냉동 보관방법에 따른 지방세포의 생존

Abstract

자가 지방이식술은 성형외과에서 보편화된 수술로 자리 잡았으며, 임상에서 점점 더 많이 이용되고 있다. 그러나 이식된 지방의 생존과 보관방법 그리고 성체줄기세포로서의 이용가능성 등 많은 의문점은 아직 풀리고 않고 있다. 이 연구의 목적은 지방흡입으로 복부에서 채취한 지방조직을 -20 ℃와 -70 ℃에서 보관하였을 때 시간에 지남에 따라 지방세포의 생존에 어떠한 차이가 있는지를 살펴보는 것이다. 성인 여성 16명(23-53세)을 대상으로 복부지방흡입수술을 시행하여 채취한 300-500 mL의 흡입지방을 이용하였다. 3000 rpm에서 2분 동안 원심분리하여 지방층을 분리한 후 각각의 시료를 -20 ℃와 -70 ℃ 냉장고에 보관한 다음 1일, 3일, 1주, 2주, 4주, 8주에 꺼내어 지방세포의 생존을 조사하였다. 보관하지 않은 신선한 지방을 대조군으로 하였다. 보관 중인 시료를 꺼내어 상온에서 녹인 다음, type I collagenase로 처리하고, incubation한 후 동일 부피의 10% FBS로 collagen 분해반응을 중지 시켰다. 200 ㎛ strainer로 걸러내고 5분 동안 gravity separation 후 상층액을 취하여 12 μM fluorescein diacetate와 1 ㎕/mL propidium iodide로 염색하여 살아 있는 성숙한 지방세포의 수를 형광현미경하에서 측정하였다. Digestion하기 전의 흡인지방(lipoaspirates)에서 GPDH의 활성도 값을 측정하고, XTT reduction assay를 시행하였다. 분리한 성숙한 지방세포와 지방전구세포에서도 XTT reduction assay를 시행하였다. 실험 결과 성숙한 지방세포의 생존율을 형광염색으로 분석하였을 때, 1일째부터 -20 ℃와 -70 ℃에서 각각 13.3 ± 7.4%, 12.6 ± 6.3%로 매우 낮은 생존율을 보였고, 두 집단 사이에 통계적 유의성은 없었으며, 8주에는 생존한 세포가 거의 없었다. GPDH 분석을 통한 세포 안정성 평가는 모든 시간조건에서 -20 ℃군보다 -70 ℃군이 높았고, 3일과 7일에 통계적 유의성이 있었으며(p < 0.05), 8주에는 각각 16.1 ± 4.0%, 18.8 ± 3.1%를 유지하였다. XTT 분석을 통한 물질대사활성도 측정결과, 지방조직은 1일째에 두 집단이 각각 30.0 ± 10.9%, 36.1 ± 12.3%로, 모두 낮은 활성도를 보였고, 7일에 두 집단간에 통계적 유의성이 있었으며(p < 0.05), 8주에는 활성도가 거의 없었다. 또한 지방세포와 지방전구세포는 1일째부터 낮은 활성도(15.4 ± 7.2/11.5 ± 5.6%), (8.0 ± 6.0/8.6 ± 3.8%)를 보였고, 8주에는 활성도가 거의 없었다. 결론적으로 지방세포는 -20 ℃와 -70 ℃에서 모두 1일째부터 생존율이 급격히 저하되었고, 이 후 시간이 지남에 따라 서서히 저하되어 8주에는 약 5%정도만 생존하였다. 이러한 결과는 기존의 냉동보관방법으로는 지방의 생존을 원하는 기간동안 유지하기가 어렵다는 것을 나타내는 것이며, 생존율을 높이기 위해서는 새로운 방법이 모색되어야 할 것으로 생각된다. The use of an autogenous fat graft has becomea common procedure in plastic surgery and in the clinic. However, questions remain concerning the surgical technique and on the viability of fat cells and preservation method of fat cells. It remains to be determined if it is possible to use fat cells as adult stem cells. The purpose of this study wasto examine the differences of viability of fat cells with time for fat tissues stored at -20 ℃ and -70 ℃ after harvest from abdominal liposuction. Sixteen female adults (aged 23 to 53 years old) were recruited for this study. Harvested aspirated fat tissues (300 - 500 mL) were obtained by abdominal liposuction. The fat layer was isolated by centrifugation of fat tissues at 3000 rpm for 2 minutes and the viability of fat cells in specimens were measured after freezing for one day, three days, one week, two weeks, four weeks and eight weeks in deep-freezers. A control group consisted of fresh specimens that were not frozen. After thawing, the fat cells at room temperature were treated with type I collagenase and the collagen degradation reaction was stopped with 10% FBS with an equal volume following incubation. The treated specimens were strained with a 200 μm strainer and the supernatant was collected after gravity separation for 5 minutes. The number of viable adult lipocytes were measured on a fluorescence microscope after staining with 12 μM fluorescein diacetate and 1 μl/mL propidium iodide. GPDH activity was measured in lipoaspirates before digestion and the XTT reduction assay was performed. For isolated lipocytes and preadipocytes, the XTT reduction assay was also performed. When the viability of adipocytes was analyzed with fluorescence staining, viability was very low for both the -20 ℃ and -70 ℃ samples from the day of storage recording (13.3 ± 7.4% and 12.6 ± 6.3%, respectively). No statistical significance between the samples stored at the two freezer temperatures was seen; there were few viable cells after eight weeks. For stability as measured with GPDH activity, samples stored at -70 ℃ had a higher activity than samples stored at -20 ℃ for all times; the difference in activity was statistically significant after storage for three and seven days (p < 0.05). The GDPH activity for specimens frozen at -20 ℃ and -70 ℃ was 16.1 ± 4.0% and 18.8 ± 3.1% for 8 weeks, respectively. When the metabolic activity was evaluated with use of the XTT assay, lipoaspirates had a low activity for samples frozen at -20 ℃ and -70 ℃ of 30.0 ± 10.9% and 36.1 ± 12.3%, respectively; the difference in activity after seven days was statistically significant (p < 0.05). There was low activity after storage for eightweeks. In addition, adipocytes had a low activity from day one of 15.4 ± 7.2% for samples frozen at -20 ℃ and 11.5 ± 5.6% for samples frozen at -70 ℃. Preadipocytes had a low activity of 8.0 ± 6.0% for samples frozen at -20 ℃ and 8.6 ± 3.8% for samples frozen at -70 ℃. Activity for samples frozen after eight weeks was very low. In conclusion, viability of adipocytes declined rapidly after frozen storage for a day at both -20 ℃ and -70 ℃, and activity decreased gradually for storage after 8 weeks where approximately 5% of the cells were alive. These findings suggest that the present fat preservation storage method using a -20 ℃ freezer is inadequate to maintain viability during the expected period, and a new method should be sought.