Journal of Animal Science and Technology

Korean Society of Animal Sciences and Technology (한국축산학회)
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Comparison between Single and Co-culture of Adipocyte and Muscle Cell Lines in Cell Morphology and Cytosolic Substances

지방과 근육 세포주의 단독 및 공동배양을 통한 세포형태학 및 세포물질 비교 연구

  • Received : 2012.02.27
  • Accepted : 2012.04.24
  • Published : 2012.04.30
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Abstract

Present study was performed to investigate the effect of single and co-culture of adipocyte and muscle cell lines on cell differentiation. 3T3-L1 (adipocyte) and L6 (muscle) cell lines were single-cultured on the condition of 10% fetal bovine serum (FBS)/Dulbeco's modified eagle's medium (DMEM) for 48 h followed by culture within 5% FBS/DMEM as a growth media. Then, the growth media was replaced by differentiation media composed of 2% FBS/DMEM without additives in single- or co-culture of the 3T3-L1 and the L6 cells to induce differentiation of both cell types. In co-culture system, the 3T3-L1 and the L6 cells were grown in separated places by being seeded on a $0.4{\mu}m$ insert membrane and on the bottom of 6 well plate, respectively. Cell differentiation was measured using morphological investigation and cytosolic analysis of glycerol-3-phosphate dehydrogenase (GPDH; for 3T3-L1) and creatine kinase (CK; for L6). Based on the GPDH results, the presence of L6 cells did not stimulate 3T3-L1 differentiation showing more differentiation of 3T3-L1 cells in the single-culture compared to the co-culture condition. In contrast, 3T3-L1 cells in the co-culture promoted differentiation of L6 cells. Enzymatic analysis supported this result showing that 3T3-L1 cells showed statistically (P<0.05) higher GPDH activity in the single-culture than the co-culture, whereas CK results of L6 cells were vice versa (P<0.05). Overall, present results may indicate that co-culture system is more reliable and precise technique compared to single-culture. Further studies on several co-culture trials including different media conditions, supplementation of differentiating substances, molecular biological analysis, etc. should be required to obtain practical and fundamental mass data.

본 연구는 기존 단독배양 위주로 이루어져온 세포배양 연구의 방법학적 한계의 극복과 대안을 제시하고자 지방과 근육세포주의 단독 및 공동배양에서 배양기법에 따른 지방 및 근육세포의 분화에 미치는 영향을 비교 조사하고자 실시하였다. 3T3-L1 (지방세포) 및 L6 (근육세포) 세포주는 성장배지인 10% FBS/DMEM (1% Pen-Strep solution 및 0.1% Fungizone 첨가) 하에서 48h 동안 단독배양 후 5% FBS/DMEM에서 배양하였다. 분화를 위한 단독 및 공동배양에서는 지방 및 근육세포 모두 분화유도물질 없이 2% FBS/DMEM으로 배양하였고, 공동배양에서는 $0.4{\mu}m$ insert membrane을 사용하여 6 well plate 하단에 L6 cell을, 상단에는 3T3-L1 cell을 공생시켰다. 지방 및 근육세포 분화정도 측정은 세포별 형태학적 측정과 glycerol-3-phosphate dehydrogenase (GPDH) 및 creatine kinase (CK) 분석을 통해 조사되었다. 형태학적으로 볼때 3T3-L1 세포주는 공동배양보다 단독배양 시 분화가 더욱 잘 일어났고 L6 세포주의 경우 역으로 같았다. 세포물질 분석에서는 분화배지 처리일(day 0)과 비교해 단독 및 공동배양 모두 지방세포 내 GPDH의 활성도가 유의적으로(P<0.05) 증가했음을 확인할 수 있었고 단독배양이 공동배양보다 유의적으로(P<0.05) 높은 수준의 GPDH 활성도를 보였다. L6 역시 마찬가지로 분화배지 처리일에 비하여 단독 및 공동배양 모두 CK 활성도가 유의적으로(P<0.05) 높았고, CK 활성도가 공동배양에서 유의적으로(P<0.05) 높게 나타남을 확인할 수 있었다. 이러한 결과는 기존 연구에서 이용된 단독 배양을 통한 세포 분화 결과 등은 생체와 비교 시 방법학적 한계로 인해 실제 생체 내에서는 그 분화정도가 매우 다를 것으로 생각되며, 이것은 앞으로 정확한 세포배양 결과 확보를 위해서는 단독배양보다는 공동배양기법을 사용해야 함을 의미한다. 향후 다양한 조건과 분화조절 물질들의 첨가를 통한 추가적인 공동배양실험이나 지방분화관련 분자생물학적 물질분석 등 다양한 실험 수행 시 보다 현실적이고 대량의 기초자료 확보가 가능할 것으로 판단된다.

Keywords

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