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Therapeutic doses of multipotent stromal cells from minimal adipose tissue.

Zhang N, Dietrich MA, Lopez MJ - Stem Cell Rev (2014)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Equine and Comparative Orthopedic Research, Equine Health Studies Program, Department of Veterinary Clinical Sciences, Louisiana State University, Baton Rouge, LA, 70803, USA.

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 10(6)-10(7) 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 7x10(6) 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.

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Photomicrographs of feline ASCs after 10 days of culture in stromal medium a, 5×; with alkaline phosphatase (ALP) staining indicating early stage osteogenesis after 10 days of culture in osteogenic medium b, 10×; with alizarin red staining of calcium deposition after 21 days of culture in osteogenic medium c, 5×; with oil red O lipid staining after 10 days of culture in adipogenic medium d, 40×; and showing early stage chondrogenesis based on alcian blue staining of unsulfated proteoglycans after 20 days of cell pellet culture in chondrogenic medium e, 64×. There is no evidence of alcian blue staining in a cell pellet cultured in stromal medium for 21 days f, 64×
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Fig5: Photomicrographs of feline ASCs after 10 days of culture in stromal medium a, 5×; with alkaline phosphatase (ALP) staining indicating early stage osteogenesis after 10 days of culture in osteogenic medium b, 10×; with alizarin red staining of calcium deposition after 21 days of culture in osteogenic medium c, 5×; with oil red O lipid staining after 10 days of culture in adipogenic medium d, 40×; and showing early stage chondrogenesis based on alcian blue staining of unsulfated proteoglycans after 20 days of cell pellet culture in chondrogenic medium e, 64×. There is no evidence of alcian blue staining in a cell pellet cultured in stromal medium for 21 days f, 64×

Mentions: Cells maintained a fibroblastic-like appearance when cultured in stromal media (Fig. 5A), and did not stain with oil red O, ALP or alizarin red. In osteogenic medium without β-glycerophosphate, cells expressed alkaline phosphatase (ALP) after 10 days of culture (Fig. 5B). Following the addition of β-glycerophosphate, granular nodules formed and stained with alizarin red (calcium deposition) after 21 days of culture (Fig. 5C). After about 4 days of culture in adipogenic medium, cells became round, and, after 21 days, robust lipid droplets that stained with oil red O were apparent (Fig. 5D). Cells were apparent within lacunae in pellets cultured in chondrogenic medium after 21 days, and unsulfated proteoglycans in the extra-cellular matrix stained with alcian blue (Fig. 5E). Pellets cultured in stromal medium did not have discernible lacunae or alcian blue staining (Fig. 5F).Fig. 5


Therapeutic doses of multipotent stromal cells from minimal adipose tissue.

Zhang N, Dietrich MA, Lopez MJ - Stem Cell Rev (2014)

Photomicrographs of feline ASCs after 10 days of culture in stromal medium a, 5×; with alkaline phosphatase (ALP) staining indicating early stage osteogenesis after 10 days of culture in osteogenic medium b, 10×; with alizarin red staining of calcium deposition after 21 days of culture in osteogenic medium c, 5×; with oil red O lipid staining after 10 days of culture in adipogenic medium d, 40×; and showing early stage chondrogenesis based on alcian blue staining of unsulfated proteoglycans after 20 days of cell pellet culture in chondrogenic medium e, 64×. There is no evidence of alcian blue staining in a cell pellet cultured in stromal medium for 21 days f, 64×
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4127443&req=5

Fig5: Photomicrographs of feline ASCs after 10 days of culture in stromal medium a, 5×; with alkaline phosphatase (ALP) staining indicating early stage osteogenesis after 10 days of culture in osteogenic medium b, 10×; with alizarin red staining of calcium deposition after 21 days of culture in osteogenic medium c, 5×; with oil red O lipid staining after 10 days of culture in adipogenic medium d, 40×; and showing early stage chondrogenesis based on alcian blue staining of unsulfated proteoglycans after 20 days of cell pellet culture in chondrogenic medium e, 64×. There is no evidence of alcian blue staining in a cell pellet cultured in stromal medium for 21 days f, 64×
Mentions: Cells maintained a fibroblastic-like appearance when cultured in stromal media (Fig. 5A), and did not stain with oil red O, ALP or alizarin red. In osteogenic medium without β-glycerophosphate, cells expressed alkaline phosphatase (ALP) after 10 days of culture (Fig. 5B). Following the addition of β-glycerophosphate, granular nodules formed and stained with alizarin red (calcium deposition) after 21 days of culture (Fig. 5C). After about 4 days of culture in adipogenic medium, cells became round, and, after 21 days, robust lipid droplets that stained with oil red O were apparent (Fig. 5D). Cells were apparent within lacunae in pellets cultured in chondrogenic medium after 21 days, and unsulfated proteoglycans in the extra-cellular matrix stained with alcian blue (Fig. 5E). Pellets cultured in stromal medium did not have discernible lacunae or alcian blue staining (Fig. 5F).Fig. 5

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Equine and Comparative Orthopedic Research, Equine Health Studies Program, Department of Veterinary Clinical Sciences, Louisiana State University, Baton Rouge, LA, 70803, USA.

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 10(6)-10(7) 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 7x10(6) 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.

Show MeSH