MDCK贴壁细胞在篮式生物反应器培养的生长代谢特征及动力学模型

Growth and metabolic characteristics and kinetic modeling of MDCK adherent cells cultured in basket bioreactors

摘要:

为解决工业化生产中篮式生物反应器取样受限的问题，本研究通过建立犬肾(Madin-Darby canine kidney, MDCK)贴壁细胞生长代谢动力学模型，预测篮式生物反应器生产过程中细胞数量、营养物和代谢产物浓度。分析了前期实验收集的MDCK贴壁细胞在5 L篮式生物反应器培养不同时间点细胞密度、葡萄糖和乳酸浓度数据，使用MATLAB软件进行非线性拟合。本文模型以Logistic方程和Luedeking-Piret方程为基础建立，并额外添加了灌流稀释项以适应灌流培养模式。通过拟合优度计算、回归分析和误差分布分析评价模型预测能力，并在40 L篮式生物反应器中对该模型的跨规模预测性能进行了检验。结果显示，MDCK细胞在5 L篮式生物反应器培养至96-108 h达到平台期，最大细胞密度为(795.13±16.22)×10⁴ cells/mL。细胞比生长速率在36 h时达到最大，为1.01/d。平台期葡萄糖的最大消耗速率是1.71 mmol/(L·h)，乳酸的最大生成速率是2.21 mmol/(L·h)。使用5 L反应器培养数据构建的MDCK细胞生长代谢动力学模型拟合优度良好，R²均大于0.95。在40 L篮式生物反应器的3批验证培养中，模型预测曲线与取样离线检测数据吻合度高，R²均大于0.95。在培养第96、120小时取样离线消化读细胞数，离线检测数值与模型预测数值没有显著差异(P=0.48, P=0.92)。结果表明基于5 L反应器建立的动力学模型可以有效地跨规模预测40 L反应器中MDCK细胞的生长和代谢状态，同时证明了5 L和40 L反应器的培养效果相似。本研究的动力学模型能够量化细胞生长、葡萄糖消耗和乳酸生成的速率，为篮式生物反应器培养工艺优化和生产操作提供理论依据，同时为疫苗企业产品质量控制提供辅助。

Abstract:

This study developed kinetic models to describe the growth and metabolism of MDCK adherent cells cultured in basket bioreactors. These models were used to predict cell density, nutrient consumption, and metabolite concentrations at key stages of the culture process in basket bioreactors, helping to overcome sampling limitations caused by environmental conditions and bioreactor design. Experimental data, including cell density and glucose and lactate levels were obtained at different time points from 5 L bioreactors. Model parameters were estimated by nonlinear fitting in MATLAB. Kinetic models were established based on the Logistic and Luedeking-Piret equations, and a perfusion dilution item was introduced considering the perfusion culture. The prediction performance of the models was evaluated based on the goodness-of-fit, regression, and error distribution. The models were then validated with independent experimental data from 40 L basket bioreactors. The results showed that MDCK cells cultured in the 5 L bioreactor entered the plateau phase between 96 h and 108 h, with the maximum cell density reaching (795.13±16.22)×10⁴ cells/mL. The specific growth rate peaked with a value of 1.01/d at the time point of 36 h. During the plateau phase, the maximum lactate production rate was 1.71 mmol/(L·h), while the glucose consumption rate reached 2.21 mmol/(L·h). The kinetic models derived from the 5 L bioreactor data showed good agreement with experimental results, with R² values exceeding 0.95. When being applied to validation data from three independent 40 L bioreactor batches, the models consistently achieved R² values above 0.95. Offline cell densities measured by digestion at 96 h and 120 h showed no significant differences from the model predictions (P=0.48, P=0.92). The results indicated that the kinetic models developed with 5 L bioreactor data could accurately predict the growth and metabolic behavior of MDCK cells in the 40 L system. In addition, the results demonstrated that the operational performance of the 40 L bioreactor was comparable to that of the 5 L system. This confirmed the reliability of the established models for scale-up applications. By quantifying key parameters, including the specific growth rate, glucose consumption rate, and lactate production rate, the models provide a sound theoretical basis for optimizing basket bioreactor processes. Moreover, they offer strong technical support for improving production efficiency and quality control in industrial-scale manufacturing.