The production method affects the efficacy of platelet derivatives to expand mesenchymal stromal cells in vitro

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.

Blood derivatives awaken in regenerative medicine strategies to modulate wound healing

“…PL is a liquid solution of biomolecules, and although its platelet content release is obtained by cellular disruption instead of α-granules degranulation, it contains most of the GFs commonly found in other platelet-rich BD [84,85]. Nevertheless, the concentration of some GFs, namely PDGF, is significantly lower in PL than in PRGF [104] ( Table 2) emphasizing the relevance of BD production method on their bioactive molecules composition.…”

Human periodontal ligament stem cells on calcium phosphate scaffold delivering platelet lysate to enhance bone regeneration

“…[21][22][23] Bernardi et al quantied the concentrations of the various growth factors in HPL. 24 They reported that 1 mL HPL contained about 0.56 Â 10 3 pg VEGF, 25.16 Â 10 3 pg PDGF-AB, 4.78 Â 10 3 pg PDGF-AA, 5.06 Â 10 3 pg PDGF-BB, 53.04 Â 10 3 pg TGF-b1, and 0.085 Â 10 3 pg FGF-basic. 24 Osteogenesis is a complex process which involves a variety of molecules, 25 thus it is tempting to predict that applying all these molecules in HPL would have synergistic effects to enhance the bone regeneration.…”

“…24 They reported that 1 mL HPL contained about 0.56 Â 10 3 pg VEGF, 25.16 Â 10 3 pg PDGF-AB, 4.78 Â 10 3 pg PDGF-AA, 5.06 Â 10 3 pg PDGF-BB, 53.04 Â 10 3 pg TGF-b1, and 0.085 Â 10 3 pg FGF-basic. 24 Osteogenesis is a complex process which involves a variety of molecules, 25 thus it is tempting to predict that applying all these molecules in HPL would have synergistic effects to enhance the bone regeneration. Indeed, a few studies indicated that HPL promoted the proliferation and osteodifferentiation of mesenchymal stem cells (MSCs), and hence HPL may represent a good therapeutic candidate for bone repair applications.…”

1H-NMR and MALDI-TOF MS as metabolomic quality control tests to classify platelet derived medium additives for GMP compliant cell expansion procedures

“…Interindividual differences between platelet donors could affect the capability to stimulate cell growth in vitro [ 7 , 8 ]: we demonstrated that, pooling together n = 16 SRGF products from single donors, standardized batches of medium additive can be obtained for applications in academic GMP facilities [ 4 , 9 ]. Nevertheless, we previously demonstrated that also the production method can affect medium additive capacity to stimulate cell growth in culture [ 10 ].Growth factors can, in fact, be extracted from platelets treating PRP by the so called freeze-and-thaw method [ 2 , 11 ], and we previously demonstrated that medium additives obtained by recalcification of PRP can promote a faster cell proliferation rate [ 10 ]. Especially when dealing with in-house manufactured medium additives, quality controls are needed also to assess and validate the capacity of the product to stimulate cell proliferation in vitro [ 9 ].…”

“…Growth factor concentration analysis is frequently used to characterize composition of platelet derived products with possible application as medium additives for cell expansion [ 2 , 12 – 15 ].Several growth factors released by platelets can affect cell proliferation rate by separate pathways [ 16 ]: thus, defined cytokines univocally affecting cell proliferation rate were not identified and validated as specific markers. Moreover, we previously demonstrated that, assessing growth factor concentrations, SRGF could not be clearly distinguished from freeze-and-thaw manufactured products [ 2 , 10 ]. Then, independently from the manufacturing method, a quick and cost effective test potentially predicting the impact of a medium additive on cell growth rate would represent a significant achievement improving both production and quality control processes.…”