RESUMEN
Mesenchymal stem/stromal cell (MSC)-based therapies have been gaining increasing attention owing to their application in various diseases and conditions. In this study, we aimed to identify the optimal condition for industrial-scale MSC manufacturing. MSCs were isolated from umbilical cord (UC) tissues by implementing the explant method (Exp) or a collagenase based-enzymatic digestion method (Col), using a good manufacturing practice-compatible serum-free medium developed in-house. Microarray analysis demonstrated that the gene expression profiles of Exp-MSCs and Col-MSCs did not significantly differ according to the method of isolation or the culture conditions used. The isolated UC-MSCs were then subjected to expansion using conventional static culture (ST) or microcarrier-based culture in stirred-tank bioreactors (MC). Metabolomic and cytokine array analyses were conducted to evaluate the biochemical status of the MSCs. However, no remarkable differences in the metabolic profile and cytokine secretome between ST-MSCs and MC-MSCs were observed. On the contrary, we observed for the first time that the hydrophobic components of ST-MSCs and MC-MSCs were different, which suggested that the cell membrane distribution of fatty acids and lipids was altered in the process of adaptation to shear stress in MC-MSCs. These results establish the flexibility of the isolation and expansion method for UC-MSCs during the manufacturing processes and provide new insights into the minor differences between expansion methods that may exert remarkable effects on MSCs. In conclusion, we demonstrated the feasibility of both Exp-MSCs and Col-MSCs and MC and ST culture methods for scale-up and scale-out of MSC production, as well as the equivalence of these cells. As for the industrialized mass production of MSCs, enzyme-based methods for isolation and cell expansion in a bioreactor were considered to be more suitable. The methods developed, which underwent comprehensive evaluation in this study, may contribute toward the provision of sufficient MSC sources and the establishment of cost-effective MSC therapies. Impact statement Our in-house-developed good manufacturing practice-grade serum-free medium could be used for both isolation (Exp and Col) and expansion (ST and MC) of umbilical cord (UC)-mesenchymal stem/stromal cells (MSCs). Characteristics of the obtained UC-MSCs were widely assessed with regard to gene expression, metabolome, and secretome. Cellular characteristics and efficacy were observed to be equivalently maintained among whichever technique was applied. In addition, our research presents the first evidence that bioreactor and microcarrier-based MSC cultures alter the fatty acid and phospholipid composition of MSCs. These results provide new insights into the differences between expansion methods that may exert remarkable effects on MSCs.
Asunto(s)
Células Madre Mesenquimatosas , Cordón Umbilical , Reactores Biológicos , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Medios de CultivoRESUMEN
Mesenchymal stromal cells (MSCs) have been isolated from numerous sources and are potentially therapeutic against various diseases. Umbilical cord-derived MSCs (UC-MSCs) are considered superior to other tissue-derived MSCs since they have a higher proliferation rate and can be procured using less invasive surgical procedures. However, it has been recently reported that 2D culture systems, using conventional cell culture flasks, limit the mass production of MSCs for cell therapy. Therefore, the development of alternative technologies, including microcarrier-based cell culture in bioreactors, is required for the large-scale production and industrialization of MSC therapy. In this study, we aimed to optimize the culture conditions for UC-MSCs by using a good manufacturing practice (GMP)-compatible serum-free medium, developed in-house, and a small-scale (30 mL) bioreactor, which was later scaled up to 500 mL. UC-MSCs cultured in microcarrier-based bioreactors (MC-UC-MSCs) showed characteristics equivalent to those cultured statically in conventional cell culture flasks (ST-UC-MSCs), fulfilling the minimum International Society for Cellular Therapy criteria for MSCs. Additionally, we report, for the first time, the equivalent therapeutic effect of MC-UC-MSCs and ST-UC-MSCs in immunodeficient mice (graft-versus-host disease model). Lastly, we developed a semi-automated cell dispensing system, without bag-to-bag variation in the filled volume or cell concentration. In summary, our results show that the combination of our GMP-compatible serum-free and microcarrier-based culture systems is suitable for the mass production of MSCs at an industrial scale. Further improvements in this microcarrier-based cell culture system can contribute to lowering the cost of therapy and satisfying several unmet medical needs.
Asunto(s)
Células Madre Mesenquimatosas , Animales , Reactores Biológicos , Técnicas de Cultivo de Célula , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Ratones , Cordón UmbilicalRESUMEN
Heavy colonization of atopic dermatitis (AD) with Staphylococcus aureus is well documented. The purpose of the present study is to examine the actions of gluco-oligosaccharide (G-OS) against S. aureus for the control of AD skin lesions infected with S. aureus. The colony counts of S. aureus cells in 0.5% sodium chloride solution supplemented with 5% G-OS (pH 4.8) were about 10-fold lower than those in 0.5% sodium chloride solution (pH 6.6; control) after incubation for 24 hours. The colony counts of S. aureus cells attached on the coverslips (pre-treatment with 1% and 5% G-OS/PBS and following treatment with plasma) were about 10-fold lower than those on the coverslips (pre-treatment with PBS and following treatment with plasma; control) in PBS after incubation for 24 hours. The materials (sugars, probably glycocalyx) that stained positively for fluorescein-isothiocyanate (FITC) -concanavalin A and were consistent with the presence of S. aureus cells were reduced when S. aureus cells attached to the coverslips treated with 5% GC-OS. In conclusion, C-OS is a promising agent that can be applied topically in a cream to clear adherent S. aureus cells from skin lesions of AD in order to prevent its exacerbation. Further, 5% C-OS can inhibit glycocalyx production by S. aureus cells and consequently have some suppressive effect on the colonization of S. aureus on the horny cells of AD lesions.