Department of Industrial Engineering, Università degli Studi di Padova, Via Marzolo 9, I-35131, Padova, Italy.
Lab Chip. 2013 Nov 21;13(22):4430-41. doi: 10.1039/c3lc50643f.
Long-term cell culture in microfluidic devices is an essential prerequisite for "on a chip" biological and physiological based studies. We investigated how medium delivery, from continuous to periodic perfusion, affects long-term cell cultures in a microfluidic platform. Computational simulations suggested that different delivery strategies result in different temporal profiles of accumulation and washing out of endogenous (EnF) and exogenous (ExF) factors, respectively. Thus, cultures exposed to the same overall amount of medium with different temporal profiles were analysed in terms of homogeneity, cell morphology and phenotype. Murine and human cell lines (C2C12 and HFF) and mouse embryonic stem cells (mESC) were cultured in microfluidic channels. An ad hoc experimental setup was developed to perform continuous and periodic medium delivery into the chip, tuning the flow rate, the perfusion time, and the interval of perfusion while using the same amount of medium volume. Periodic medium delivery with a short perfusion pulse ensured cell homogeneity compared to standard cell culture. Conversely, a continuous flow resulted in cell heterogeneity, with abnormal morphology and vesiculation. Only dramatic and unfeasible increasing of perfused medium volume in the continuous configuration could rescue normal cell behaviour. Consistent results were obtained for C2C12 and HFF. In order to extend these results to highly sensitive cells, mESC were cultured for 6 days in the microfluidic channels. Our analysis demonstrates that a periodic medium delivery with fast pulses (with a frequency of 4 times per day) resulted in a homogeneous cell culture in terms of cell viability, colony morphology and maintenance of pluripotency markers. According to experimental observations, the computational model provided a rational description of the perfusion strategies and of how they deeply shape the cell microenvironment in microfluidic cell cultures. These results provide new insight to define optimal strategies for homogeneous and robust long-term cell culture in microfluidic systems, an essential prerequisite for lab on chip cell-based applications.
长期细胞培养是“片上”生物和生理学研究的必要前提。我们研究了从连续灌流到周期性灌流的介质输送如何影响微流控平台中的长期细胞培养。计算模拟表明，不同的输送策略分别导致内源性（EnF）和外源性（ExF）因子的积累和冲洗的时间分布不同。因此，以不同的时间分布暴露于相同的总体培养基量的培养物，在均匀性、细胞形态和表型方面进行了分析。使用微流控通道培养了小鼠和人细胞系（C2C12 和 HFF）和小鼠胚胎干细胞（mESC）。开发了一种特定的实验装置来进行连续和周期性介质输送到芯片中，调整流速、灌注时间和灌注间隔，同时使用相同的培养基体积。与标准细胞培养相比，短脉冲周期性介质输送可确保细胞均匀性。相反，连续流动导致细胞异质性，出现异常形态和囊泡化。只有在连续配置中大幅且不切实际地增加灌注培养基的体积，才能恢复正常的细胞行为。C2C12 和 HFF 得到了一致的结果。为了将这些结果扩展到高度敏感的细胞，将 mESC 在微流控通道中培养了 6 天。我们的分析表明，快速脉冲（每天 4 次）的周期性介质输送可实现细胞存活率、集落形态和多能性标志物维持方面的均匀细胞培养。根据实验观察，计算模型提供了对灌流策略的合理描述，以及它们如何深刻地塑造微流控细胞培养中的细胞微环境。这些结果为定义微流控系统中均匀且稳健的长期细胞培养的最佳策略提供了新的见解，这是片上细胞应用的必要前提。
