Low serum and serum-free culture of multipotential human adipose stem cells

Adipose tissue is an abundant source of stem cells. However, liposuction cannot yield cell quantities sufficient for direct applications in regenerative medicine. Therefore, the development of GMP-compliant ex vivo expansion protocols is required to ensure the production of a “cell drug” that is safe, reproducible, and cost-effective. Thus, we developed our own basal defined xeno- and serum-free cell culture medium (UrSuppe), specifically formulated to grow human adipose stem cells (hASCs). With this medium, we can directly culture the stromal vascular fraction (SVF) cells in defined cell culture conditions to obtain hASCs. Cells proliferate while remaining undifferentiated, as shown by Flow Cytometry (FACS), Quantitative Reverse Transcription PCR (RT-qPCR) assays, and their secretion products. Using the UrSuppe cell culture medium, maximum cell densities between 0.51 and 0.80 × 105 cells/cm2 (=2.55–4.00 × 105 cells/mL) were obtained. As the expansion of hASCs represents only the first step in a cell therapeutic protocol or further basic research studies, we formulated two chemically defined media to differentiate the expanded hASCs in white or beige/brown adipocytes. These new media could help translate research projects into the clinical application of hASCs and study ex vivo the biology in healthy and dysfunctional states of adipocytes and their precursors. Following the cell culture system developers’ practice and obvious reasons related to the formulas’ patentability, the defined media’s composition will not be disclosed in this study.

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Development of a Biodegradable Microcarrier for the Cultivation of Human Adipose Stem Cells (hASCs) with a Defined Xeno- and Serum-Free Medium

Applied Sciences ◽

10.3390/app11030925 ◽

2021 ◽

Vol 11 (3) ◽

pp. 925

Author(s):

Francesco Muoio ◽

Stefano Panella ◽

Matias Lindner ◽

Valentin Jossen ◽

Yves Harder ◽

...

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Culture Medium ◽

Free Cell ◽

Adipose Stem Cells ◽

Cell Culture Medium ◽

Serum Free Medium ◽

Free Medium ◽

Serum Free ◽

Human Adipose Stem Cells

Stirred single-use bioreactors in combination with microcarriers (MCs) have established themselves as a technology that has the potential to meet the demands of current and future cell therapeutic markets. However, most of the published processes have been performed using fetal bovine serum (FBS) containing cell culture medium and non-biocompatible MCs. This approach has two significant drawbacks: firstly, the inevitable potential risks associated with the use of FBS for clinical applications; secondly, non-biocompatible MCs have to be removed from the cell suspension before implantation, requiring a step that causes loss of viable cells and adds further costs and complications. This study aimed to develop a new platform based on a chemically defined xeno- and serum-free cell culture medium and biodegradable MC that can support the growth of human adipose stem cells (hASCs) while still preserving their undifferentiated status. A specific combination of components and manufacturing parameters resulted in a MC prototype, called “BR44”, which delivered the desired functionality. MC BR44 allows the hASCs to stick to its surface and grow in a chemically defined xeno- and serum-free medium (UrSuppe). Although the cells’ expansion rate was not as high as with a commercial non-biodegradable standard MC, those cultured on BR44 maintained a better undifferentiated status in both static and dynamic conditions than those cultured on traditional 2D surfaces.

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Human Adipose Stem Cells (hASCs) Grown on Biodegradable Microcarriers in Serum- and Xeno-Free Medium Preserve Their Undifferentiated Status

Journal of Functional Biomaterials ◽

10.3390/jfb12020025 ◽

2021 ◽

Vol 12 (2) ◽

pp. 25

Author(s):

Francesco Muoio ◽

Stefano Panella ◽

Valentin Jossen ◽

Matias Lindner ◽

Yves Harder ◽

...

