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.
Highlights
Mesenchymal 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
The Population doublings (PD) for MSCs cultured in equine platelet lysate (ePL) was 4.17 ± 0.25 at day 4, 6.35 ± 0.49 at day 8, 7.04 ± 0.32 at day 12, 6.97 ± 0.4 at day 16, 7.26 ± 0.43 at day 20, 7.09 ± 0.47 at day 24, 7.17 ± 0.63 at day 28, and 7.14 ± 0.11 at day 32, whereas PD for MSCs cultured in FBS was 4.43 ± 0.95 at day 4, 6.36 ± 0.92 at day 8, 6.58 ± 0.84 at day 12, 6.51± 0.71 at day 16, 6.91 ± 0.62 at day 20, 7.02 ± 0.72 at day 24, 6.66 ± 0.73 at day 28 and 6.70 ± 0.66 at day 32 (Fig. 2a)
Cell viability MSCs cultured with ePL culture medium exhibited similar percentages of viable cells (64.6 ± 7.67) compared with MSCs cultured in FBS culture medium (61.83 ± 10.42), as evaluated by flow cytometry following extensive washes with phosphate-buffered saline (PBS) (Fig. 3a)
Summary
Mesenchymal 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. There is concerning evidence to show that FBS contains endotoxins (such as lipopolysaccharide (LPS)) and xenogeneic antigens that may alter the phenotype of MSCs grown in FBS, rendering these cells immunogenic [10,11,12] This may prompt the immune system to reject MSCs following introduction into the recipient, even when the delivered MSCs are autologous to the host. These facts, together with the rising cost of FBS and ethical concerns related to the manufacturing of FBS, underpin the rationale behind the development of FBS-free media to support the expansion of MSCs for clinical purposes
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