Successful Use of Human AB Serum to Support the Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cell in a Microcarrier-Based Platform

Abstract

Mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and for the promotion of tissue repair, hence the increase of clinical trials in a worldwide scale. In particular, adipose tissue-derived stem/stromal cells (AT MSC) present easy accessibility and a rather straightforward process of isolation, providing a clear advantage over other sources. The high demand of cell doses (millions of cells/kg), needed for infusion in clinical settings, requires a scalable and efficient manufacturing of AT MSC under xenogeneic(xeno)-free culture conditions. Here we describe the successful use of human AB serum [10%(v/v)] as a culture supplement, as well as coating substrate for the expansion of these cells in microcarriers using (i) a spinner flask and (ii) a 500-mL mini-bioreactor (Applikon TM Biotechnology). Cells were characterized by immunophenotype and multilineage differentiation potential. Upon an initial cell adhesion in the spinner flask of 35 ± 2.5%, culture reached a maximal cell density of 2.6 ± 0.1 × 10 5 at day 7, obtaining a 15 ± 1-fold increase. The implementation of the culture in the 500-mL mini-bioreactor presented an initial cell adhesion of 22 ± 5%, but it reached maximal cell density of 2.7 ± 0.4 × 10 5 at day 7, obtaining a 27 ± 8-fold increase. Importantly, in both stirred systems, cells retained their immunophenotype and multilineage differentiation potential (osteo-, chondro-and adipogenic lineages). Overall, the scalability of this microcarrier-based system presented herein is of major importance for the purpose of achieving clinically meaningful cell numbers.

Influence of Microenvironment on Mesenchymal Stem Cell Therapeutic Potency: From Planar Culture to Microcarriers

“…Based on literature, hMSC attachment on microcarriers can achieve 70% to 90% under the static or dynamic condition in serum-containing medium, whereas the efficiency significantly decreases to 22% to 23% in serum- and xeno-free medium (dos Santos et al, 2011 ; Timmins et al, 2012 ). But there is also evidence that hMSCs can grow fast and achieve the same cell density in a xeno-free serum compared to serum-containing culture despite the low adhesion efficiency (Moreira et al, 2020 ). Currently, most studies on screening microcarriers for bioprocessing only emphasize the seeding efficiency and expansion fold; however, the recovery efficiency or yield is equally critical for the large-scale production (Schnitzler et al, 2012 ; Timmins et al, 2012 ; Goh et al, 2013 ; Rafiq et al, 2016 ; Moloudi et al, 2019 ).…”

Impact of Bioreactor Geometry on Mesenchymal Stem Cell Production in Stirred‐Tank Bioreactors

“…2) and in some cases we worked with assumptions. Our literature survey produced five important conclusions: most studies used an up‐pumping axial stirrer or 45° 3‐SPB device, as well as one setup with a combined 3‐SPB/Rushton turbine ( N P = 0.3–3.1), the d S / D T ratio ranged from 0.3–0.5, in most cases, the flow regime was transitional, meaning Re < 10 000 for baffled STRs and Re < 60 000 for non‐baffled STRs, the mean energy dissipation was low ( = 0.2–4.8 mW kg −1 ) resulting in a low mean shear stress of 0.01–0.06 N m −2 , and the final hMSC concentration was 0.4–44 · 10 5 cells mL −1 84–92. …”

Bioprocessing of Human Mesenchymal Stem Cells: From Planar Culture to Microcarrier-Based Bioreactors

“…This indicates the power input per unit volume needed to maintain microcarriers in suspension and is proportional to agitation speed [28,68]. Therefore, the agitation speed typically increases along with the culture time to offset the heavier microcarriers loaded with propagating cells, larger working volume or multi-microcarrier aggregation [28,31,36,45,48]. Another factor related to mixing is the impeller tip speed, at the point of which is the highest shear stress in the entire bioreactor, and thus the most severe potential damage to cells.…”

“…Similar to planar culture, the most common medium used for hMSC expansion in microcarrier-based bioreactors is DMEM or αMEM supplemented with FBS. Recent interest in pursuing xeno-free culture to diminish the influence of undefined animal-derived components, human serum [45] or platelet lysate [41] have been tested and demonstrated successful replacement of FBS for xeno-free microcarrier suspension cultures in bioreactors. Moreover, the potential of these cultures for therapeutic purposes has led to the development of several commercially available media specific for hMSC expansion in microcarrier-based culture systems, including MesenCultTM-XF [33], PRIME-XV MSC Expansion SFM [42], StemPro ® MSC SFM XenoFree [29], MSCGMTM BulletKit TM [48].…”