Isolation und Charakterisierung von multipotenten Vorläuferzellen aus murinem Fettgewebe mithilfe eines klinisch zugelassenen Aufbereitungssystems

 

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Zusammenfassung

Einleitung: Das Potenzial von multipotenten Stamm-/Vorläuferzellen aus Fettgewebe (ASPCs) für die regenerative Medizin ist gut belegt. Problematisch für den klinischen Einsatz von ASPCs ist jedoch die Zellisolation, welche oft Expansionsschritte außerhalb des OPs beinhaltet. Eine neue Generation von klinisch zugelassenen, kommerziell erhältlichen Aufbereitungssystemen kommt ohne Expansionsschritte aus, wobei nicht geklärt ist, ob insbesondere für das autologe Kleintiermodell ausreichend Zellmaterial von suffizienter Qualität gewonnen werden kann. Ziel der vorliegenden Studie war es daher, mit dem klinisch zugelassenen ARC™-Aufbereitungssystems von InGeneron adipogene Vorläuferzellen aus dem Leistenfettpolster der Ratte zu isolieren und zu charakterisieren.

Material und Methoden: Die Zellisolation erfolgte nach Herstellerangabe aus dem inguinalen Fettpolster von 3 männlichen Wistar-Ratten. Der Einfluss der atmosphärischen Sauerstoffkonzentration auf die Zellen wurde minimiert indem die Zellsuspension direkt nach Isolation zur Hälfte unter Hypoxie (2% O₂) kultiviert wurde. Die andere Hälfte wurde als Kontrolle unter klassischer Normoxie (21% O₂) mitgeführt. Neben der Ermittlung des molekularen Oberflächenprofils (CD90, CD29, CD45 und CD11b/c) mittels FACS wurden die osteogene und adipogene Differenzierbarkeit der ASPCs geprüft. Weiterhin wurde das kumulative Wachstum, das klonogene Potenzial (CFU assay) sowie die metabolische Aktivität (WST-1 assay) der ASPCs bestimmt.

Ergebnisse: Die Dauer der Aufbereitung betrug 90 (± 12) min. Pro 1 g Fett konnten ca. 60 000 adhärente Zellen isoliert werden. In beiden Gruppen zeigte sich eine hohe Expression der mesenchymalen Marker CD90 und CD29 und eine Fehlen der Leukozytenmarker CD45 und CD11b/c. Für alle ASPCs konnte ein starkes osteogenes und ausreichendes adipogenes Differenzierungspotenzial nachgewiesen werden. Hypoxisch kultivierte ASPCs zeigten eine gesteigerte Proliferation, eine erhöhte CFU-Effizienz sowie eine signifikant gesteigerte metabolischen Aktivität.

Schlussfolgerung: Die Gewinnung von multipotenten ASPCs aus dem Leistenfettpolster von Wistar-Ratten mithilfe des ARC™ Aufbereitungssystems von InGeneron ist in ausreichender Quantität und Qualität möglich. Die hypoxische Kultivierung erhöht das Stammzellpotenzial der Zellen. Unsere Studie hat somit nachfolgenden Studien den Weg geebnet, die eine Klinik-analoge, autologe Transplantation von ASPCs im Rattenmodell anstreben.

Abstract

Introduction: 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.

Materials and 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.

Conclusion: 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.

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