Growth and functional harvesting of human mesenchymal stromal cells cultured on a microcarrier‐based system

Abstract

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.

Production of oncolytic adenovirus and human mesenchymal stem cells in a single‐use, Vertical‐Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier‐based cell culture processes

“…However, the differences observed in cell volumetric concentrations between PBS‐VW and ST bioreactors throughout culture time (Figure C) were not statistically significant; in both bioreactor systems hMSC displayed similar maximum growth rates (0.012 h −1 in PBS‐VW vs. 0.010 h −1 in ST bioreactor) and expansion factors (12 and 11, respectively). The maximum cell concentration achieved in both bioreactor systems (∼3 × 10 5 cell/mL) were lower than other reports in the literature . The difference in the cell growth profile may reflect the distinct cell origins (hMSC were isolated from the bone marrow of different donors), and the different culture conditions such as the microcarrier type, medium formulation, culture system, and operation mode.…”

Stirred tank bioreactor culture combined with serum‐/xenogeneic‐free culture medium enables an efficient expansion of umbilical cord‐derived mesenchymal stem/stromal cells

“…Upon the first 24 h of cell adhesion to the microcarriers in the bioreactor, no lag phase was observed and exponential growth was achieved reaching a maximal cell density of 1.4 (± 0.33) × 10 5 cells/mL) ( µ max = 1.1 day ‐1 ) at day 4 corresponding to 115 million cells. Overall, this culture system allowed the production of a higher amount of cells in a shorter period of time (four days), instead of seven to ten days of culture (for stirred systems) described in the literature , which means a more cost‐effective expansion process. To date, and to our knowledge, there are no reports in the literature describing the expansion of UCM MSC in stirred systems, namely stirred‐tank reactors, using serum‐/xeno‐free culture medium.…”

“…Despite the high proliferative capacity, the number of MSC isolated from a single umbilical cord unit is limited and in vitro expansion is essential to achieve the number of cells required for therapeutic application (10 7 –10 9 cells) . Since MSC are adherent cells, their expansion at a larger scale has been commonly performed using microcarriers and stirred culture systems . Alternative technologies using fully closed, disposable and automated systems have also been described, such as the Wave bioreactor , Hollow fibers and the Aastrom Replicell ® system, a customized bioreactor designed for patient‐specific MSC/multicellular therapy, with applications in several clinical trials .…”

Mesenchymal stem cells – A new hope for radiotherapy-induced tissue damage?

“…To avoid the use of xenogeneic components such as fetal bovine serum for culturing MSCs, several alternatives have been proposed, among them platelet lysates and human plasma and serum [121][122][123]. In order to obtain large numbers of usable cells from small bone marrow or tissue samples, several closed-system bioreactor techniques have been proposed to proliferate MSCs according to GMP requirements, among them microcarrier-based approaches [124,125] and fixed-bed culturing systems [126,127].…”