The effect of early rounds of ex vivo expansion and cryopreservation on the adipogenic differentiation capacity of adipose-derived stromal/stem cells

Multipotent adipose-derived stromal/stem cells (ASCs) are candidates for use in cellular therapies for the treatment of a variety of conditions/diseases. Ex vivo expansion of freshly isolated ASCs may be necessary prior to clinical application to ensure that clinically relevant cell numbers are administered during treatment. In addition, cryopreserving cells at early passages allows for storage of freshly isolated cells for extended periods of time before expanding these cells for clinical usage. There are however several concerns that these laboratory-based procedures may alter the characteristics of the cells and in so doing decrease their regenerative potential. In this study we report on the impact of early rounds of cryopreservation (P0) and ex vivo expansion (P0 to P5) on the phenotypic characteristics and adipogenic differentiation potential of ASCs. Our results show that ASCs that upregulate CD36 expression during adipogenic differentiation gradually decrease with increasing expansion rounds. The consequent decrease in adipogenic differentiation capacity was evident in both gene expression and flow cytometry-based phenotypic studies. Successive rounds of expansion did not however alter cell surface marker expression of the cells. We also show that early cryopreservation of ASCs (at P0) does not affect the adipogenic differentiation potential of the cells.The South African Medical Research Council in terms of the SAMRC’s Flagship Award Project [SAMRC-RFAUFSP-01-2013/STEM CELLS], the SAMRC Extramural Unit for Stem Cell Research and Therapy, and the Institute for Cellular and Molecular Medicine of the University of Pretoria.http://www.nature.com/scientificreportsam2020Immunolog

Human platelet lysate to substitute fetal bovine serum in hMSC expansion for translational applications: a systematic review

Foetal bovine serum (FBS), is the most commonly used culture medium additive for in vitro cultures, despite its undefined composition, its potential immunogenicity and possible prion/zoonotic transmission. For these reasons, significant efforts have been targeted at finding a substitute, such as serum free-media or human platelet-lysates (hPL). Our aim is to critically appraise the state-of-art for hPL in the published literature, comparing its impact with FBS. In June 2019 a systematic search of the entire Web of Science, Medline and PubMed database was performed with the following search terms: (mesenchymal stem cells) AND (fetal bovine serum OR fetal bovine calf) AND (human platelet lysate). Excluded from this search were review articles that were published before 2005, manuscripts in which mesenchymal stem cells (MSCs) were not from human sources, and when the FBS controls were missing. Based on our search algorithm, 56 papers were selected. A review of these papers indicated that hMSCs cultured with hPL showed a spindle-shaped elongated morphology, had higher proliferation indexes, similar cluster of differentiation (CD) markers and no significant variation in differentiation lineage (osteocyte, adipocyte, and chondrocyte) compared to those cultured with FBS. Main sources of primary hMSCs were either fat tissue or bone marrow; in a few studies cells isolated from alternative sources showed no relevant difference in their response. Despite the difference in medium choice and a lack of standardization of hPL manufacturing, the majority of publications support that hPL was at least as effective as FBS in promoting adhesion, survival and proliferation of hMSCs. We conclude that hPL should be considered a viable alternative to FBS in hMSCs culture-especially with a view for their clinical use