Comparative Analysis of MSC-Derived Exosomes Depending on Cell Culture Media for Regenerative Bioactivity

Abstract

BACKGROUND: In order to produce and isolate the exosome derived from the cell of interests, a serum free environment (starvation) has been essential for excluding the unknown effect from serum-derived exosomes. Recently, serum-free culture media have been developed as a substitute for serum supplemented media so that MSC proliferates with maintaining the original characteristics of the cells in a serum free condition. Due to the different properties of the exosomes representing the states and characteristics of the origin cells, a study is needed to compare the properties of the cell-derived exosomes according to the cell culture media. METHODS: To compare the cell culture condition on exosomes, human umbilical cord mesenchymal stem cells (UCMSCs) were cultured with two different media, serum containing media, 10% FBS supplemented DMEM (NM) and serum-free chemically defined media, CellCor TM CD MSC (CDM). To remove FBS-derived exosomes from UCMSC cultured with NM, the medium was replaced with FBS-free DMEM for starvation during exosome isolation. The production yield and expression levels of angiogenic and pro-inflammatory factors were compared. And, the subpopulations of exosome were classified depending on the surface properties and loaded cytokines. Finally, the wound healing and angiogenic effects have been evaluated using in vitro assays. RESULTS: The UCMSC-derived exosomes under two different cell culture media could be classified into subpopulations according to the surface composition and loaded cytokines. Especially, exosome derived from UCMSC cultured with CDM showed higher expression levels of cytokines related to regenerative bioactivities which resulted in enhanced wound healing and angiogenesis. CONCLUSION: CDM has the advantages to maintain cell proliferation even during the period of exosome isolations and eliminate unknown side effects caused by serumderived exosomes. Additionally, exosomes derived from UCMSC cultured with CDM show better wound healing and angiogenic effects due to a lot of regeneration-related cytokines and less pro-inflammatory cytokines compared to with NM.

Inhaled Placental Mesenchymal Stromal Cell Secretome from Two- and Three-Dimensional Cell Cultures Promotes Survival and Regeneration in Acute Lung Injury Model in Mice

“…An investigation into different administration routes to determine the optimal delivery method and frequency of administration would help maximize benefits. The last important limitation is the presence of residual proteins and EVs from FCS in the studied CM, which could potentially distort and even reduce the therapeutic efficacy of the MMSC secretome [ 44 ]. However, we used identical cell culture conditions, so we assume that the contribution of FCS-derived EVs was the same for all cell cultures.…”

“…This is consistent with recent studies showing that the regenerative potential of EVs from fetal serum is significantly lower than vesicles from MMSCs [ 45 ]. Interestingly, EVs collected from the MMSC cultured in fetal serum–containing medium had greater wound healing and angiogenic effects for endothelial cells compared with FBS-derived EVs [ 44 ]. Based on the literature, the MMSC secretome examination may benefit from the use of chemically defined, serum-free, and xeno-free medium that is not only optimized for cell growth and viability for a variety of cell types, but is also free of exogenous contaminating fetal serum-derived EVs and extracellular protein/RNA species [ 31 ].…”

The Limbal Niche and Regenerative Strategies

“…The isolation and purification of these vesicles is not uniform across different studies, and, thus, advantages seen using a single isolation method might not be present in other populations of MSC-derived exosomes [ 130 ]. One study showed that varying the conditions under which exosomes are isolated led to a significant difference in the expression of pro-inflammatory cytokines within the different exosome populations [ 131 ]. Exosomes’ ability to modulate inflammation should be further explored in the context of LSCD; however, investigators should rigorously adhere to standardized isolation techniques to derive a homogenous population of vesicles.…”

Human Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Neural Differentiation of Neural Progenitor Cells

“…Recent studies have demonstrated that transplanted stem cells can differentiate into neuronal cells and glial cells and that substantial functional recovery is mostly due to paracrine signaling effects provided by grafted stem cells [7][8][9]. However, several safety issues, including immunogenic risk, still remain [10].…”

“…EVs exhibit tetraspanins CD63, CD81, and CD9 on the surface and present the endo-lysosomal regulatory transport protein (ALIX) [11,12]. MSC-derived EVs have been regarded to have a similar therapeutic potency as MSCs without the potential safety risks [10]. For the regeneration of neurological disorders, EV-based therapy is also promising as it is a neuroprotective paracrine cargo containing abundant proteins and microRNAs [13].…”