Optimization of the Cultivation of Donor Mesenchymal Stromal Cells for Clinical Use in Cellular Therapy

Vanessa Valim,Annelise Pezzi,Maria Aparecida Lima Da Silva,Lucia Silla,Bruna Amorin,Ana Paula Alegretti

doi:10.4236/cellbio.2014.31003

Abstract

Mesenchymal stromal cells(MSC)have shown their benefits in graft-versus-host disease(GVHD), with five unsettled matters: 1) MSCs expansion in medium with Fetal Bovine Serum(FBS)and its risk of xenoreaction; 2) The number of cells indicated for therapy that is relatively high, with the need to optimize the expansion, number and time wise; 3) The utilization of third party donors; 4) Culture passage number(P);and 5) Source of the cells. This study was designed to determine the superiority of the Platelet Lysates(PL)over FBS on the expansion of MSC, the optimal cell’ plating density and days between each pass, and to investigate if donor total nucleated cells(TNC)obtained from the washouts of discharged bags and filters of hematopoietic stem cell transplantation(HSCT)can be expanded to be used at clinical grade. TNC were removed, plated and after the first passage were cultivated in different concentrations with FBS or PL, and the number of days to reach 80% of confluence was observed. Next, cultures with the same plating density were fed either with PL or with FBS and after seven days counted to analyze how much they had grown in that period. The proliferation of mesenchymal stromal cells in the presence of PL and SFB was averaged 11.88 and 2.5 times, respectively, in a period of 7 days. The highest concentration of plating cells using PL took less time to reach confluence as compared with the three lower ones. This study suggests that the PL is the best choice as a supplement to expand MSC and to allow the proliferation of enough number of MSC at P2 for clinical use.

Highlights

Mesenchymal stem cells (MSCs), referred to mesenchymal stromal cells, are considered as non-hematopoietic multipotent stem cells with self-renewal properties and ability to differentiate into mesoderm tissues
Despite the suggested role in regenerative medicine as a broadly applicable stem cell source [4], there are mounting evidences of predominantly paracrine activity in the setting of allogeneic hematopoietic stem cell transplantation where the beneficial effects have been shown on steroid resistant acute graft versus host disease [5]-[7], on enhancing HSC engraftment [8]-[10], on conditioning regimen damaged tissue repair [11], and on bone marrow stromal repair [12]
We describe here our experience in ex vivo expansion of platelets lysates (PL) supplemented MSC, compare its expanding potential to that obtained with fetal bovine serum (FBS) supplementation, and test different culture cell densities to obtain clinical grade MSC cultures in the shorter period of time

Summary

Introduction

Mesenchymal stem cells (MSCs), referred to mesenchymal stromal cells, are considered as non-hematopoietic multipotent stem cells with self-renewal properties and ability to differentiate into mesoderm tissues. The best known source of mesenchymal stem cells is the bone marrow [18], but it has been isolated from other organs and tissues, such as skeletal muscle and dermis [19], adipose tissue [16], synovium [20], umbilical vein endothelium [21] and saphenous vein [22], kidney [23], umbilical cord blood and placenta [24] [25], cartilage [26], ligamentt [27] and lung [28]

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Results

Conclusion