A novel technique to propagate primary human preadipocytes without loss of differentiation capacity.
OBJECTIVE
The study of human preadipocytes is hampered by the limited availability of adipose tissue and low yield of cell preparation. Proliferation of preadipocytes using common protocols, including fetal bovine serum (FBS), results in a markedly reduced differentiation capacity. Therefore, we were interested in developing an improved culture system that allows the proliferation of human preadipocytes without loss of differentiation capacity.


METHODS
Adipose tissue samples were taken from subjects undergoing elective abdominal surgery. Cells were seeded at various densities and cultured using different formulations of proliferation media including factors such as fibroblast growth factor-2 (basic fibroblast growth factor), epidermal growth factor, insulin, and FBS either alone or in combination. Cells were counted and induced to differentiate after confluence. After complete differentiation, cells were harvested, and glycerol-3-phosphate dehydrogenase activity was measured. Cells were subcultured for up to five passages.


RESULTS
The proliferation medium with 4 growth factors (PM4), consisting of 2.5% FBS, 10 ng/mL epidermal growth factor, 1 ng/mL basic fibroblast growth factor, and 8.7 muM insulin, resulted in lower doubling times at all seeding densities tested (0.05 x 10(4) to 1.5 x 10(4)) compared with medium supplemented with 10% FBS. In contrast to cells in FBS medium, cells grown with PM4 medium retained full differentiation rate (glycerol-3-phosphate dehydrogenase activity, 493 +/- 215 vs. 41 +/- 17 mU/mg, p < 0.01). Differentiation capacity was fully retained at least for up to three passages in PM4 medium.


CONCLUSIONS
The use of PM4 medium results in substantial proliferation of human preadipocytes with preserved differentiation capacity. This novel technique represents a valuable tool for the study of human adipose tissue development and function starting from small samples.
Skurk
Thomas
T
Technische Universität München, Else Kröner-Fresenius-Centre for Nutritional Medicine, Am Forum 5, D-85350 Freising-Weihenstephan, Germany. nutritional.medicine@wzw.tum.de
Ecklebe
Silke
S
Hauner
Hans
H
D017667:Adipocytes / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D000328:Adult; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D008875:Middle Aged; D008934:Mitogens / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013997:Time Factors
D003470:Culture Media; D007328:Insulin; D008934:Mitogens; D016222:Fibroblast Growth Factor 2; D012710:Serum Albumin, Bovine; D004815:Epidermal Growth Factor