From 3D to 3D: isolation of mesenchymal stem/stromal cells into a three-dimensional human platelet lysate matrix

Abstract

Mesenchymal stem/stromal cells (MSCs) are considered an important candidate in cell therapy and tissue engineering approaches. The culture of stem cells in a 3D environment is known to better resemble the in vivo situation and to promote therapeutically relevant effects in isolated cells. Therefore, the aim of this study was to develop an approach for the isolation of MSCs from adipose tissue into a 3D environment. Furthermore, the use of cryoprotective medium for cryopreservation of whole adipose tissue was evaluated. For the isolation of MSCs, a novel human platelet lysate-based hydrogel was used as matrix and the migration, yield, viability and metabolic activity of cells from the 3D matrix were compared to cells from 2D explant culture. Also, the surface marker profile and differentiation capacity of MSCs from the 3D matrix were evaluated and compared to MSCs from isolation by enzymatic treatment. We found that cryopreservation of whole adipose tissue is feasible, and therefore adipose tissue can be stored and is available for MSC isolation on demand. Also, we demonstrate the isolation of MSCs into the 3D matrix and that cells from this condition display a similar phenotype and differentiation capacity like MSCs derived by traditional isolation procedure. The presented approach allows, for the first time, to isolate MSCs directly into a soft 3D hydrogel environment, avoiding any contact to a 2D plastic culture surface. 3D explant isolation of MSCs 3 Significance Statement:In this paper we present a new method for the isolation of mesenchymal stem cells. Usually, these cells grow on two-dimensional plastic surfaces which is far away from their physiologic environment. Our new method allows for the first time the direct outgrowth of cells from primary tissue into a three-dimensional environment, avoiding any contact to a two-dimensional plastic surface. In future, this will allow an entirely three-dimensional in vitro cultivation of stem cells.Using 3D isolated cells will probably also increase the physiologic relevance of in vitro models. 3D explant isolation of MSCs4