Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells

Paola Iudicone,Luigi Frati,Marianna Nuti,Luca Pierelli,Giuseppina Bonanno,Daniela Fioravanti,Michelina Miceli,Pierangela Totta,Claudio Lavorino

doi:10.1186/1479-5876-12-28

Abstract

BackgroundSupplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC.MethodsPL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation.ResultsPI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic medium was irrelevant as compared to PL and PI-PL.ConclusionThe replacement of animal additives with human supplements is a basic issue in MSC ex vivo production. PI-PL represents a standardized, plasma-poor, human preparation which appears as a safe and good candidate to stimulate MSC growth in clinical-scale cultures.

Highlights

Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells
The concentration of platelet derived growth factor AB (PDGF-AB), vascular endothelial growth factor (VEGF), basic-fibroblast growth factor (bFGF) and TGF-β1 released in Platelet lysate (PL) were comparable between PL control and pathogen inactivation (PI)-PL lots and among the different preparations, suggesting that pooling of 12 PLT units in a single unit may balance the individual variability of PLT growth factor release into the final product (Table 1)
The use of animal sera for clinical-scale MSC expansion raises concerns because of the risk to transmit prions, viruses or to induce immunological reactions in recipients following infusion and for these reasons these products are not allowed in good manufacturing practice (GMP) applied to cell therapy

Summary

Introduction

Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. We describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC. Most clinical-scale MSC production protocols use cocktails which contain serum of animal origin as supplement. These products maintain the potential risk of pathogen transmission and immunological reactions related to the different species origin. The technology shows efficacy in inactivating viruses, bacteria, protozoa and eventual residual leucocytes Starting from these concepts, we adopted a PLT pooling procedure followed by an additional step of photochemical treatment necessary for PI to produce a plasma-poor, pathogen-free PL in a closed sterile system. This preparation was named MesengenTM by a trademark associated with the registration of the international patent application of this product (PCT/IB2012/055062)

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Results

Conclusion