Large-scale Cell Expansion Research Articles

An efficient serum-free medium for ex vivo expansion of myoblasts from Larimichthys crocea for cultured meat production

The large-scale production of cultured meat has been restricted by high cost of serum, batch difference and unclear composition, thus it is important to develop serum-free media for cell growth. Here we obtained a serum-free medium (SFM), which was suitable for ex vivo culturing of myoblasts from larimichthys crocea. It offered simple composition and high effectiveness that supporting myoblast attachment, expansion and differentiation. Our findings revealed that the optimal SFM consisted of DMEM/F12 base medium, 5 mg/mL bovine serum albumin (BSA), 20 mmol HEPES, 1 % penicillin/streptomycin mixture (PS) and the exogenous additive hydrocortisone. The living cells of SFM could occupy about 65 % of the serum-containing medium (SM). Additionally, differentiation assays showed that myoblasts cultured without serum still differentiated into more muscle tubes, with a higher fusion index than those cultured with serum. Finally, transcriptome sequencing was conducted to demonstrate that hydrocortisone might act as a ligand binding to G protein-coupled receptors, triggering the downstream PI3K/AKT signaling pathway and leading to cell proliferation. Our findings will provide a basis for large-scale cell expansion and cell culture meat production in the future.

Large-Scale Expansion of Human Liver Stem Cells Using Two Different Bioreactor Systems.

The assessment of human liver stem cells (HLSCs) as cell therapeutics requires scalable, controlled expansion processes. We first focused on defining appropriate process parameters for HLSC expansion such as seeding density, use of antibiotics, optimal cell age and critical metabolite concentrations in conventional 2D culture systems. For scale-up, we transferred HLSC expansion to multi-plate and stirred-tank bioreactor systems to determine their limitations. A seeding density of 4000 cells cm-2 was needed for efficient expansion. Although growth was not significantly affected by antibiotics, the concentrations of lactate and ammonia were important. A maximum expansion capacity of at least 20 cumulative population doublings (cPDs) was observed, confirming HLSC growth, identity and functionality. For the expansion of HLSCs in the multi-plate bioreactor system Xpansion (XPN), the oxygen supply strategy was optimized due to a low kLa of 0.076 h-1. The XPN bioreactor yielded a final mean cell density of 94 ± 8 × 103 cells cm-2, more than double that of the standard process in T-flasks. However, in the larger XPN50 device, HLSC density reached only 28 ± 0.9 × 103 cells cm-2, while the glucose consumption rate increased 8-fold. In a fully-controlled 2 L stirred-tank bioreactor (STR), HLSCs expanded at a comparable rate to the T-flask and XPN50 processes in a homogeneous microenvironment using advanced process analytical technology. Ultimately, the scale-up of HLSCs was successful using two different bioreactor systems, resulting in sufficient numbers of viable, functional and undifferentiated HLSCs for therapeutic applications.

Efficient expansion and CRISPR-Cas9-mediated gene correction of patient-derived hepatocytes for treatment of inherited liver diseases

Cell-based exvivo gene therapy in solid organs, especially the liver, has proven technically challenging. Here, we report a feasible strategy for the clinical application of hepatocyte therapy. We first generated high-quality autologous hepatocytes through the large-scale expansion of patient-derived hepatocytes. Moreover, the proliferating patient-derived hepatocytes, together with the AAV2.7m8 variant identified through screening, enabled CRISPR-Cas9-mediated targeted integration efficiently, achieving functional correction of pathogenic mutations in FAH or OTC. Importantly, these edited hepatocytes repopulated the injured mouse liver at high repopulation levels and underwent maturation, successfully treating mice with tyrosinemia following transplantation. Our study combines exvivo large-scale cell expansion and gene editing in patient-derived transplantable hepatocytes, which holds potential for treating human liver diseases.

