Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process

Abstract

Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot‐sizes required for commercial production. The use of animal‐derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot‐to‐lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large‐scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum‐free hMSC manufacturing process. Human bone‐marrow derived hMSCs were expanded on fibronectin‐coated, non‐porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 105 cells.mL−1 in serum‐free medium. The hMSCs were successfully harvested by our recently‐developed technique using animal‐free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post‐harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony‐forming potential. The hMSCs were held in suspension post‐harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum‐free vehicle solution using a controlled‐rate freezing process. Post‐thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component‐free hMSC production process from expansion through to cryopreservation. Biotechnol. Bioeng. 2015;112: 1696–1707. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Application of Bioreactor in Stem Cell Culture

“…Thus, serum free culture is a direction in the future standardized production of biological products with bioreactors [51,52]. It is necessary to investigate the metabolic characteristics of SCs in large scale expansion of cells, design the key components in the serum free medium and optimize the physical and chemical properties of the medium, which may develop individualized culture medium [53].…”

Cell and gene therapy manufacturing: the necessity for a cost-based development approach

“…However for many clinical indications, achieving lot sizes of several hundred billion to trillions of cells will be imperative for commercial success; utilizing high-density bioreactor manufacturing platforms, such as stirred-tank bioreactors in conjunction with microcarriers, is likely to be the only way to accomplish this [21][22][23][24][25].…”

Developing an Automated Robotic Factory for Novel Stem Cell Therapy Production

“…The cell density achieved was ~ 1.7 x 10 5 cells/mL, although recent studies by the group and others have demonstrated cell densities approaching clinically-relevant numbers of ~ 1 x 10 6 cells/mL [15,16]. It is likely with further bioprocess development and parameter optimisation, cell densities will continue to increase, as has been the case with traditional biopharmaceutical development.…”