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Title: Development and characterisation of a cytokine supplemented serum free medium formulation for bone marrow derived mesenchymal stromal cells
Authors: Wilkinson, Steven Andrew
Issue Date: 2018
Publisher: Newcastle University
Abstract: Background: The discovery and subsequent characterisation of adult human-derived stem cells appears poised to help revolutionise the fields of regenerative medicine and tissue engineering, offering clinicians the opportunity to develop fully functional replacement tissues, alongside holding significant promise as next generation gene or protein delivery vehicles. Despite their apparent potential however, current stem cell-based therapeutics typically rely on delivering massive doses of cells to sites of injury in order to help ensure adequate cell survival in the highly detrimental microenvironments presented by damaged and degrading biological material. In order to produce the cell numbers required for these types of treatments, relatively modest donor cell populations are subjected to extended periods of in vitro expansion, within highly regulated GMP culture conditions. One crucially important aspect of this manufacturing framework is the requirement for xeno-free expansion systems, including the use of serum-free culture medium. Unfortunately, whilst a number of functional serum-free media formulations are commercially available at the present time, their proprietary nature makes them both highly expensive and wholly unsuitable for use in academic research. Aims: The goal of this project is to begin development of a characterised cytokinesupplemented serum-free medium formulation using a design of experiments-based methodology. Methods: Primary bone marrow-derived mesenchymal stromal cells (BM-MSCs) were isolated, characterised and used to screen a series of selected cytokines and growth supplements for their ability to successfully support cell proliferation and continued survival in the absence of serum. Cells cultured in the resultant serum-free formulation were then compared to those grown in conventional medium in regard to genetic, metabolic and morphological factors. In addition, the impact of batch-to-batch variability on BM-MSC growth and metabolic activity was assessed as a means of determining the potential impact of raw material variation on cell quality and any related manufacturing processes. Finally, a number of different extracellular matrix proteins were also screened for the purpose of mediating cell-surface interactions in serum-free conditions. ii Results: We successfully identified a cytokine supplemented medium preparation capable of supporting the proliferation of BM-MSCs during serum-free culture. Evaluation of cells expanded in this medium provided evidence of altered secretory and genetic characteristics leading to shifted therapeutic potential. Furthermore, the identification of a combination of different extracellular matrix proteins able to enhance cell adhesion in the absence of serum served to provide the beginnings of a complete serum-free formulation. In addition, FBS batch variability was shown to have significant effects on cell proliferation and gene expression, including a number of genetic markers linked to differentiation potential and lineage specificity. Conclusions: We offer a new serum-free medium formulation for use in the expansion of primary BM-MSCs, alongside providing evidence of the impact of raw material variability on the therapeutically relevant characteristics of these cells.
Description: Eng D Thesis
Appears in Collections:School of Chemical Engineering and Advanced Materials

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