Methods in Molecular Biology (2022) 2436: 83–111

DOI 10.1007/7651_2021_426

© Springer Science+Business Media, LLC 2021

Published online: 06 October 2021

Chemically Defined, Xeno-Free Expansion of Human

Mesenchymal Stem Cells (hMSCs) on Benchtop-Scale

Using a Stirred Single-Use Bioreactor

Misha Teale

, Valentin Jossen

, Dieter Eibl

, and Regine Eibl

Abstract

In recent years, the use of hMSCs, which may be isolated from adipose tissue among others, for the

treatment of diseases has increased significantly. The cell quantities required for such therapeutic

approaches, between 10¹² and 10¹³, have thus far been predominantly produced using commercially

available multi-tray systems, such as the Cell Factory (Thermo Fisher Scientific) or HYPERStack (Corning),

which can be purchased with up to 40 layers. However, the handling of these planar multilayer systems is

difficult, and process monitoring opportunities remain limited. Here, automated stirred single-use bio-

reactors provide a viable alternative to the time-consuming multiplication of cells using such planar systems,

while still managing to achieve the desired clinically relevant quantities. In these stirred single-use systems,

adherent cells are predominantly cultivated in suspension up to pilot scale using carrier materials, also

referred to as microcarriers (MCs).

This chapter describes the steps which need to be realized to guarantee successful hMSC expansion

within a stirred single-use bioreactor (Eppendorf’s BioBLU® 0.3c) operated using MCs under serum- and

xeno-free conditions at benchtop scale. The cultivations were performed using an immortalized human

adipose-derived mesenchymal stem cell (hASC) line, hence referred to as hASC52telo, and a new chemically

defined, xeno-free medium, hence referred to as the UrSuppe formulation. Spinner flask cultivations were

performed under comparable process conditions.

Key words BioBLU® 0.3c bioreactor, Cell expansion, hTERT immortalized ASC5Telo cells,

Microcarrier, Single-use, UrSuppe

1

Introduction

Human mesenchymal stem cells are plastic-adherent cells with

multipotent differentiation capacity in vitro [1]. They are charac-

terized not only by their high safety [2] but also by their remarkable

therapeutic function [3–5], which includes their paracrine activity,

ability to transfer mitochondria and organelles to other cells, and

their application in achieving therapeutic molecule transfer using

exosomes secreted during cultivation. Thus, it is not at all

surprising that currently more than 1000 clinical trials are

registered on www.ClinicalTrials.gov for the treatment of autoim-

mune and metabolic diseases, orthopedic diseases, as well as

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