Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate

Thomas R.J Heathman,Alexandra Stolzing,Claire Fabian,Qasim A Rafiq,Karen Coopman,Alvin W Nienow,Bo Kara,Christopher J Hewitt

doi:10.1016/j.jcyt.2016.01.007

Abstract

Background aimsThe selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. MethodsIn this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. ResultsThe introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 105 and 1.02 ± 0.005 × 105 cells/mL compared with 0.79 ± 0.05 × 105 and 0.36 ± 0.04 × 105 cells/mL in FBS-containing medium. ConclusionsThis study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up.

Highlights

The growing field of regenerative medicine (RM) aims to treat unmet clinical indications such as cardiovascular disease and neurological disorders by restoring or maintaining cell or tissue function
Human MSCs were isolated from bone-marrow aspirate purchased from Lonza obtained from two healthy donors with informed consent: BM-human mesenchymal stromal cell (hMSC) 1 and BM-hMSC 2.These two BM-hMSC donors were selected after pre-screening of five potential donors because of their differing growth and characteristics [13], representing the two extremes in terms of culture performance (±5.2 population doublings over 30 days of expansion).The local ethics committee approved the use of the samples for research
It can be seen from Figure 1(A,B) that BM-hMSCs expanded in human platelet lysate (HPL) have significantly higher (P < 0.05) growth rate for both donors compared with fetal bovine serum (FBS).This is most apparent over the first three passages (18 days culture) before the cells expanded in HPL undergo a senescent phase at approximately 10 cumulative population doublings in the monolayer expansion process

Summary

Introduction

The growing field of regenerative medicine (RM) aims to treat unmet clinical indications such as cardiovascular disease and neurological disorders by restoring or maintaining cell or tissue function. The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BMhMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 105 and 1.02 ± 0.005 × 105 cells/ mL compared with 0.79 ± 0.05 × 105 and 0.36 ± 0.04 × 105 cells/mL in FBS-containing medium. This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up

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Conclusion