Okonkowski Jessica, Balasubramanian Uma, Seamans Craig, Fries Serena, Zhang Jinyou, Salmon Peter, Robinson David, Chartrain Michel
Merck Research Laboratories, Bioprocess R&D, PO Box 2000, RY80Y-105, Rahway, NJ 07065, USA.
J Biosci Bioeng. 2007 Jan;103(1):50-9. doi: 10.1263/jbb.103.50.
We report the successful cultivation of cholesterol dependent NS0 cells in linear low-density polyethylene (LLDPE) Wave Bioreactors when employing a low ratio of cyclodextrin to cholesterol additive mixture. While cultivation of NS0 cells in Wave Bioreactors was successful when using a culture medium supplemented with fetal bovine serum (FBS), cultivation with the same culture medium supplemented with cholesterol-lipid concentrate (CLC), which contains lipids and synthetic cholesterol coupled with the carrier methyl-beta-cyclodextrin (mbetaCD), proved to be problematic. However, it was possible to cultivate NS0 cells in the medium supplemented with CLC when using conventional cultivation vessels such as disposable polycarbonate shake-flasks and glass bioreactors. A series of experiments investigating the effect of the physical conditions in Wave Bioreactors (e.g., rocking rate/angle, gas delivery mode) ruled out their likely influence, while the exposure of the cells to small squares of Wave Bioreactor film resulted in a lack of growth as in the Wave Bioreactor, suggesting an interaction between the cells, the CLC, and the LLDPE contact surface. Further experiments with both cholesterol-independent and cholesterol-dependent NS0 cells established that the concurrent presence of mbetaCD in the culture medium and the LLDPE film was sufficient to inhibit growth for both cell types. By reducing the excess mbetaCD added to the culture medium, it was possible to successfully cultivate cholesterol-dependent NS0 cells in Wave Bioreactors using a cholesterol-mbetaCD complex as the sole source of exogenous cholesterol. We propose that the mechanism of growth inhibition involves the extraction of cholesterol from cell membranes by the excess mbetaCD in the medium, followed with the irreversible adsorption or entrapment of the cholesterol-mbetaCD complexes to the LLDPE surface of the Wave Bioreactor. Controlling and mitigating these negative interactions enabled the routine utilization of disposable bioreactors for the cultivation of cholesterol-dependent NS0 cell lines in conjunction with an animal component-free cultivation medium.
我们报告了在使用环糊精与胆固醇添加剂混合物的低比例时，胆固醇依赖性NS0细胞在线性低密度聚乙烯（LLDPE）波浪生物反应器中成功培养的情况。虽然在使用补充有胎牛血清（FBS）的培养基时，NS0细胞在波浪生物反应器中的培养是成功的，但使用补充有胆固醇-脂质浓缩物（CLC）的相同培养基进行培养时却出现了问题，CLC中含有脂质和与载体甲基-β-环糊精（mbetaCD）偶联的合成胆固醇。然而，当使用传统的培养容器如一次性聚碳酸酯摇瓶和玻璃生物反应器时，在补充有CLC的培养基中培养NS0细胞是可行的。一系列研究波浪生物反应器中物理条件（如摇晃速率/角度、气体输送模式）影响的实验排除了它们可能的影响，而细胞暴露于波浪生物反应器薄膜的小方块导致生长缺乏，如同在波浪生物反应器中一样，这表明细胞、CLC和LLDPE接触表面之间存在相互作用。对胆固醇非依赖性和胆固醇依赖性NS0细胞进行的进一步实验表明，培养基中同时存在mbetaCD和LLDPE薄膜足以抑制两种细胞类型的生长。通过减少添加到培养基中的过量mbetaCD，可以使用胆固醇-mbetaCD复合物作为外源性胆固醇的唯一来源，在波浪生物反应器中成功培养胆固醇依赖性NS0细胞。我们提出，生长抑制机制涉及培养基中过量的mbetaCD从细胞膜中提取胆固醇，随后胆固醇-mbetaCD复合物不可逆地吸附或截留到波浪生物反应器的LLDPE表面。控制和减轻这些负面相互作用使得一次性生物反应器能够常规用于结合无动物成分培养基培养胆固醇依赖性NS0细胞系。
