Herrera-Herrera M L, Zapata-Bustos R, Salazar-Olivo L A
División de Biología Molecular, Instituto Potosino de Investigación Cientifica y Tecnológica, San Luis Potosí, México.
Cytotherapy. 2009;11(1):52-60. doi: 10.1080/14653240802495963.
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.
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).
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.
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.
脂肪组织已成为成体干细胞的一个有前景的来源。为寻找最佳培养条件，我们评估了一种无气体交换的培养基L15支持小鼠3T3-F442A细胞、人正常前脂肪细胞（HNPA）和脂肪瘤来源的前脂肪细胞（HLPA）增殖及脂肪生成的能力。
比较了3T3-F442A、HNPA和HLPA细胞在短期培养中以及在含5%二氧化碳气氛的杜氏改良 Eagle培养基（DMEM）或DMEM-F12中，或在无气体交换气氛的L15培养基中多次传代培养时的增殖情况。通过定量脂质积累和甘油-3-磷酸脱氢酶（GPDH）活性，并通过逆转录聚合酶链反应（RT-PCR）检测脂肪生成标志物的表达，来评估这些细胞的脂肪生成。
在添加血清的L15或DMEM中，3T3前脂肪细胞以相似的速率生长，但L15诱导这些细胞产生更高的集落形成效率。HNPA和HLPA在L15中比在DMEM-F12中连续10代以上生长更活跃，且在L15培养基中达到更高的集落形成效率。另一方面，虽然高糖DMEM和添加1 g/L葡萄糖的L15诱导3T3细胞产生相似水平的脂肪生成，但通过脂质积累、GPDH活性以及脂肪生成标志物C/EBPα、GLUT-4、LPL和aP2的表达测定发现，不添加葡萄糖的L15相对于DMEM-F12增加了HNPA和HLPA的脂肪生成。
无气体交换培养基L15比DMEM或DMEM-F12在更大程度上支持小鼠3T3细胞以及人正常和脂肪瘤来源的皮下前脂肪细胞的增殖和脂肪生成。常规使用L15将优化脂肪细胞的转化应用。
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