Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells

Abstract

BackgroundMesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity.MethodsPlatelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α.ResultsOur results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed the release of TNF-α when exposed to LPS-stimulated monocytes similar to those cultured in FBS.ConclusionePL has the potential to be used for the expansion of MSCs before clinical application, avoiding the concerns associated with the use of FBS.

Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

“…It was obtained from hPCs via a standardized platelet apheresis technique. This technique is thought to be significantly advantageous owing to the high concentration of platelets and low level of leukocyte contamination that are associated with it [ 56 ]. Freezing and thawing is known to be the simplest method, resulting in a mechanical disruption of hPCs.…”

The effects of human platelet lysate versus commercial endothelial growth medium on the endothelial differentiation potential of human amniotic fluid mesenchymal stem cells

“…Flow cytometry analysis revealed that hAF-MSCs were positive for CD44, CD73, CD90 and HLA-ABC and negative for CD31, CD34, CD45, and HLA-DR. The surface antigen markers used for immunophenotyping were the same as those used in previous studies [ 25 , 26 , 27 , 36 ]. The negative expression values of CD31, CD34, and CD45 confirmed that the cells were clearly discriminated from endothelial cells (CD31) and hematopoietic stem cells (CD34 and CD45) [ 25 ].…”

“…The hPL was obtained from platelet concentrates via a standardized platelet apheresis technique that provided a high concentration of platelets and a low level of leukocyte contamination [ 36 ]. To lysis the platelet concentrates, repeated freeze/thaw cycles were selected in the present study.…”

Veterinary Regenerative Medicine for Musculoskeletal Disorders: Can Mesenchymal Stem/Stromal Cells and Their Secretome Be the New Frontier?

“…Supplementation of the medium is performed to mimic in vivo conditions in order to sustain cell growth. Foetal bovine serum (FBS) or platelet lysate [35,36] are used in the cell culture medium as a source of growth factors [37], which supports attachment and further expansion of MSCs. The use of antibiotics is important in order to prevent bacterial contaminations, and the most commonly used are penicillin and streptomycin.…”