Abstract
Low yield of adult adipose-derived multipotent stromal cells (ASC) can limit autologous cell therapy in individuals with minimal adipose tissue. In this study, ASC isolation was optimized from approximately 0.2 g of feline epididymal adipose tissue for a treatment dose of 106–107 ASCs/kg. The ASC yield was determined for three digestions, 0.1 % collagenase in medium for 30 min (Classic), 0.3 % collagenase in buffer for 30 min (New) and 0.3 % collagenase in buffer for 1 h (Hour). After isolation by the new tissue digestion, continuously cultured ASCs (fresh) and cells recovered and expanded after cryostorage at P0 (revitalized) were characterized up to cell passage (P) 5. Outcomes included CD9, CD29, CD44, CD90 and CD105 expression, cell doublings and doubling times, fibroblastic, adipogenic and osteogenic colony forming unit (CFU) frequency percentages and lineage-specific target gene expression after induction. The New digestion had the highest CFU yield, and about 7x106 ASCs/kg were available within three cell passages (P2). Compared to earlier passages, target surface antigen expression was lowest in fresh P5 cells, and fresh and revitalized P3–5 cells had slower expansion. Fresh and revitalized P1 ASCs had higher CFU frequency percentages and lineage-specific gene expression than P3. The New method described in this study was most efficient for feline epididymal ASC isolation and did not alter in vitro cell behavior. Fresh and revitalized P0-P2 feline ASCs may be most effective for preclinical and clinical trials. This study offers a potential option for ASC isolation from limited adipose tissue resources across species.
Highlights
Adipose-derived multipotent stromal cells (ASCs) are an appealing cell option for numerous regenerative medicine therapies due, in part, to culture expansion, multipotentiality, immune privilege, trophic effects and higher in vitro proliferation compared to undifferentiated cells from bone marrow [1,2,3]
Species-specific information about ASC isolation and culture expansion supports repeatable results with the greatest potential value for comparison among species [47]. This study addresses these necessities by optimizing feline ASC isolation and expansion to test the hypothesis that 106–107 ASCs/kg are available from individual cat epididymal adipose tissue within 3 cell passages before and after ASC cryopreservation
One of three adipose digestion methods was found to yield sufficient feline ASCs from minimal tissue to give a therapeutic dose within three cell passages of fresh or cryopreserved cells, both of which retained in vitro MSC characteristics
Summary
Adipose-derived multipotent stromal cells (ASCs) are an appealing cell option for numerous regenerative medicine therapies due, in part, to culture expansion, multipotentiality, immune privilege, trophic effects and higher in vitro proliferation compared to undifferentiated cells from bone marrow [1,2,3]. Feline epididymal adipose tissue is a promising source of WAT ASCs for feline therapeutic applications and intra-species comparisons. Existing reports support the potential therapeutic value of feline MSCs, including neurogenic and cardiogenic capabilities of adult bone marrow-derived multipotent stromal cells (BMSCs)[8,9,10,11] and treatment of chronic kidney disease by intra-renal injection of BMSCs and ASCs [12]. About 0.2–2 % of cats in the United States suffer from diabetes mellitus, Stem Cell Rev and Rep (2014) 10:600–611 lymphoma or retinal disease [13,14,15,16]which affect considerable numbers of other species, including humans [17,18,19]. There are reports of cell-based therapies for these conditions in humans and animals [17,18,19,20,21], but, to date, information surrounding comparable therapies for the conditions in cats is scarce
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