Keyword(s):

Stem Cells ◽

Adipose Stem Cells ◽

Marker Genes ◽

Free Medium ◽

Serum Free ◽

Cell Densities ◽

Short Time ◽

Serum Free Conditions ◽

Human Adipose Stem Cells

Human adipose stem cells (hASCs) are promising candidates for cell-based therapies, but they need to be efficiently expanded in vitro as they cannot be harvested in sufficient quantities. Recently, dynamic bioreactor systems operated with microcarriers achieved considerable high cell densities. Thus, they are a viable alternative to static planar cultivation systems to obtain high numbers of clinical-grade hASCs. Nevertheless, the production of considerable biomass in a short time must not be achieved to the detriment of the cells’ quality. To facilitate the scalable expansion of hASC, we have developed a new serum- and xeno-free medium (UrSuppe) and a biodegradable microcarrier (BR44). In this study, we investigated whether the culture of hASCs in defined serum-free conditions on microcarriers (3D) or on planar (2D) cell culture vessels may influence the expression of some marker genes linked with the immature degree or the differentiated status of the cells. Furthermore, we investigated whether the biomaterials, which form our biodegradable MCs, may affect cell behavior and differentiation. The results confirmed that the quality and the undifferentiated status of the hASCs are very well preserved when they grow on BR44 MCs in defined serum-free conditions. Indeed, the ASCs showed a gene expression profile more compatible with an undifferentiated status than the same cells grown under standard planar conditions.

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Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation

Journal of Plastic Reconstructive & Aesthetic Surgery ◽

10.1016/j.bjps.2007.01.045 ◽

2007 ◽

Vol 60 (4) ◽

pp. S11

Author(s):

K. Hemmrich ◽

M. Wosnitza ◽

A. Groger ◽

S. Gräber ◽

N. Pallua

Keyword(s):

Stem Cells ◽

Adipose Stem Cells ◽

Endothelial Differentiation ◽

Human Adipose Stem Cells

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Microvesicles from human adipose stem cells promote wound healing by optimizing cellular functions via AKT and ERK signaling pathways

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1152-x ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 34

Author(s):

Sen Ren ◽

Jing Chen ◽

Dominik Duscher ◽

Yutian Liu ◽

Guojun Guo ◽

...

Keyword(s):

Stem Cells ◽

Wound Healing ◽

Signaling Pathways ◽

Adipose Stem Cells ◽

Erk Signaling ◽

Cellular Functions ◽

Promote Wound Healing ◽

Human Adipose Stem Cells

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Modulation of Human Adipose Stem Cells’ Neurotrophic Capacity Using a Variety of Growth Factors for Neural Tissue Engineering Applications: Axonal Growth, Transcriptional, and Phosphoproteomic Analyses In Vitro

Cells ◽

10.3390/cells9091939 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1939

Author(s):

Katharina M. Prautsch ◽

Alexander Schmidt ◽

Viola Paradiso ◽

Dirk J. Schaefer ◽

Raphael Guzman ◽

...

Keyword(s):

Stem Cells ◽

Growth Factors ◽

Neurotrophic Factor ◽

Axonal Growth ◽

Cellular Level ◽

Adipose Stem Cells ◽

Transcriptional Analysis ◽

Axonal Outgrowth ◽

Human Adipose Stem Cells

We report on a potential strategy involving the exogenous neurotrophic factors (NTF) for enhancing the neurotrophic capacity of human adipose stem cells (ASC) in vitro. For this, ASC were stimulated for three days using NTF, i.e., nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), NT4, glial cell-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF). The resulting conditioned medium (CM) as well as individual NTF exhibited distinct effects on axonal outgrowth from dorsal root ganglion (DRG) explants. In particular, CM derived from NT3-stimulated ASC (CM-NT3-ASC) promoted robust axonal outgrowth. Subsequent transcriptional analysis of DRG cultures in response to CM-NT3-ASC displayed significant upregulation of STAT-3 and GAP-43. In addition, phosphoproteomic analysis of NT3-stimulated ASC revealed significant changes in the phosphorylation state of different proteins that are involved in cytokine release, growth factors signaling, stem cell maintenance, and differentiation. Furthermore, DRG cultures treated with CM-NT3-ASC exhibited significant changes in the phosphorylation levels of proteins involved in tubulin and actin cytoskeletal pathways, which are crucial for axonal growth and elongation. Thus, the results obtained at the transcriptional, proteomic, and cellular level reveal significant changes in the neurotrophic capacity of ASC following NT3 stimulation and provide new options for improving the axonal growth-promoting potential of ASC in vitro.

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