Abstract
OBJECTIVE: Adipose tissue-derived stem cells (ASCs) are currently being investigated for a number of uses in regenerative medicine. Certain clinical approaches would require controlled and reproducible methods of production high number of cells, and here we describe the isolation and expansion of ASCs using such a workflow.
MATERIALS AND METHODS: Lipoaspirate from 3 different human donors was processed in the operating theatre to obtain stromal vascular fraction (SVF) using an automated device. Isolation and expansion of ASCs was done using an automated hollow fiber bioreactor. The cells of the SVF went through 3.53±0.90 doublings over 10.7±1.1 days in the bioreactor, similar to results from parallel cultures of SVF in flasks (n=3 p=0.34). The resulting cellular population was identified as ASCs by plastic adherence, tri-lineage differentiation capability, and surface marker expression. ASCs were then expanded in the bioreactor, yielding 4.94x108±1.61x108 ASCs after 6 days of incubation.
RESULTS: Cell doubling count in the bioreactor was similar to ASCs expanded in flasks (n=3 p=0.25). Levels of soluble factors released by ASCs were not significantly different between cells grown in a bioreactor or flasks. Levels of these factors were also similar through passaging of the cells.
CONCLUSIONS: Finally, we show biological activity of the expanded ASCs by injecting them into streptozotocin-induced diabetic mice, resulting in a significantly lower fasting glucose and improved glucose tolerance test (p<0.05). The large number of ASCs produced by this workflow allows the storage of a large population of similar, low passaged cells for in vitro or in vivo experiments, and our workflow can also potentially be used to generate ASCs for future clinical trials.
MATERIALS AND METHODS: Lipoaspirate from 3 different human donors was processed in the operating theatre to obtain stromal vascular fraction (SVF) using an automated device. Isolation and expansion of ASCs was done using an automated hollow fiber bioreactor. The cells of the SVF went through 3.53±0.90 doublings over 10.7±1.1 days in the bioreactor, similar to results from parallel cultures of SVF in flasks (n=3 p=0.34). The resulting cellular population was identified as ASCs by plastic adherence, tri-lineage differentiation capability, and surface marker expression. ASCs were then expanded in the bioreactor, yielding 4.94x108±1.61x108 ASCs after 6 days of incubation.
RESULTS: Cell doubling count in the bioreactor was similar to ASCs expanded in flasks (n=3 p=0.25). Levels of soluble factors released by ASCs were not significantly different between cells grown in a bioreactor or flasks. Levels of these factors were also similar through passaging of the cells.
CONCLUSIONS: Finally, we show biological activity of the expanded ASCs by injecting them into streptozotocin-induced diabetic mice, resulting in a significantly lower fasting glucose and improved glucose tolerance test (p<0.05). The large number of ASCs produced by this workflow allows the storage of a large population of similar, low passaged cells for in vitro or in vivo experiments, and our workflow can also potentially be used to generate ASCs for future clinical trials.
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