The effect of culture media on large-scale expansion and characteristic of adipose tissue-derived mesenchymal stromal cells

Abstract

Background Adipose tissue-derived mesenchymal stromal cells (ASCs) have been shown to exhibit some promising properties of their use in regenerative medicine as advanced therapy medicinal products (ATMP). However, different sources of their origin, methods of isolation, and expansion procedures cause the laboratory and clinical results difficult to compare. Methods ASCs were isolated from lipoaspirates and cultured in three different medium formulations: αMEM and DMEM as a basal medium supplemented with 10% of human platelet lysate (hPL) and DMEM supplemented with 20% fetal bovine serum (FBS) and bFGF as a gold standard medium. Subsequently, the impact of culture media on ASCs growth kinetics, their morphology and immunophenotype, ability to differentiate, clonogenic potential, and secretion profile was evaluated. Results All cultured ASCs lines showed similar morphology and similar clonogenic potential and have the ability to differentiate into three lines: adipocytes, osteoblasts, and chondroblasts. The immunophenotype of all cultured ASCs was consistent with the guidelines of the International Society for Cell Therapy (ISCT) allowing to define cells as mesenchymal stromal cell (MSC) (≥ 95% CD105, CD73, CD90 and ≤ 2% CD45, CD34, CD14, CD19, HLA-DR). The immunophenotype stabilized after the second passage and did not differ between ASCs cultured in different conditions. The exception was the ASCs grown in the presence of FBS and bFGF, which expressed CD146 antigens. The secretion profile of ASCs cultured in different media was similar. The main secreted cytokine was IL-6, and its level was donor-specific. However, we observed a strong influence of the medium formulation on ASCs growth kinetics. The proliferation rate of ASCs in medium supplemented with hPL was the highest. Conclusions Culture media that do not contain animal-derived antigens (xeno-free) can be used to culture cells defined as MSC. Xeno-free medium is a safe alternative for the production of clinical-grade MSC as an advanced therapy medicinal product. Additionally, in such culture conditions, MSC can be easily expanded in accordance with the Good Manufacturing Process (GMP) requirements to a desired amount of cells for clinical applications.

Biological Considerations in Scaling Up Therapeutic Cell Manufacturing

“…In another study, a higher immunosuppressive effect was observed for BM-MSCs expanded in HPL-supplemented media (Gottipamula et al, 2012). Other studies comparing cell expansion in HPL-or FBS-supplemented media have reported no difference in the immunosuppressive effects of BM-MSCs (Bieback et al, 2009), or in the secretion profiles of A-MSCs (Czapla et al, 2019). Chromosomal stability appeared to be the same if not better for cells grown in HPL (Shih and Burnouf, 2015;Astori et al, 2016).…”

“…HPL provides strong growth-promoting activity to support the expansion of a variety of cells (Choi et al, 1980;Eastment and Sirbasku, 1980;Hara et al, 1980). In fact, there are now ample studies demonstrating that proliferation of MSCs from various tissue sources is higher when HPL is used (Schallmoser et al, 2007;Bieback et al, 2009;Gottipamula et al, 2012;Gottipamula et al, 2013;Gottipamula et al, 2016;Czapla et al, 2019;Kakudo et al, 2019) and generally studies utilizing HPL for in vitro expansion of MSCs have found it to be an acceptable alternative to FBS in terms of maintaining cellular features for clinical applications (Fekete et al, 2012;Becherucci et al, 2018). However, studies on the effects of HPL on the immunosuppressive capacity of MSCs have been contradictory.…”

Selection of basal medium for culturing human umbilical cord mesenchymal stem cells in combination with human platelet lysate

“…In a recent study, αMEM and DMEM as basal media supplemented with 10% of human platelet lysate (hPL) and DMEM supplemented with 20% fetal bovine serum (FBS) and bFGF were compared in culturing adipose tissue-derived mesenchymal stromal cells (ASCs). The results indicate thatαMEM supplemented with 10% hPL yield the highest proliferation rate and rate of clonal genic potential, while no differences in adipocyte, osteocyte, and chondroblast differentiation [20]. This study was done with ASCs.…”

Inactivated Platelet Lysate Supports the Proliferation and Immunomodulant Characteristics of Mesenchymal Stromal Cells in GMP Culture Conditions

“…A variety of protocols are described for GMP MSC production, some of these using selected FBS, others using xeno-free components, HPL, or plasma. As MSCs are considered ATMPs, qualified protocols with standardised characteristics are needed so that MSCs can be developed in large-scale quality and at a relatively low-cost of production using xeno-free media for clinical-grade expansion [ 25 , 26 ]. In recent years, we have been establishing methods to isolate and expand MSCs for clinical use, and we have also demonstrated that iHPLs prepared in-house from a large pool of donor platelets, that undergo pathogen inactivation using psoralen, are safer and more efficient than FBS in isolating BM-MSCs when following GMP guidelines [ 8 , 27 ].…”