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.

Keywords: Adipose tissue-derived mesenchymal stromal cells; Advanced therapy medicinal products; Human platelet lysate; Interleukin-6.

Fig. 1

ASCs surface markers expression. The plots show the phenotype of ASCs obtained directly after ASCs isolation (before seeding on plates) and in the subsequent passages. Passage “0” indicates cells seeded after isolation that were trypsinizated when obtained 80–90% confluency. The cell phenotype stabilizes from passage 2 (expression of surface markers according to the guidelines of ISCT). The graphs summarized the surface marker expression on cells obtained from six isolation from different donors (n = 6)

Fig. 2

The morphology of ASCs cultured in three different medium formulations. a Phase-contrast images of ASCs at passage 1 (40% of confluency, 3–5 days of culture) parallel cultivated in either FBS- or hPL-supplemented basal medium. b Slight differences in the morphology between ASCs cultured in three different medium formulations were confirmed by flow cytometry gating of forward vs side scatter (FSC vs SSC). ASCs cultured in media supplemented with hPL were smaller and exhibited less complexity (granularity)

Fig. 3

Differentiation analysis of ASCs. ASCs cultured in three different media were harvested and plated in differentiation media. ASCs differentiated in vitro into adipocytes (FABP4, green, n = 4, magnification × 10), osteoblasts (Alizarin Red, red, n = 4, magnification × 4), and chondroblasts (Safranin O, dark red, n = 4, magnification × 20)

Fig. 4

Clonogenic potential of ASCs. ASCs cultured in three different medium formulations exhibit similar clonogenic potential (n = 4). However statistically insignificant, more clones were obtained from single cells cultured on αMEM+10% hPL medium

Fig. 5

Secretome of ASCs. a During 48 h culture, ASCs secreted into the culture medium mainly IL-6 and small amounts of RANTES, osteoprotegerin, MCP-1, and GRO particles. The graph shows an averaged level of secreted cytokines by ASCs (n = 3). b The secretion of the most abundant cytokine—IL-6—was donor-specific, and the medium formulation did not influence its secretion. The amount of secreted IL-6 based on the ELISA assay (n = 4) performed in respective cell culture

Fig. 6

The evaluated number of cells that may be obtained in three different culture conditions. The highest yield of seeded cells may be achieved in cell culture carried out in αMEM supplemented with 10% hPL. After approximately 4 weeks, from 1 million of isolated ASCs, almost 50 million of cells may be obtained