Abstract 5252: Expansion of human T cells and NK cells for chimeric antigen receptor based therapies using human platelet lysate

Abstract T cells and NK cells expressing chimeric antigen receptors (CAR) have demonstrated potent clinical efficacy in patients with hematological malignancies. One of the issues to overcome in the treatment of solid tumors using CARs is the lack of a process that generates large amounts of CAR-T cells, reduces cell exhaustion and differentiation, and improves long term survival after infusion into patients. Previously, Torres Chavez et al. demonstrated in a series of in vitro and in vivo experiments, performed in both hematologic and solid tumor models, the profound qualitative impact on CAR-T cell performance after using human platelet lysate (hPL) as a cell growth supplement. T cells expanded with their hPL showed a good proliferative capacity and enhanced long-term in vivo persistence compared to Fetal Bovine Serum (FBS) or human AB Serum (ABS), resulting in superior anti-tumor effects. Additionally, this group demonstrated that the transduction of T cells to generate the CAR-T cells was significantly improved by the use of hPL. Mill Creek’s human platelet lysate (hPL) is produced using expired human platelets obtained from accredited blood banks in the United States. These platelets were originally intended for use in patient transfusion. The safety of platelets used in transfusion is managed by the U.S. Food & Drug Administration (FDA), as well as the Association for the Advancement of Blood & Biotherapies (AABB). These organizations set standards, including testing for transmissible diseases. The United States record for blood safety is well established, with extremely low rates of disease transmission, making the platelet units used for hPL manufacture low risk. The Covid-19 pandemic has increased awareness of emerging infectious diseases, even though transmission of Covid-19 via blood transfusion has not been documented. For that reason, we developed a gamma irradiated version of our products, which offers an additional safety measure in the clinic. Our data from experiments performed using human CD3+ and purified NK cells from donor peripheral blood suggests that our human platelet lysates offer an improved cell phenotype and expansion capacities versus serum derived products, but also versus other platelet lysates on the market. PLTGold® and PLTGold®-GI efficiently promote cell expansion, with higher cell yields and lower cell exhaustion rate. Additionally, our hPLs are suitable for suspension culture, potentially facilitating the large-scale expansion of allogeneic CAR cells. The use of our platelet lysates has the potential of significantly improving CAR-T and CAR-NK manufacture, but also the outcome of the therapy itself by improving the quality and potency of the end product. Citation Format: Vanesa Alonso-Camino, William Mirsch. Expansion of human T cells and NK cells for chimeric antigen receptor based therapies using human platelet lysate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5252.

Skin repair and immunoregulatory effects of myeloid suppressor cells from human cord blood in atopic dermatitis.

Previously, we achieved large-scale expansion of bone marrow-derived suppressor cells (MDSCs) derived from cluster of differentiation (CD)34+ cells cultured in human umbilical cord blood (hUCB) and demonstrated their immunomodulatory properties. In the present study, we assessed the therapeutic efficacy of hUCB-MDSCs in atopic dermatitis (AD). Dermatophagoides farinae (Df)-induced NC/Nga mice (clinical score of 7) were treated with hUCB-MDSCs or a control drug. The mechanisms underlying the therapeutic effects of hUCB-MDSCs were evaluated. hUCB-MDSCs demonstrated immunosuppressive effects in both human and mouse CD4+ T cells. hUCB-MDSCs significantly reduced the clinical severity scores, which were associated with histopathological changes, and reduced inflammatory cell infiltration, epidermal hyperplasia, and fibrosis. Furthermore, hUCB-MDSCs decreased the serum levels of immunoglobulin E, interleukin (IL)-4, IL-5, IL-13, IL-17, thymus- and activation-regulated chemokines, and thymic stromal lymphopoietin. Additionally, they altered the expression of the skin barrier function-related proteins filaggrin, involucrin, loricrin, cytokeratin 10, and cytokeratin 14 and suppressed the activation of Df-restimulated T-cells via cell-cell interactions. hUCB-MDSCs promoted skin recovery and maintained their therapeutic effect even after recurrence. Consequently, hUCB-MDSC administration improved Df-induced AD-like skin lesions and restored skin barrier function. Our findings support the potential of hUCB-MDSCs as a novel treatment strategy for AD.

Characterization of Proliferation Medium and Its Effect on Differentiation of Muscle Satellite Cells from Larimichthys crocea in Cultured Fish Meat Production

To find a suitable medium for muscle satellite cells of Larimichthys crocea, herein, the effect of different basal media and coating materials on the proliferation of piscine satellite cells (PSCs) was explored. Firstly, two basal media, namely F10 and DMEM/F12, were selected as experimental materials, and high-sugar DMEM was the main basal culture medium used with fish muscle cells as a control. The results showed that the PSCs proliferated better in F10 than in DMEM/F12 or DMEM. Secondly, the effects of rat tail collagen, polylysine and matrix coatings, as compared with no coating, on the proliferation and later differentiation of PSCs were also investigated. Our results indicated that there was no significant difference between coating and no coating on the proliferation of PSCs in the F10-based medium. Meanwhile, it was found that the myotubes were washed out, and only those under matrix-coated conditions remained intact in the process of differentiation. The results also suggested that PSCs could still differentiate into myotubes without their stemness being affected after proliferation in the F10-based medium. Hence, this study identified an efficient proliferation medium based on F10 basal medium that could shorten the culture time and maintain the stemness of PSCs, thus providing a basis for large-scale cell expansion and cell-culture-based meat production in the future.

