세 종류의 수생 동물로부터 유래된 세포군들의 체외 배양기술 최적화

Abstract

최근의 동물세포 배양은 줄기세포를 이용한 치료용 세포 생산, 생리활성물질의 개발 및 생산, 천연생리활성물질의 유도체 제작 등 다양한 영역에서 활용되고 있다. 하지만 수생동물의 확립된 세포주의 대부분은 하나의 종으로부터 유래된 다른 조직의 세포이며, 일부 세포주는 짧은 기간 배양한 세포이고, 대부분의 세포주가 동결보존 가능여부를 확인하지 않은 상태이다. 따라서 본 연구에서는 수생 종의 세포 배양 기술을 최적화 시키는 것을 목표로 시베리안 철갑상어 (Acipenser baerii), 참전복 (Haliotis discus hannai), 바다송사리 (Oryzias dancena)를 대상으로 실험을 진행 하였다. 먼저, A baerii 심장 유래 세포를 분리, 배양 한 후 특성 분석을 진행 하였다. 우선 배양세포를 확보하기 위해서 조직으로부터 세포를 분리하는 방법, 초기 세포배양밀도, 기본배지 종류를 실험 조건으로 하였다. 세포분리 방법은 물리적 또는 효소적 방법을 사용하였는데, 효소적 방법을 이용하였을 때 효과적인 세포분리가 가능 하였고. 초기 세포배양밀도는 5.5 × 105 cells/cm2 이상의 밀도에서 높은 세포 생존율과 부착률을 보였고, 기본배지는 Leibovitz's L-15 기본배지에서 오랜 기간 배양 가능하였다. 배양된 세포를 대상으로 특성분석한 결과 모두 정상적인 이배체 염색체를 가지고 있었고, 두 개의 다른 형태의 세포로 특정 유전자 발현과 단백질 발현을 확인한 결과 긴 형태의 세포는 섬유아세포로 둥근 형태의 세포는 혈관내피세포로 추정되었다. 그리고 14일간 동결하여 해동 후 생존율을 확인한 결과 모든 세포군이 해동 후 안정적으로 배양 가능함을 확인 하였다. 다음으로, H. discus hannai 치설조직에서 분리한 세포로 다양한 초기배양 조건을 실험 하였다. 효소적 방법으로 세포를 분리하였을 때 9.95±2.37%의 낮은 생존율을 보였고, 초기세포밀도와 염분도 그리고 여러 성장인자를 첨가한 배지를 이용하여 여러 실험 조건으로 나누어 초기 부착률을 확인 하였을 때 성장 인자를 첨가한 35 psu의 배지에서 100% (8/8)의 초기부착과 높은 50-70% 의 부착률을 확인 하였다. 마지막으로, O. dancena 배아세포주의 동결보존 조건을 최적화 하였다. Dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), trehalose를 다양한 농도로 첨가하여 만든 동결배지로 오랜 기간 배양한 배아세포를 7일 동안 동결한 결과 10% DMSO, 20% FBS, 0.1 M trehalose의 동결배지에서 가장 높은 해동 후 세포생존율이 나타났고, 동결 전 세포와 형태적인 외형, 성장속도 모두 유사하였다. 이후 위 조건을 사용하여 57일 동안 동결한 배아세포와 7일간 동결한 다른 배치의 짧은 기간 배양된 배아세포의 해동 후 생존율을 비교하였을 때 안정적인 해동 후 생존율이 나타났고, 형태적인 외형, 성장속도 또한 유사한 결과를 보였다. 따라서 본 연구결과는 여러 수생동물로부터 세포배양 기술을 확립 하는데 있어 유용한 정보로 이용 할 수 있고, 확립된 세포는 체외 모델의 개발, 형질전환 개체의 생산, 생물학적 연구의 기초 재료와 기반기술을 제공하는데 활용될 수 있다고 예상된다.

Cell culture is one of the major techniques used in a variety of biological researches. In contrast to terrestrial mammalian species, however, cell culture system for aquatic species have been poorly established in spite of the usefulness of cultured cells from aquatic species for research and industrial purposes. Therefore, this study was conducted to optimize cell culture system for three cell populations derived from different aquatic species including Siberian sturgeon (Acipenser baerii), ablaone (Haliotis discus hannai), and marine medaka (Oryzias dancena), all of which are important experimentally or industrially. The first experiment was conducted to establish the efficient condition for stable derivation of heart-derived cell culture in Siberian sturgeon (Acipenser baerii). Three factors including isolation methods, cell densities in initial seeding, and basal media were evaluated for the derivation of heart-derived cell culture. As the results, enzymatic isolation was more efficient than mechanical isolation in both cell retrieval and further culture. Total 48 trials of culture employing low and middle cell densities of less than 5.5 × 104 cells/cm2 in initial seeding did not induce cell survivals (0%, 0/48), but the trials in high cell density of more than 5.5 × 105 cells/cm2 could induce cell survival and primary cell attachment on the plate (88.9%, 24 in 27 trials). When all initially attached cell populations were continuously cultured in two different media, only five cell populations that were enzymatically isolated and cultured under Leibovitz’s L-15 medium could grow up to more than 40th subculture. Each cell population was stably cultured according to its own growth rate and all showed normal diploid DNA contents. Two morphologically different cell types that has an elongated shape or a round shape were identified in culture, which was subsequently identified that two cell types are considered as a fibroblast (an elongated shape) and a vascular endothelial cell (a round shape) on the basis of the results of gene and protein expression analysis. Additionally, the sufficient number of viable cells could be successfully retrieved after freezing and thawing from all five cell populations suggesting the feasibility of long-term cryopreservation of the cells. The second experiment was conducted to identify various initial culture conditions for primary cell populations derived from Haliotis discus hannai radula tissue. The survival rate after cell isolation procedures using the enzymatic method was as low as 9.95±2.37%. Based on three different experimental conditions for H. discus hannai radula-derived cell culture, I found that the salinity of the media and the presence of growth-promoting factors were important to support radula-derived primary cell populations during the initial culture. The growth factor-containing media adjusted to 35 psu salinity could induce 100% (8 out of 8 trials) initial cell attachment, and the rate of cell attachment reached 50–70%. The last experiment was conducted to identify optimal medium composition for freezing Oryzias dancena embryonic cell lines. Different freezing media consisting of various concentration of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and trehalose were prepared and long-term cultured embryonic cell line was frozen in each freezing medium by conventional slow freezing program for 7 days. Through measurement of viability and growth of post-thaw cells frozen in each freezing medium, it was determined that optimal composition of three components was 10% DMSO, 20% FBS, and 0.1 M trehalose. The post-thaw cells frozen in optimal freezing medium showed similar morphology and growth rate with non-frozen cells. Next, this condition was applied to two different sets of experiment; (1) freezing of the same cells during expanded period (57 days) and (2) freezing of short-term cultured cells from other batches for 7 days. The viability of post thaw cells was significantly low and comparable in set 1 and 2, respectively, when compared with the result of long term-cultured cells frozen in optimal freezing medium for 7 days and similar morphology and growth rate with non-frozen counterparts were detected in the post-thaw cells from both sets. From this study, I established optimal culture conditions for three cell populations derived from different aquatic animals. The results suggest that species-specific condition for cell culture should be established and further optimized for development of stable in vitro system in aquatic animals. The data from this study will provide useful information for cell line development and cryopreservation of the cultured cells from various aquatic species. In addition, the cells derived from this study will contribute to development of in vitro model for basic biological studies and biotechnological application.