manner. Controlled stirred-tank bioreactor systems under xenogeneic (xeno)-free culture conditions offer ideal settings to develop and optimize cell manufacturing to meet the standards and needs of human MSC for cellular therapies. Herein we describe two microcarrier-based stirred culture systems using spinner flasks and controlled stirred-tank bioreactors under xeno-free conditions for the efficient ex vivo expansion of human bone marrow and adipose tissue-derived MSC.

Expansion of Human Mesenchymal Stem Cells in a Microcarrier Bioreactor.

Human mesenchymal stem cells (hMSCs) are considered as a primary candidate in cell therapy owing to their self-renewability, high differentiation capabilities, and secretions of trophic factors. In clinical application, a large quantity of therapeutically competent hMSCs is required that cannot be produced in conventional petri dish culture. Bioreactors are scalable and have the capacity to meet the production demand. Microcarrier suspension culture in stirred-tank bioreactors is the most widely used method to expand anchorage dependent cells in a large scale. Stirred-tank bioreactors have the potential to scale up and microcarriers provide the high surface-volume ratio. As a result, a spinner flask bioreactor with microcarriers has been commonly used in large scale expansion of adherent cells. This chapter describes a detailed culture protocol for hMSC expansion in a 125 mL spinner flask using microcarriers, Cytodex I, and a procedure for cell seeding, expansion, metabolic sampling, and quantification and visualization using microculture tetrazolium (MTT) reagent.

Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells.

In recent times, the study and use of induced pluripotent stem cells (iPSC) have become important in order to avoid the ethical issues surrounding the use of embryonic stem cells. Therapeutic, industrial and research based use of iPSC requires large quantities of cells generated in vitro. Mammalian cells, including pluripotent stem cells, have been expanded using 3D culture, however current limitations have not been overcome to allow a uniform, optimized platform for dynamic culture of pluripotent stem cells to be achieved. In the current work, we have expanded mouse iPSC in a spinner flask using Cytodex 3 microcarriers. We have looked at the effect of agitation on the microcarrier survival and optimized an agitation speed that supports bead suspension and iPS cell expansion without any bead breakage. Under the optimized conditions, the mouse iPSC were able to maintain their growth, pluripotency and differentiation capability. We demonstrate that microcarrier survival and iPS cell expansion in a spinner flask are reliant on a very narrow range of spin rates, highlighting the need for precise control of such set ups and the need for improved design of more robust systems.

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture.

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10-30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 10(9) BHK-21 cells from 4 × 10(6) cells in 13 day with 1,051 mL culture medium.