[Isolation and Characterization of Multipotent Precursor Cells from Murine Adipose Tissue using a Clinically Approved Cell Separation System].
BACKGROUND
Recent studies underscored the clinical potential of adipose-derived multipotent stem-/precursor cells (ASPCs). One of the main hurdles en route to clinical application was to isolate cells without having to perform expansion cultures outside the OR. A new generation of clinically approved, commercially available cell separation systems claims to provide ASPCs ready for application without further expansion cultures. However, it is unclear if the new systems yield sufficient cells of adequate quality for the use in autologous murine models. The aim of this study was to isolate and characterize adipose-derived precursor cells taken from the inguinal fat pat of wistar rats using InGeneron's clinically approved ARC™-cell separation system.


METHODS
We isolated cells from the inguinal fat pad of 3 male Wistar rats according to the manufacturer's protocol. In order to reduce the influence of the atmospheric oxygen on the multipotent precursor cells, one half of the cell suspension was cultivated under hypoxia (2% O2) simulating physiological conditions for ASPCs. As a control, the other half of the cells were cultivated under normoxia (21% O2). Cell surface markers CD90, CD29, CD45 and CD11b/c were analyzed by FACS, and osteogenic and adipogenic differentiation of the ASPCs was performed. Finally, cellular growth characteristics were assessed by evaluation of the cumulative population doublings and CFU assay, and metabolic activity was evaluated by WST-1 assay.


RESULTS
Processing time was 90 (± 12) min. 1 g of adipose tissue yielded approximately 60 000 plastic adhering cells. Both groups showed a high expression of the mesenchymal stem cell markers CD90 and CD29 while they were negative for the leucocyte markers CD45 and CD11b/c. A strong osteogenic differentiation and a sufficient adipogenic differentiation potential was proven for all ASPCs. Under hypoxia, ASPCs showed increased proliferation characteristics and CFU efficiency as well as a significantly increased metabolic activity.


CONCLUSIONS
This study showed that sufficient multipotent ASPCs of appropriate quality can be isolated from the inguinal fat pad of Wistar rats using the ARC™-cell separation system. As shown in previous studies, cultivation of cells under hypoxic conditions increased their stemness. Our findings will enable future studies that focus on autologous transplantation of ASPCs in a rat model, which most closely resembles a possible clinical application.
Abteilung für Handchirurgie, Plastische Chirurgie, Ästhetische Chirurgie der Ludwig-Maximilians-Universität München, Campus Innenstadt und Campus Großhadern.
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Krug
Abteilung für Handchirurgie, Plastische Chirurgie, Ästhetische Chirurgie der Ludwig-Maximilians-Universität München, Campus Innenstadt und Campus Großhadern.
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Beer
Labor für Experimentelle Chirurgie und Regenerative Medizin (ExperiMed), LMU Klinikum - Klinik für Allgemeine, Unfall- und Wiederherstellungschirurgie, Ludwig-Maximilians-Universität, München.
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Saller
Labor für Experimentelle Chirurgie und Regenerative Medizin (ExperiMed), LMU Klinikum - Klinik für Allgemeine, Unfall- und Wiederherstellungschirurgie, Ludwig-Maximilians-Universität, München.
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Aszodi
Abteilung für Handchirurgie, Plastische Chirurgie, Ästhetische Chirurgie der Ludwig-Maximilians-Universität München, Campus Innenstadt und Campus Großhadern.
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Holzbach
Abteilung für Handchirurgie, Plastische Chirurgie, Ästhetische Chirurgie der Ludwig-Maximilians-Universität München, Campus Innenstadt und Campus Großhadern.
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Giunta
Abteilung für Handchirurgie, Plastische Chirurgie, Ästhetische Chirurgie der Ludwig-Maximilians-Universität München, Campus Innenstadt und Campus Großhadern.
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Volkmer
D017667:Adipocytes / Q000166:cytology*; D050156:Adipogenesis / Q000502:physiology; D000818:Animals; D002452:Cell Count; D018929:Cell Culture Techniques / Q000295:instrumentation; D002454:Cell Differentiation / Q000502:physiology; D002469:Cell Separation / Q000295:instrumentation*; D004867:Equipment Design; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010012:Osteogenesis / Q000502:physiology; D010101:Oxygen Consumption; D051381:Rats; D017208:Rats, Wistar