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Rapid enrichment of adipose derived mesenchymal stromal cells for use in cellular therapies

Angela K. Muise Lisa M. White Michael S. Badowski David T. Harris

*Corresponding author: David T. Harris davidh@email.arizona.edu


Background: Mesenchymal stromal cells (MSC) are multipotent cells that can be isolated from many tissues and are the subject of multiple clinical investigations. While adipose tissue is a reliable source of MSC, it still requires enrichment to increase potency and decrease volume prior to use in autologous cellular therapy. Prolonged in vitro culture can enrich and expand MSC but may also induce functional changes and stem cell senescence. Methods: The stromal vascular fraction (SVF) obtained by enzymatic digestion of lipoaspirated human adipose tissue was plated at a maximum density of 100,000–200,000 cells/cm2 onto tissue culture flasks. A method which involved washing every 24 hours to remove non-adherent cells (Method A) was compared to a method that involved harvesting and re-plating cells in fresh cell culture flasks (Method B). Adherent cell populations containing the purified MSC could then be harvested and assayed at various time points. Results: MSC from the SVF were enriched from 16% of cells positive for CD73 at day 0 to 39% at day 1 to 59% within 2 days, with minimal (1–2 cell doublings) cellular expansion for Method B. Flow cytometric analysis demonstrated co-expression of known MSC phenotypic markers (CD73, CD105, and CD90) along with maintenance of functional capabilities (e.g., cell growth, colony formation and directed differentiation). After enrichment MSC can be harvested and concentrated for resuspension in minimal volumes for clinical use. Discussion: The maximum density plating and short-term culture approach described herein represents a simple and novel method to rapidly isolate MSC for clinical therapies, with minimal costs and time, which can also be performed in a closed culture system.

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