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Chemically Defined Xeno- and Serum-Free Cell Culture Medium to Grow Human Adipose Stem Cells.

ABSTRACT: 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.

SUBMITTER: Panella S

PROVIDER: S-EPMC7926673 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Chemically Defined Xeno- and Serum-Free Cell Culture Medium to Grow Human Adipose Stem Cells.

Panella Stefano S Muoio Francesco F Jossen Valentin V Harder Yves Y Eibl-Schindler Regine R Tallone Tiziano T

Cells 20210222 2

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 (<i>UrSuppe</i>), specifically formulated to grow human adipose stem cells (hASCs ...[more]

PMID: 33671568

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Project description:Cell therapy is witnessing a notable shift toward cell-free treatments based on paracrine factors, in particular, towards small extracellular vesicles (sEV), that mimic the functional effect of the parental cells. While numerous sEV-based applications are currently in advanced preclinical stages, their promised translation depends on overcoming the manufacturing hurdles posed by the large-scale production of purified sEV. Unquestionably, the culture medium used with the parental cells plays a key role in the sEV's secretion rate and content. An essential requisite is the use of a serum-, xeno-, and blood-free medium to meet the regulatory entity requirements of clinical-grade sEV's production. Here, we evaluated OxiumTMEXO, a regulatory complying medium, with respect to production capacity and conservation of the EV's characteristics and functionality and the parental cell's phenotype and viability. A comparative study was established with standard DMEM and a commercially available culture medium developed specifically for sEV production. Under similar conditions, OxiumTMEXO displayed a three-fold increase of sEV secretion, with an enrichment of particles ranging between 51 and 200 nm. These results were obtained through direct quantification from the conditioned medium to avoid the isolation method's interference and variability and were compared to the two culture media under evaluation. The higher yield obtained was consistent with several harvest time points (2, 4, and 6 days) and different cell sources, incluiding umbilical cord-, menstrual blood-derived mesenchymal stromal cells and fibroblasts. Additionally, the stem cell phenotype and viability of the parental cell remained unchanged. Furthermore, OxiumTMEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The in vitro internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV in vivo indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumTMEXO medium sustains an efficient and robust production of large quantities of sEV, conserving the classic functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of EVs for the development of cell-free therapies.

Dataset Information

Chemically Defined Xeno- and Serum-Free Cell Culture Medium to Grow Human Adipose Stem Cells.

Publications

Chemically Defined Xeno- and Serum-Free Cell Culture Medium to Grow Human Adipose Stem Cells.

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