Enhanced viability and neural differential potential in poor post-thaw hADSCs by agarose multi-well dishes and spheroid culture

Abstract

Human adipose-derived stem cells (hADSCs) are potential adult stem cells source for cell therapy. But hADSCs with multi-passage or cryopreservation often revealed poor growth performance. The aim of our work was to improve the activity of poor post-thaw hADSCs by simple and effective means. We describe here a simple method based on commercially available silicone micro-wells for creating hADSCs spheroids to improve viability and neural differentiation potential on poor post-thaw hADSCs. The isolated hADSCs positively expresse d CD29, CD44, CD105, and negatively expressed CD34, CD45, HLA-DR by flow cytometry. Meanwhile, they had adipogenic and osteogenic differentiation capacity. The post-thaw and post-spheroid hADSCs from poor growth status hADSCs showed a marked increase in cell proliferation by CKK-8 analysis, cell cycle analysis and Ki67/P27 quantitative polymerase chain reaction (qPCR) analysis. They also displayed an increase viability of anti-apoptosis by annexin v and propidium iodide assays and mitochondrial membrane potential assays. After 3 days of neural induction, the neural differentiation potential of post-thaw and post-spheroid hADSCs could be enhanced by qPCR analysis and western blotting analysis. These results suggested that the spheroid formation could improve the viability and neural differentiation potential of bad growth status hADSCs, which is conducive to ADSCs research and cell therapy.

Microtissues Enhance Smooth Muscle Differentiation and Cell Viability of hADSCs for Three Dimensional Bioprinting

“…Currently, various spheroid culture methods have emerged to form cell aggregates and study their effect on MSCs, including the use of non-adherent plates or microfabrication-based non-adhesive surfaces, the hanging-drop method, spinner or rotary dynamic culture systems, or the use of agarose multi-well dishes from rubber micro-molds (Song et al, 2004; Nyberg et al, 2005; Bogdanova-Jatniece et al, 2014; Rettinger et al, 2014; Guo et al, 2015; Nakagawa et al, 2016). Compared to other methods, the hanging-drop method is relatively economical and convenient.…”

“…Calabrese et al fabricated a new Collagen I-based 3D scaffold and assessed its chondrogenic potential in vitro , which promoted the early chondrogenic differentiation of hADSCs (Calabrese et al, 2017). Guo et al reported that spheroid formations may enhance the neural differentiation potential of post-thaw hADSCs (Guo et al, 2015). Other research has shown that hypoxia reinforces proliferation and VEGF expression of hADSCs (Lee et al, 2009) suggesting that the stemness of hADSCs might be intensified via a hypoxic environment in the inner parts of MTs.…”

Differentiation of human adipose derived stem cells into Leydig‐like cells with molecular compounds

“…ADSCs were conducted the adipogenic induction at passage 2 (P2) as the reported protocols . ADSCs (1 × 10 6 cells/well) were seeded into six‐well cell culture dishes.…”

“…Flow cytometry was conducted as the reported method . Briefly, cell samples were fixed using 4% paraformaldehyde in PBS, and permeabilized using 0.1% TritonX‐100 (Sigma).…”

“…Although several researches had attempted to induce stem cells, such as induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), and mesenchymal stem cells (MSCs) into steroidogenic cells using exogenous gene transcription factor transfection, it is not so safe for further clinical application. Human adipose derived stem cells (ADSCs) have been investigated as ideal seed cells because of the ease of obtaining, low immunogenicity and higher differentiation capacity . Currently, the differentiation of ADSCs into LCs using molecular compounds but not bringing in the exogenous genes had not been reported.…”