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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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Fluorescent photomicrographs of SVF a, P0 b, P1 c and P3 d feline pididymal ASCs with cytoskeleton (actin, green) and nuclear (DNA, blue) staining (Acti-stain™ 488, actin; Hoechst dye, DNA; 20×)
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Fig4: Fluorescent photomicrographs of SVF a, P0 b, P1 c and P3 d feline pididymal ASCs with cytoskeleton (actin, green) and nuclear (DNA, blue) staining (Acti-stain™ 488, actin; Hoechst dye, DNA; 20×)

Mentions: Five days after seeding, the majority of adherent SVF cells had the spindle shaped morphology typical of ASCs, though small, polygonal cells were apparent (Fig. 4A). The cell population was more homogenous and organized at P0 and 1 with the same spindle shaped morphology (Figs. 4B, C). By P3, the majority of cells were polygonal and poorly organized (Fig. 4D).Fig. 4


Therapeutic doses of multipotent stromal cells from minimal adipose tissue.

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

Fluorescent photomicrographs of SVF a, P0 b, P1 c and P3 d feline pididymal ASCs with cytoskeleton (actin, green) and nuclear (DNA, blue) staining (Acti-stain™ 488, actin; Hoechst dye, DNA; 20×)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Fluorescent photomicrographs of SVF a, P0 b, P1 c and P3 d feline pididymal ASCs with cytoskeleton (actin, green) and nuclear (DNA, blue) staining (Acti-stain™ 488, actin; Hoechst dye, DNA; 20×)
Mentions: Five days after seeding, the majority of adherent SVF cells had the spindle shaped morphology typical of ASCs, though small, polygonal cells were apparent (Fig. 4A). The cell population was more homogenous and organized at P0 and 1 with the same spindle shaped morphology (Figs. 4B, C). By P3, the majority of cells were polygonal and poorly organized (Fig. 4D).Fig. 4

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