Expansion of functional personalized cells with specific transgene combinations

Abstract

Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

“…Cells of the immortalised mesenchymal stromal cell line N-KM (normal bone marrow) [41] were cultured in 6- or 24-well plates in 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin and 100 U/mL glutamine (Life Technologies) supplemented IMDM (Lonza, Cologne, Germany) until they reached approximately 70% confluency.…”

Generation and characterization of a functional human adipose‐derived multipotent mesenchymal stromal cell line

“…With regard to the establishment of cell culture models, it is most important to obtain cells with a physiological and functional phenotype which should be as close as possible to primary cells. In this line, a functionality of immortalized cells displaying similar sets of integrated transgenes has already been demonstrated for different cell types, including human alveolar epithelial cells and murine hepatocytes (Kuehn et al, ; Lipps et al, ). Furthermore, MSC immortalization is increasingly performed using combinations of different factors to improve the characteristics of the resulting cell lines and overcome limitations of hTERT‐immortalized MSC (Liu et al, ; Tang et al, ; Tátrai et al, ).…”

“…The technique for MSC immortalization used in the current study was recently described as a promising approach widely applicable to obtain expandable and functional cells from diverse tissue origins (Lipps et al, ). Here, we describe its applicability to human adipose‐derived MSC.…”

“…The cells were allowed to adapt to culture conditions for 48 hr. Then, they were randomly transduced overnight with a gene library composed of 33 different expansion genes, as described previously (Lipps et al, ). Transduction of the primary cells was done in the presence of polybrene (8 µg/ml; Sigma‐Aldrich).…”

“…For the identification of the genes that were integrated in the selected MSC clone (K5 iMSC) and thus facilitated immortalization, a PCR protocol was used, as previously described (Lipps et al, ). Briefly, the genomic DNA of the cell line was isolated and for each expansion gene from the gene library, an independent PCR reaction was done.…”