Development of large-scale manufacturing of adipose-derived stromal cells for clinical applications using bioreactors and human platelet lysate

Abstract

In vitro expanded adipose-derived stromal cells (ASCs) are a useful resource for tissue regeneration. Translation of small-scale autologous cell production into a large-scale, allogeneic production process for clinical applications necessitates well-chosen raw materials and cell culture platform. We compare the use of clinical-grade human platelet lysate (hPL) and fetal bovine serum (FBS) as growth supplements for ASC expansion in the automated, closed hollow fibre quantum cell expansion system (bioreactor). Stromal vascular fractions were isolated from human subcutaneous abdominal fat. In average, 95 × 10⁶ cells were suspended in 10% FBS or 5% hPL medium, and loaded into a bioreactor coated with cryoprecipitate. ASCs (P0) were harvested, and 30 × 10⁶ ASCs were reloaded for continued expansion (P1). Feeding rate and time of harvest was guided by metabolic monitoring. Viability, sterility, purity, differentiation capacity, and genomic stability of ASCs P1 were determined. Cultivation of SVF in hPL medium for in average nine days, yielded 546 × 10⁶ ASCs compared to 111 × 10⁶ ASCs, after 17 days in FBS medium. ASCs P1 yields were in average 605 × 10⁶ ASCs (PD [population doublings]: 4.65) after six days in hPL medium, compared to 119 × 10⁶ ASCs (PD: 2.45) in FBS medium, after 21 days. ASCs fulfilled ISCT criteria and demonstrated genomic stability and sterility. The use of hPL as a growth supplement for ASCs expansion in the quantum cell expansion system provides an efficient expansion process compared to the use of FBS, while maintaining cell quality appropriate for clinical use. The described process is an obvious choice for manufacturing of large-scale allogeneic ASC products.

Keywords:

• Adipose-derived stromal cells
• bioreactor
• clinical application
• closed cultivation systems
• cryoprecipitate
• human platelet lysate
• mesenchymal stromal cells
• stem cells

Acknowledgements

The authors thank Terumo BCT with special tanks to Brent Rice for support and guidance; Sofie Lykke Larsen and Sonja K. Brorsen for technical assistance; Lisbeth D. Lund and Ellen M. Johansen for constructive discussions. We are grateful to Andreas Printzlau for supplying the liposuction aspirates and the patients for consenting to participate, and the Blood Bank, Department of Immunology, Rigshospitalet University Hospital, Denmark for supplying the cryoprecipitate.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by Aase and Ejnar Danielsens Foundation [10-001242], Arvid Nilssons Foundation [2014], and Capital Regional Research Foundation [R139-A4945 2015].