초록
최근 단일클론항체의 상업적 이응가치가 증대하면서 그 수요량 또한 급격히 증가, 이를 뒷받침할 수 있는 대량생산 기술이 요구되고 있다. 본 연구에서는 쥐 하이브리도마 Alps 25-3의 대량배양을 위해 우선적으로 무혈청 배지 (serum-free media)의 개발을 시도하였다. 동물세포 배양액 IMDM과 Ham's F-12를 1:1로 혼합한 배지를 기본으로하여 ITES 용액 (insulin 10$\mu\textrm{g}$/$m\ell$, transferrin 10$\mu\textrm{g}$/$m\ell$, ethanolamine 10$\mu$M and selenium 30nM)을 첨 가하여 일차적으로 결정한 EBM(enriched basal medium)에 steroid hormones, vltamins, lipid, mineral 및 여러성분들을 각각 첨가하여 혈청과 대치될 수 있는 성분들의 최적농도를 조사하였다. 각 성분들의 개별효과와 함께 그들을 혼합하였을 때의 상호효과를 통해 혈청배지와 거의 동일한 성장을 보이는 무혈청배지 KM3(EBM with BSA 100$\mu\textrm{g}$/$m\ell$, mineral solution and 0.05% PEG)를 결정할 수 있었으며 이 배지에서의 최고 항체 생성량은 혈청배지의 약 80% 로 나타났다. 또한 무혈청 배지에서 여러 cell line의 성장과 그들의 항체 생성을 조사함으로써 다른 쥐 하이브리도마 세포배양에도 적응시킬 수 있음을 확인할 수 있었다.
A serum-free medium that could be used for the large-scale culture of mouse hybridoma to produce monoclonal antibodies was developed. The medium was based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's F-12, supplemented with insulin 10$\mu\textrm{g}$/$m\ell$, transferrin 10$\mu\textrm{g}$/$m\ell$, ethanolamine 10$\mu$M and selenium 30nM (designated EBM (enriched basal medium) with the supplements). The effect of various supplements of steroid hormones, vitamins, lipid and mineral salts was investigated and their optimal concentration was determined to replace fetal calf serum (PCS). These components were added respectively and then added by way of two or three combination to discern of which component combination was effective to the culture of hybridoma. As a result, serum-free medium KM3 (EBM with BSA 100$\mu\textrm{g}$/$m\ell$, mineral cocktail and 0.05% PEG) was deter-mined. The hybridoma Alps 25-3 cultured in this medium showed almost the same growth rate as in medium added with 2% fetal bovine serum. However, the antibody concentration from KM3 cultures was 80% of that obtained from culture with FCS. KM3 was also examined for the culture of other mouse hybridomas, KW, A4W & HCGK, and it was confirmed that it could support the growth of these hybridomas and the production of monoclonal antibodies.
