Minamoto Y, Ogawa K, Abe H, Iochi Y, Mitsugi K
Central Research Laboratories, Ajinomoto Co., Kawasaki, Japan.
Cytotechnology. 1991;5 Suppl 2:S35-51.
We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium. As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37 degrees C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640 + 10% FBS. It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively. Insulin has also proved to be heat stable in a solution of Fe-gluconate. We thus established a new serum-free medium, all the components of which could be heat-sterilizable. Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast. Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line. The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin. The cell density reached as high as 2 x 10(8)/ml in the serum-free medium. Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day. The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.
我们试图基于我们的无血清培养基建立一种新的无血清且可热灭菌的培养基，在该无血清培养基中，许多淋巴母细胞和杂交瘤能够生长，其生长情况与在传统含血清培养基中一样良好。众所周知，L-谷氨酰胺（L-Gln）是细胞生长最不耐热但又必不可少的成分之一。作为L-Gln的替代品，发现二肽如甘氨酰-L-谷氨酰胺或L-丙氨酰-L-谷氨酰胺即使在高压灭菌后也相当稳定，可用于哺乳动物细胞生长。含L-Gln二肽的无血清培养基即使在37℃储存4个月后在溶液中仍相当稳定。在含L-丙氨酰-L-谷氨酰胺的无血清培养基中，小鼠杂交瘤能够生长并产生比在RPMI 1640 + 10%胎牛血清中更多的抗体。已证明，同样不耐热但对各种细胞系生长必不可少的牛血清白蛋白（BSA）和转铁蛋白，可分别被热稳定的α-环糊精和胆固醇以及葡萄糖酸铁替代。胰岛素在葡萄糖酸铁溶液中也被证明是热稳定的。因此，我们建立了一种新的无血清培养基，其所有成分都可进行热灭菌。此外，通过添加表皮生长因子（EGF）和BSA，但不包含胎牛血清中的黏附因子，发现该无血清培养基能够支持人二倍体成纤维细胞的长期连续培养。最后，使用这种可高压灭菌的无血清培养基在灌注培养装置中，我们能够连续培养人淋巴母细胞系。通过添加富含葡萄糖、碳酸氢盐、L-半胱氨酸和抑肽酶的无血清培养基，发现IgM的产生速率显著提高。在无血清培养基中细胞密度高达2×10⁸/ml。尽管反应器中的工作体积仅为1升，但IgM的产生速率达到480毫克/天。这种新的可热灭菌无血清培养基有几个优点，因为L-Gln肽是L-Gln的热稳定且可用的前体。
