Comparison of the formation, adipogenic maturation, and retention of human adipose‐derived stem cell spheroids in scaffold‐free culture techniques

Abstract

While three‐dimensional spheroids outperform traditional two‐dimensional monolayer culture for human adipose‐derived stem cells (hASCs), there is not a consensus on the most successful method for enhancing their adipogenic differentiation and minimizing the loss of physiologically relevant, fatty spheroids during culture. To this end, we compared three culture methods, namely, elastin‐like polypeptide‐polyethyleneimine (ELP‐PEI) coated surfaces, ultra‐low attachment static culture, and suspension culture for their ability to form and retain productive hASC spheroids. The ELP‐PEI coatings used the ELP conjugated to two molecular weights of PEI (800 or 25,000 g/mol). FTIR spectroscopy, atomic force microscopy, and contact angle goniometry revealed that the ELP‐PEI coatings had similar chemical structures, surface topography, and hydrophobicity. Time‐lapse microscopy showed that increasing the PEI molecular weight resulted in smaller spheroids. Measurement of triglyceride content showed that the three methods induced comparable differentiation of hASCs toward the adipogenic lineage. DNA content and morphometric analysis revealed merging of spheroids to form larger spheroids in the ultra‐low attachment static culture and suspension culture methods. In contrast, the retention of hASC spheroid sizes and numbers with a regular spheroid size (~100 μm) were best atop the ELP‐PEI800 coatings. Overall, this research shows that the spheroid culture atop the ELP‐PEI coatings is a suitable cell culture model for future studies involving long‐term, three‐dimensional culture of mature adipocytes derived from hASCs.

Elastin-Collagen Based Hydrogels as Model Scaffolds to Induce Three-Dimensional Adipocyte Culture from Adipose Derived Stem Cells

“…In addition to the scaffold mechanical properties, chemical composition of the scaffold materials and the porosity of the scaffolds also affect the adipogenic differentiation of hASCs. We and others have previously shown that changing the surface amine content can affect the size of the hASC spheroids, the stability of the spheroids over the culture period, and their differentiation capability [ 28 , 29 , 30 ]. While the chemistry experienced by the hASCs in the present study is different for the collagen-only (2C and 6C) scaffolds, the ELP containing (2C+E and 2C+E_c) scaffolds, and the crosslinked (2C_c, 2C+E_c, and 6C_c) scaffolds, our scaffold formation process did not introduce any additional amine groups into our scaffolds.…”

“…The effects of hydrogel scaffold chemistry, structure (e.g., porosity), and properties (e.g., modulus) on the ultimate function of the encapsulated cells remains an active area of research. Presence of positively charged amine groups have been shown to induce hASC spheroid formation, which led to an enhanced differentiation along adipogenic and osteogenic lineages [ 28 , 29 , 30 ]. Porosity and pore size also play an important role in supporting adipogenic differentiation of hASCs [ 31 , 32 ].…”

Human Adipose-Derived Stromal/Stem Cell Culture and Analysis Methods for Adipose Tissue Modeling In Vitro: A Systematic Review

“…Finally, none of the screened articles exclusively studied 2D culture; therefore, all articles included in this analysis report 3D culture methods. Data and analysis hereafter exclusively refer to the 27 articles [4,5,[8][9][10][11][12][13][15][16][17][19][20][21][22][23][24][25][26]28,30,31,33,38,44,45,48] identified to be most relevant to the central goal of this systematic review. Out of the 27 relevant articles, 26 articles used adipogenic medium to differentiate hASC into adipocytes in vitro.…”

“…Gelatin was identified as one of the most commonly used scaffold materials as it naturally contains RGD peptides found in many ECM components that promote cell adhesion [13,16,17,21]. Collagen is another commonly used scaffold material as it is the most abundant protein in human adipose tissue ECM and, therefore, closely recapitulates the native tissue environment [10,31]. hASC attachment and adipocyte maturation have been shown to impact scaffold structure [10,16].…”

“…Collagen is another commonly used scaffold material as it is the most abundant protein in human adipose tissue ECM and, therefore, closely recapitulates the native tissue environment [10,31]. hASC attachment and adipocyte maturation have been shown to impact scaffold structure [10,16]. Additionally, scaffold material and properties have been shown to impact hASC adipogenesis [16].…”