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers.

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To address the primary shortcomings of the two-dimensional (2D) planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on a GMP-grade, dissolvable, and porous microcarrier for the 3D culture of adherent cells, including human mesenchymal stem/stromal cells (hMSCs), HEK293T cells, and Vero cells. To achieve large-scale expansion, a two-stage expansion was conducted with 5 L and 15 L stirred-tank bioreactors to yield 1.1 x 1010 hMSCs with an overall 128-fold expansion within 9 days. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous-flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Compared with 2D planar culture, there are no significant differences in the quality of hMSCs harvested from 3D culture. We have also applied these dissolvable porous microcarriers to other popular cell types in the CGT sector; specifically, HEK293T cells and Vero cells have been cultivated to peak cell densities of 1.68 x 107 cells/mL and 1.08 x 107 cells/mL, respectively. This study provides a protocol for using a bioprocess engineering platform harnessing the characteristics of GMP-grade dissolvable microcarriers and advanced closed equipment to achieve the industrial-scale manufacturing of adherent cells.

Preparation of thermosensitive PNIPAm- based copolymers with tertiary amino and hydroxyl monomer for cell attachment and detachment study

Thermo-responsive microcarriers are essential for large-scale expansion of anchorage-dependent cells in a suspension culture. In this study, the authors prepared four different N-isopropylacrylamide (NIPAm)-based thermosensitive copolymers by copolymerizing with N-hydroxyethyl acrylamide (HEAA), 2-Hydroxyethyl methacrylate (HEMA), 2-(Diethylamino) ethylmethacrylate (DEAEMA), and 2-(Dimethylamino) ethylmethacylate (DMAEMA) through free radical polymerization (FRP) and used them for surface modification of Cytodex-3 microcarrier. The thermosensitive characteristic of the copolymers enabled microcarriers to attach and detach cells in response to temperature changes. Herein, NIH 3T3 cells were used as a model cell line and it was found that the cells' adhesion rate on Cytodex-3 microcarriers modified with different formulations of thermosensitive copolymers was greater than unmodified microcarriers. The microcarriers coated with 5 wt% of the copolymer showed better cell attachment than 3 wt% and 10 wt% copolymer coated microcarriers. Particularly, 5 wt% P(NIPAm-5% HEMA) copolymer coated microcarriers showed excellent cell attachment than other formulations. More importantly, as the temperature was reduced to 4 °C, the cells cultured on copolymer modified microcarriers were completely detached as a cell sheet from it withoutproteolytic enzymetreatment. Besides preserving the functionality and integrity of cellular structures, the thermal-induced detachment of cells from modified microcarriers was much higher (∼9.9 x 104 cells/mL) than cells detached from the original Cytodex-3 (∼7.5x103 cells/mL) in 1 h at 4 °C. Therefore, this study verified that microcarriers modified with thermosensitive copolymers exhibited enhanced cell attachment and detachment with temperature change, while maintaining cell functionality, thereby providing adequate number of viable cells for large scale production.

Comparison of the different anti-CD16 antibody clones in the activation and expansion of peripheral blood NK cells

Natural killer (NK) cells are promising tool for cancer treatment. Methods have been developed for large-scale NK cell expansion, including feeder cell-based methods or methods involving stimulation with NK cell activating signals, such as anti-CD16 antibodies. Different clones of anti-CD16 antibodies are available; however, a comprehensive comparison of their differential effects on inducing NK cell activation and expansion has not been conducted among these various clones under the same experimental conditions. Herein, we found that the NK cell expansion rate differed depending on the various anti-CD16 antibodies (CB16, 3G8, B73.1, and MEM-154) coated on microbeads when stimulated with genetically engineered feeder cells, K562‑membrane-bound IL‑18, and mbIL‑21 (K562‑mbIL‑18/-21). Only the CB16 clone combination caused enhanced NK cell expansion over K562‑mbIL‑18/-21 stimulation alone with similar NK cell functionality. Treatment with the CB16 clone once on the initial day of NK cell expansion was sufficient to maximize the combination effect. Overall, we developed a more enhanced NK expansion system by merging a feeder to effectively stimulate CD16 with the CB16 clone.

