Microcarrier culture of mesenchymal stem cells with platelet lysate as a serum substitute

Abstract

Concerns about the use of animal derived products for the production of therapies in humans has resulted in the search for alternatives that can replace fetal bovine serum (FBS). One of these possible substitutes is platelet lysate (PL). For the production of clinically relevant numbers of mesenchymal stromal cells (MSCs) for cell therapy in humans, an upgrade to large-scale culture is necessary to acquire therapeutic cell quantities. Large-scale culture with microcarriers is effective in the presence of FBS, whereas large microcarrier aggregates are formed in the presence of PL. Therefore, a pre-treatment of PL to stop the aggregates from forming is essential. Here, we present two methods to use PL in microcarrier culture. Human MSCs were isolated from bone marrow aspirates and cultured in αMEM with 5% platelet lysate or 15% FBS. Two types of PL culture medium was prepared: 1) heat-inactivation of PL (HIPL); and 2) pre-gelling of PL (PGPL). MSCs were seeded at 400 cells/cm2 and cultured for 6 passages, or cultured for 21 days in spinner flasks (Cytodex1, 2 g/L) Growth kinetics were determined for both types of culture. Cells were characterized using FACS. HIPL cultured MSCs were smaller and more elongated and PGPL cultured MSCs were smaller, more condensed with multiple dendrites per cell when compared with the FBS cultured MSCs (Fig 1). Analysis of cell growth showed that PGPL resulted in the highest cell numbers for both monolayer and microcarrier culture (Fig.2). FACS analysis showed comparable expression of the typical MSC surface markers (Fig.3). Alizarin Red, Safranin O and Oil Red O staining showed that all MSCs could be induced into the osteo-, chondro- and adipogenic lineage, respectively, regardless the serum supplement. Based on the results we conclude that with the correct pre-treatment, it is possible to use PL in a microcarrier culture. Therefore, PL could be used for large-scale cultivation of MSCs for cell therapy.