Simplified culture techniques for growth and differentiation of murine and human pre-adipocytes for translational applications.
BACKGROUND
Adipose tissue has become a promising source of adult stem cells. Looking for optimal culture conditions, we evaluated the ability of L15, a free-gas exchange culture medium, to support cell proliferation and adipogenesis of murine 3T3-F442A and human normal (HNPA) and lipoma-derived (HLPA) pre-adipocytes.


METHODS
3T3-F442A, HNPA and HLPA cell proliferation were compared in short-term cultures and along multiple passages in Dulbecco's modified Eagle medium (DMEM) or DMEM-F12 under a 5% CO(2) atmosphere or L15 medium under a free-gas exchange atmosphere. Adipogenesis in these cells was evaluated by quantifying lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity, and by assaying the expression of adipogenic markers by reverse transcriptase-polymerase chain reaction (RT-PCR).


RESULTS
3T3 pre-adipocytes grew at similar rates in serum-supplemented L15 or DMEM, but L15 induced higher colony-forming efficiency in these cells. HNPA and HLPA grew more actively in L15 than in DMEM-F12 for more than 10 successive passages and reached higher colony-forming efficiency in L15 medium. On the other hand, while high-glucose DMEM and L15 supplemented with glucose 1 g/L induced similar levels of 3T3 adipogenesis, L15 with no added glucose increased HNPA and HLPA adipogenesis with respect to DMEM-F12, as measured by lipid accumulation, GPDH activity and expression of adipogenic markers C/EBPalpha, GLUT-4, LPL and aP2.


CONCLUSIONS
The free-gas exchange medium L15 supports cell proliferation and adipogenesis of murine 3T3 and normal and lipoma-derived human subcutaneous pre-adipocytes to a greater extent than DMEM or DMEM-F12. The routine use of L15 will optimize translational applications of adipose cells.
Herrera-Herrera
M L
ML
División de Biología Molecular, Instituto Potosino de Investigación Cientifica y Tecnológica, San Luis Potosí, México.
Zapata-Bustos
R
R
Salazar-Olivo
L A
LA
D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000502:physiology*; D050156:Adipogenesis / Q000187:drug effects / Q000502:physiology*; D000273:Adipose Tissue / Q000166:cytology*; D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D003470:Culture Media / Q000494:pharmacology; D046508:Culture Techniques; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008067:Lipoma; D051379:Mice; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*
D003470:Culture Media