Abstract 1775: Expansion of human T cells using xenogeneic free and gamma irradiated human platelet lysate

Abstract Commercially available human platelet lysate (hPL) is produced using expired human platelets obtained from accredited blood banks in the United States. These platelets were originally intended for use in patient transfusion. The safety of platelets used in transfusion is managed by the U.S. Food & Drug Administration (FDA), as well as the American Association of Blood Banks (AABB). These organizations set standards, including testing for transmissible diseases. The United States record for blood safety is well established, with extremely low rates of disease transmission, making the platelet units used for hPL manufacture low risk. The Covid-19 pandemic has increased awareness of emerging infectious diseases, even though transmission of Covid-19 via blood transfusion has not been documented. For that reason, gamma irradiated hPL offers an additional safety measure in the clinic. Chimeric Antigen Receptor (CAR) expressing T-cells have demonstrated potent clinical efficacy in patients with hematological malignancies. In addition, there are several phase I clinical trials evaluating the use of CAR-T-cells for targeting of solid tumor-associated antigens. Some of the challenging issues found during production of CAR-T cells are the efficiency of T cell transduction to generate CAR-T cells, the expansion of T cells to clinically relevant numbers and the long-term survival in vivo of the therapeutic cells. The use of human platelet lysate has been demonstrated to improve these issues. Our data from experiments performed using human CD3+ from donors demonstrates that human platelet lysates offer an improved performance on T cell expansion versus serum derived products. hPL efficiently promotes T cell expansion, with higher cell yields and lower cell exhaustion rate. Additionally, we efficiently developed a protocol for suspension culture of T cells, which could facilitate the large-scale expansion of allogeneic CAR-T cells. Citation Format: Vanesa Alonso-Camino. Expansion of human T cells using xenogeneic free and gamma irradiated human platelet lysate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1775.

A Multi-Stage Bioprocess for the Expansion of Rodent Skin-Derived Schwann Cells in Computer-Controlled Bioreactors

Regenerative therapies for the treatment of peripheral nerve and spinal cord injuries can require hundreds of millions of autologous cells. Current treatments involve the harvest of Schwann cells (SCs) from nerves; however, this is an invasive procedure. Therefore, a promising alternative is using skin-derived Schwann cells (Sk-SCs), in which between 3-5 million cells can be harvested from a standard skin biopsy. However, traditional static planar culture is still inefficient at expanding cells to clinically relevant numbers. As a result, bioreactors can be used to develop reproducible bioprocesses for the large-scale expansion of therapeutic cells. Here, we present a proof-of-concept SC manufacturing bioprocess using rat Sk-SCs. With this integrated process, we were able to simulate a feasible bioprocess, taking into consideration the harvest and shipment of cells to a production facility, the generation of the final cell product, and the cryopreservation and shipment of cells back to the clinic and patient. This process started with 3 million cells and inoculated and expanded them to over 200 million cells in 6 days. Following the harvest and post-harvest cryopreservation and thaw, we were able to maintain 150 million viable cells that exhibited a characteristic Schwann cell phenotype throughout each step of the process. This process led to a 50-fold expansion, producing a clinically relevant number of cells in a 500 mL bioreactor in just 1 week, which is a dramatic improvement over current methods of expansion.

Large-scale expansion of human umbilical cord-derived mesenchymal stem cells using PLGA@PLL scaffold

Mesenchymal stem cells (MSCs) are highly important in biomedicine and hold great potential in clinical treatment for various diseases. In recent years, the capabilities of MSCs have been under extensive investigation for practical application. Regarding therapy, the efficacy usually depends on the amount of MSCs. Nevertheless, the yield of MSCs is still limited due to the traditional cultural methods. Herein, we proposed a three-dimensional (3D) scaffold prepared using poly lactic-co-glycolic acid (PLGA) nanofiber with polylysine (PLL) grafting, to promote the growth and proliferation of MSCs derived from the human umbilical cord (hUC-MSCs). We found that the inoculated hUC-MSCs adhered efficiently to the PLGA scaffold with good affinity, fast growth rate, and good multipotency. The harvested cells were ideally distributed on the scaffold and we were able to gain a larger yield than the traditional culturing methods under the same condition. Thus, our cell seeding with a 3D scaffold could serve as a promising strategy for cell proliferation in the large-scale production of MSCs. Moreover, the simplicity and low preparation cost allow this 3D scaffold to extend its potential application beyond cell culture.Graphical

