ホルモン産生ヒト下垂体腺腫細胞の長期培養およびホルモン分泌維持のための工夫

Inventions for Preservation of Hormonal Function in Long-Term Culture of Human Functioning Pituitary Adenoma

　ホルモン分泌能を維持しながらヒト下垂体腺腫細胞の長期培養が可能な新しいin vitroの実験系を得る目的で，イオン・蛋白質・その他高分子を自由に透過させる膜を底部に張った培養カップ（ミリセル—CM）を用いてGHおよびPRL産生下垂体腺腫細胞の培養を試みた。手術時摘出した腺腫細胞を一旦従来のディッシュに播種して数日間培養後，機械的に剥離させ，大小の細胞集合体を含む浮遊液を基底膜成分でコートしたミリセル—CMに入れて4〜6ヵ月間培養し，経時的に培地のGHとPRL値を測定し，光顕と電顕更に免疫組織学的検討も行った。培地の量を調整する事により，細胞集合体を含めて播種した全ての細胞を膜に付着させる事が可能で，6ヵ月後でもホルモン分泌能が維持され，超微構造的にも細胞内小器官の発達も良好で，多数の分泌顆粒も認められた。特に2ヵ月頃までは培養開始時に近い状態が保たれており，この培養方法はin vitroの実験系として有用と思われた。

This study was designed to establish in vitro model systems in human hormone-producing pitui-tary adenomas that are analogous to the in vivo cellular environment.

Mechanically dispersed cells composed of single cells and aggregates from 6 pituitary adenomas (3 GH producing adenomas and 3 prolactinomas) were cultured on microporous membrane cell culture inserts (Millicell-CM) coated with Basement Mem-brane Matrigel for up to 6 months. Growth hormone or prolactin in the medium was measured during the culture, and morphological feature in vitro was also compared with that of the original tumor at inter-vals.

Not only single cells but also large aggregated cells which usually float in the medium when seeded on conventional plastic, were flattened and firmly attached to coated microporous membrane under the control of medium volume in culture. In both type adenomas, especially prolactinomas, surviving aggregated adenoma cells revealed preserved hor-mone activity and no dedifferentiation of cell cha-racteristics after 6 months in culture. Particularly during the first 2 months in culture, close similarity exsisted between in vivo and in vitro conditions with regard to cell morphology and hormone release.

These results indicate that this new culture method may further aid the investigation of in vitro cellular structure and function in human pituitary adenomas under conditions which closely mimic the in vivo cellular enviroment.