Abstract
BackgroundThe use of fetal bovine serum (FBS) as a media supplement for the ex vivo expansion of bone-marrow derived mesenchymal stromal cells (BM-MSC) has been discouraged by regulatory agencies, due to the risk of transmitting zoonoses and to elicit immune reactions in the host once transplanted. Platelet derivatives are valid FBS substitutes due to their content of growth factors that can be released disrupting the platelets by physical methods or physiological stimuli. We compared platelet derivatives produced by freezing/thawing (platelet lysates, PL) or after CaCl2 activation (platelet releasate surnatant rich in growth factors, PR-SRGF) for their content in growth factors and their ability to support the ex vivo expansion of BM-MSC.MethodsThe cytokine content in the two platelet derivatives was evaluated. BM-MSC were expanded in complete medium containing 10, 7.5 and 5% PL or PR-SRGF and the cell phenotype, clonogenic capacity, immunomodulation properties and tri-lineage differentiation potential of the expanded cells in both media were investigated.ResultsThe concentration of PDGF-AB, PDGF-AA, PDGF-BB in PR-SRGF resulted to be respectively 5.7×, 1.7× and 2.3× higher compared to PL. PR-SRGF promoted a higher BM-MSC proliferation rate compared to PL not altering BM-MSC phenotype. Colony forming efficiency of BM-MSC expanded in PR-SRGF showed a frequency of colonies significantly higher than cells expanded in PL. BM-MSC expanded in PL or PR-SRGF maintained their immunomodulatory properties against activated lymphocytes even if BM-MSC expanded in FBS performed significantly better.ConclusionsThe method used to release platelet factors significantly affects the enrichment in growth factors and overall product performance. The standardization of the production process of platelet derivatives and the definition of their release criteria requires further investigation.
Highlights
The use of fetal bovine serum (FBS) as a media supplement for the ex vivo expansion of bone-marrow derived mesenchymal stromal cells (BM-MSC) has been discouraged by regulatory agencies, due to the risk of transmitting zoonoses and to elicit immune reactions in the host once transplanted
Cell proliferation platelet releasate surnatant rich in growth factors (PR-SRGF) promoted a higher proliferation rate compared to platelet lysate (PL) and FBS resulting in a greater cumulative PD (cPD) until P8 at any supplement concentration
At P5 the cPD of bone marrow (BM)-MSC expanded in presence of platelet releasate (PR)-SRGF was significantly higher compared to PL at each concentration (P < 0.05 for the 5% and P < 0.01 for the 7.5 and 10% conditions)
Summary
The use of fetal bovine serum (FBS) as a media supplement for the ex vivo expansion of bone-marrow derived mesenchymal stromal cells (BM-MSC) has been discouraged by regulatory agencies, due to the risk of transmitting zoonoses and to elicit immune reactions in the host once transplanted. Platelet derivatives are valid FBS substitutes due to their content of growth factors that can be released disrupting the platelets by physical methods or physiological stimuli. We compared platelet derivatives produced by freezing/thawing (platelet lysates, PL) or after CaCl2 activation (platelet releasate surnatant rich in growth factors, PR-SRGF) for their content in growth factors and their ability to support the ex vivo expansion of BM-MSC. Release of growth factors from platelets can be obtained through physical methods by freezing-thawing the platelet rich plasma obtained from the platelet apheresis or from the buffy coat. After pooling and one or more centrifugation/filtration steps in order to remove cellular debris, PL is ready for being added to the growth media [10]
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