Photo-mediated zwitterionic hydrogel as “blocked” platform for expansion and maintaining stemness of stem cells

Three-dimensional cell culture based on hydrogels has been a promising method for offering sufficient and effective seed cells for clinical stem cell therapy. However, the development of a required platform is always a great challenge for large expansion and stemness maintenance of stem cells. In this study, a photoresponsive zwitterionic copolymer (PSC)-based hydrogel is designed and prepared, comprising of sulfobetaine methacrylate (SBMA) and coumarin derivative (CMA) segments. By incorporating CMA segments, the PSC hydrogel possesses a comfortable gelation process and rapid photocleavage behavior under different UV radiation for cell encapsulation and harvesting. The SBMA segments provide a clean microenvironment for the stemness maintenance of stem cells via protein resistance. In addition, the cellular recognition peptide CGRGDS immobilized to hydrogel also improves the specific adhesion and proliferative capacity of stem cells. Finally, the well-defined zwitterionic hydrogel can act as a “blocked” platform for cell expansion. Both L929 cells and adipose-derived stem cells (ADSCs) encapsulated in the hydrogel show high viability and expansion. More importantly, zwitterionic hydrogels can maintain the stemness expression of ADSCs regardless of differentiation mediums. Overall, the novel “blocked” platform provided in this work has promising prospects in the large-scale expansion of stem cells.

Large-Scale Expansion of Porcine Adipose-Derived Stem Cells Based on Microcarriers System for Cultured Meat Production

Cultured meat is an innovative meat-production technology that does not rely on animal husbandry. As a new food component, cultured fat is of great significance to cultured meat. In this study, we isolated adipose-derived stem cells (ADSCs) and identified the purity by immunofluorescence staining of ADSC-specific surface marker proteins CD44 and CD29 and showed that most of the cells were positive for CD29 and CD44. In addition, we detected the expression of FABP4 and Plin1 to confirm that ADSCs differentiated into mature adipocytes at 10 days post-induction. Subsequently, the culture conditions of ADSCs on microcarriers (MCs) were optimized and showed that cell density of living cells reached their highest after 5 days when continuously stirring at 50 rpm. Finally, the expression of FABP4 and PPARγ was detected to confirm the adipogenic differentiation ability of ADSCs on 2D and 3D culture systems and showed that ADSCs maintained their adipogenic differentiation ability after expansion on MCs. In conclusion, this research demonstrated that reliance on MCs to expand ADSCs was a promising approach for production of cultured fat.

Senescence recovering by dual drug-encapsulated liposomal nanoparticles for large-scale human cell expansion.

Although regenerative therapy and bioartificial tissues and organs require a sufficient number of human cells, current cell expansion processes are accompanied by accumulation of senescent cells that are related to deterioration of cellular functions and induction of senescence-associated secretory phenotype (SASP). Therefore, suppression of replicative senescence during expansion is one of the crucial issues for dissemination of regenerative medicine. We herein developed dual drug-encapsulated liposomal nanoparticles (LNPs) to suppress cellular senescence in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and natural killer (NK) cells by removal of dysfunctional mitochondria from the senescent cells. We found that LNP treatment reduced senescent makers; downregulation of p21 expression and reduction of SA-β-Gal activity in both cells provably due to mitophagy reactivation in the cells. Moreover, SASP secretion in hAT-MSCs and tumor cytotoxicity in NK cells were also improved upon LNP treatments. These findings may contribute to the production of highly effective expanded cells for regenerative medicine and bioartificial tissues and organs.

Large-Scale Expansion of Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Stirred Suspension Bioreactor Enabled by Computational Fluid Dynamics Modeling.

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) hold great potential to generate novel and curative cell therapy products. However, the current large-scale cultivation of hUCMSCs is based on empirical geometry-dependent methods, limiting the generation of high-quantity and high-quality hUCMSCs for clinical therapy. Herein, we develop a novel scale-up strategy based on computational fluid dynamics (CFD) to effectively expand the hUCMSCs in a 3D tank bioreactor. Using a standardized hUCMSCs line on microcarriers, we successfully translated and expanded the hUCMSCs from a 200 mL spinner flask to a 1.5 L computer-controlled bioreactor by matching the shear environment and suspending the microcarrier. Experimental results revealed that the batch-cultured hUCMSCs in bioreactors with an agitation speed of 40 rpm shared a more favorable growth and physiological state, similar to that run at 45 rpm in a 200 mL spinner flask, showing comparability in both culture systems. Notably, the maximum cell density reached up to 27.3 × 105 cells/mL in fed-batch culture, 2.9 folds of that of batch culture and 20.2 times of seeding cells. As such, efficient process optimization and scale-up expansion of hUCMSCs were achieved in the microcarrier-based bioreactor system by the developed CFD simulation strategy, which provided an alternative toolbox to generate massive and standardized curative cell therapy products.

