Abstract
3D cell culture technologies have recently shown very valuable promise for applications in regenerative medicine, but the most common 3D culture methods for mesenchymal stem cells still have limitations for clinical application, mainly due to the slowdown of inner cell proliferation and increase in cell death rate. We previously developed a new 3D culture of adipose-derived mesenchymal stem cells (ASCs) based on its self-feeder layer, which solves the two issues of ASC 3D cell culture on ultra-low attachment (ULA) surface. In this study, we compared the 3D spheroids formed on the self-feeder layer (SLF-3D ASCs) with the spheroids formed by using ULA plates (ULA-3D ASCs). We discovered that the cells of SLF-3D spheroids still have a greater proliferation ability than ULA-3D ASCs, and the volume of these spheroids increases rather than shrinks, with more viable cells in 3D spheroids compared with the ULA-3D ASCs. Furthermore, it was discovered that the SLF-3D ASCs are likely to exhibit the abovementioned unique properties due to change in the expression level of ECM-related genes, like COL3A1, MMP3, HAS1, and FN1. These results indicate that the SLF-3D spheroid is a promising way forward for clinical application.
Highlights
For 3D stem cell culture, the 3D cell environment can be manipulated to mimic what a cell experiences in vivo and provide more accurate information on cell interactions, metabolic profiling, and other cell normal physiology data; this culture has more valuable applications in stem cell research, drug discovery, tumor therapy, and regenerative medicine (Duval et al, 2017)
We further studied the possible mechanisms for the difference between self-feeder 3D (SLF-3D) and ultra-low attachment (ULA)-3D based on the expression level of extracellular matrix (ECM) genes
We developed a novel and economical self-feeding 3D spheroid (SLF-3D) culture method for adipose stem cells in which the semi-suspended spheroids are formed on adherent adipose-derived mesenchymal stem cells (ASCs) attached to plastic plates, where the adherent cells play roles in support of the semi-suspended 3D-ASC spheroids
Summary
For 3D stem cell culture, the 3D cell environment can be manipulated to mimic what a cell experiences in vivo and provide more accurate information on cell interactions, metabolic profiling, and other cell normal physiology data; this culture has more valuable applications in stem cell research, drug discovery, tumor therapy, and regenerative medicine (Duval et al, 2017). Several studies have been devised to culture 3D cell spheroids of ASCs (Di Stefano et al, 2020; Hoefner et al, 2020; Sung et al, 2020; Yin et al, 2020) In these studies, ULA3D culture has been more widely used, and in addition to its simplicity and inexpensive nature, this method has the following advantages: it is compatible with many cell lines, initiates by self-assembly, and consists of natural cells and their deposited extracellular matrix (ECM) (Vu et al, 2021). The 3D spheroids based on a self-feeder layer (SLF-3D) have been proven to have multilineage differentiation potential and “stemness” properties (Luo et al, 2021; Supplementary Figure S1); they can survive for at least 10 passages and still could proliferate This situation is very different from that of 3D cell spheroids generated by the ULA method. We hope that the self-feeder layer 3D culture can be used as a simple and effective ASC culture optimization strategy to meet clinical needs
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