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OBJECTIVE: Mesenchymal stem cell (MSC) extracellular vesicles (EVs) have emerged as a biotherapeutic for osteoarthritis; however, manufacturing large quantities is not practical using traditional monolayer (2-D) culture. We aimed to examine the effects of 3-D and 2-D culture 2 types of media: Dulbecco modified Eagle medium and a commercially available medium (CM) on EV yield. ANIMALS: Banked bone marrow-derived MSCs (BM-MSCs) from 6 healthy, young horses were used. METHODS: 4 microcarriers (collagen-coated polystyrene, uncoated polystyrene, collagen-coated dextran, and uncoated dextran) were tested in static and bioreactor cultures, and the optimal microcarrier was chosen. The BM-MSCs were inoculated into a bioreactor with collagen-coated dextran microcarriers at 5,000 cells/cm2 or onto culture dishes at 4,000 cells/cm2 in either Dulbecco modified Eagle medium or CM media. Supernatants were obtained for metabolite and pH analysis. The BM-MSCs were expanded until confluent (2-D) or for 7 days (3-D) when the 48-hour EV collection period commenced using EV-depleted media. Extracellular vesicles were isolated and characterized via nanoparticle tracking analysis, Western blot, transmission electron microscopy, and protein quantification. The BM-MSCs were harvested, quantified, and immunophenotyped. RESULTS: The number of EVs isolated was not improved by 3-D culture or CM media, however, the CM 3-D condition improved the number of EVs produced per BM-MSC over the CM 2-D condition (mean ± SD: 306 ± 99 vs 37 ± 22, respectively). Glucose decreased and lactate and ammonium accumulated in 3-D culture. Surface markers of stemness exhibited reduced expression in 3-D culture. CLINICAL RELEVANCE: Optimization of our 3-D culture methods could improve BM-MSC expansion and thus EV yield.

Assuntos

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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.

Assuntos

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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.

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Recently, stem cell-based therapies have been proposed as an alternative for the treatment of many diseases. Stem cells (SCs) are well known for their capacity to preserve themselves, proliferate, and differentiate into multiple lineages. These characteristics allow stem cells to be a viable option for the treatment of diverse diseases. Traditional methodologies based on 2-dimensional culture techniques (T-flasks and Petri dishes) are simple and well standardized; however, they present disadvantages that limit the production of the cell yield required for regenerative medicine applications. Lately, microcarrier (MC)-based culture techniques have emerged as an attractive platform for expanding stem cells in suspension systems. Although the use of stem cell expansion on MCs has recently shown significant increase, their implementation for medical purposes is been hampered by bottlenecks in upstream and downstream processing. Therefore, there is an urgent need in the development of bioprocesses that simplify stem cell cultures under xeno-free conditions and detachment from MCs without diminishing their pluripotency and viability. A critical analysis of the factors that impact the up and downstream bioprocessing on MC-based stem cell cultures is presented in this review. This analysis aims to raise the awareness of the current drawbacks that limit MC-based stem cell bioprocessing in regenerative medicine and propose alternatives to overcome them.

Assuntos

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Human mesenchymal stromal cells (hMSCs) cells are attractive for applications in tissue engineering and cell therapy. Because of the low availability of hMSCs in tissues and the high doses of hMSCs necessary for infusion, scalable and cost-effective technologies for in vitro cell expansion are needed to produce MSCs while maintaining their functional, immunophenotypic and cytogenetic characteristics. Microcarrier-based culture systems are a good alternative to traditional systems for hMSC expansion. The aim of the present study was to develop a scalable bioprocess for the expansion of human bone marrow mesenchymal stromal cells (hBM-MSCs) on microcarriers to optimize growth and functional harvesting. In general, the results obtained demonstrated the feasibility of expanding hBM-MSCs using microcarrier technology. The maximum cell concentration (n = 5) was ~4.82 ± 1.18 × 10(5) cell mL(-1) at day 7, representing a 3.9-fold increase relative to the amount of inoculated cells. At the end of culture, 87.2% of the cells could be harvested (viability = 95%). Cell metabolism analysis revealed that there was no depletion of important nutrients such as glucose and glutamine during culture, and neither lactate nor ammonia byproducts were formed at inhibitory concentrations. The cells that were recovered after the expansion retained their immunophenotypic and functional characteristics. These results represent an important step toward the implementation of a GMP-compliant large-scale production system for hMSCs for cellular therapy.

Assuntos

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We present kinetic and physiological data regarding the culturing of rCHO-K1 cells on various microcarriers, to evaluate the potential of this culture strategy for mass production of these cells and expression of a recombinant disintegrin. Cultures were performed in 500 mL spinner flasks in DMEM culture medium with 10 percent v/v fetal calf serum, gently shaken at 37 C, pH 7.4, in a 10 percent v/v CO2 atmosphere. The following values were obtained, respectively, for the adhesion time-constant Ka (h) and specific growth rate micron max (d-1) on each microcarrier: Cytodex 1 (0.91, 0.45), Cultispher S (0.28, 0.34), Immobasil FS (0.85, 0.52) and Pronectin F (5.12, 0.67). Metabolic characteristics showed some variation among the cultures with the four microcarriers, the most significant being the higher production of ammonia with microcarriers coated with adhesive molecules (Cultispher S and Pronectin F) relative to the uncoated carriers (Cytodex 1 and Immobasil FS). Experiments where the DMEM medium was gradually replaced by the serum-free medium (CHO-SFM-II) revealed important advantages over media containing serum, not only for assay purposes, but also for purification of the disintegrin. Altogether these results demonstrate that cultures on microcarriers, especially on Pronectin F, show good potential for larger scale cultures of rCHO-K1 cell.

Assuntos