Substrate topography regulates extracellular matrix component secretion by bone marrow-derived mesenchymal stem cells

The extracellular matrix (ECM) has a complicated nano- and macro-architecture. We prepared poly-l-lactic acid (PLLA)-ECM substrates derived from mesenchymal stem cells (MSCs) by culturing cells on PLLA electro-spun fiber membranes of three different diameters. After this, PLLA membranes made with electro-spun nano-fibers were more suitable than traditional tissue culture films for cell proliferation capacity and morphology. In addition, membranes comprising these fibers promoted the gene expression of biomolecules related to ECM composition and production when compared with gene expression using the conventional substrate culture method. MSC proliferation on nano-fibrous PLLA-ECM substrates showed excellent biocompatibility. Moreover, these MSCs also expressed higher levels of the MSC markers, CD44, CD90, and CD105, as indicated by flow cytometry. Thus, PLLA electro-spun fibers are promising substrates for MSCs and promote ECM secretion, suggesting this method may be used for large-scale stem cell expansion.

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 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

Validation of an automated cell counting method for cGMP manufacturing of human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) must be manufactured as advanced therapy medicinal products (ATMPs) for innovative tissue replacement clinical applications. Yet, production of hiPSCs under current Good Manufacturing Practice (cGMP) presents many hurdles, such as the large-scale cell expansion needed to reach therapeutically-relevant hiPSC doses. For the monitoring of this phase, a fast and reliable cell counting method should be used. Conventional manual cell counting by the hemocytometer method is dependent on the operator's expertise and is time-consuming. Therefore, automation of sample preparation and analysis is needed to improve precision and rapidity of hiPSC cell counting. We investigated whether an automated cell counting method could be validated for use with hiPSCs, in comparison with a reference cell counting method included in the European Pharmacopeia, 10th edition. The proposed method was the fluorescence imaging-based NucleoCounter NC-100 system, whereas the reference method was manual cell counting using a Bürker hemocytometer. The validation strategy complied with EudraLex cGMP regulations for ATMP manufacturing and ICH Q2(R1) indications for validation of analytical methods. The use of the NucleoCounter NC-100 system for automated cell counting was validated, focusing on accuracy, specificity, intra- and inter-operator reproducibility, range and linearity, showing higher precision than the manual method. The automated method can be used more effectively than the manual one for hiPSC cell counting. Thus, this piece of work paves the way for all cGMP facilities that want to pursue hiPSC manufacturing for clinical use.

Facile bead-to-bead cell-transfer method for serial subculture and large-scale expansion of human mesenchymal stem cells in bioreactors.

The conventional planar culture of adherent cells is inefficient for large‐scale manufacturing of cell and gene therapy products. We developed a facile and efficient bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells (hMSCs) with microcarriers in bioreactors. We first compared culture medium with and without nucleosides and found the former maintained the expression of surface markers of hMSCs during their prolonged culture and enabled faster cell proliferation. Subsequently, we developed our bead‐to‐bead cell transfer method to subculture hMSCs and found that intermittent agitation after adding fresh microcarriers to cell‐populated microcarriers could promote spontaneous cell migration to fresh microcarriers, reduce microcarrier aggregation, and improve cell yield. This method enabled serial subculture of hMSCs in spinner flasks from passage 4 to passage 9 without using proteolytic enzymes, which showed faster cell proliferation than the serial planar cultures undergoing multiple enzyme treatment. Finally, we used the medium containing nucleosides and our bead‐to‐bead cell transfer method for cell culture scale‐up from 4‐ to 50‐L cultures in single‐use bioreactors. We achieved a 242‐fold increase in the number of cells to 1.45 × 1010 after 27‐day culture and found that the cells harvested from the bioreactors maintained proliferation ability, expression of their surface markers, tri‐lineage differentiation potential and immunomodulatory property. This study shows the promotive effect of nucleosides on hMSC expansion and the potential of using our bead‐to‐bead transfer method for larger‐scale manufacturing of hMSCs for cell therapy.