细胞图型及微流控芯片上的细胞培养和分化
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摘要
微流控和图型化技术在细胞生物学领域中的应用具有重要的意义。本论文首先讨论几种新的细胞图型化技术。其一为PDMS模负压吸附法,由此形成的物理或化学印迹被用于胚胎纤维细胞的图型化;其二为高分辨率压印刻蚀法,通过紫外光软膜纳米压印技术可制作高分辨率生物分子图型,细胞的贴附情况表明此法有效地改善了生物分子图型的稳定性;另外微流控芯片中单细胞阵列也被实现,并且细胞可以长时间的培养观察。微流控技术首先被用于基底材料对胚胎干细胞的培养和成神经细胞分化影响的研究,结果表面,尽管胚胎干细胞可在多种基底上生长,细胞贴附力较强的基底更利于分化实验的实现。微流控芯片也被用于研究骨髓间充质干细胞的培养及相应的成脂肪细胞和神经细胞的分化。且由微接触印刷法制备的图型被用于诱导骨髓间充质干细胞的成神经细胞分化。又制作了用于老鼠胚胎干细胞的神经分化的微井图型。最后,尝试了神经细胞电极芯片,用于信号传导的测量。
Microfluidics and patterning technologies are now widely used for cell biology studies. Of particular interest is to use these new technologies to control more precisely the stem cell culture and differentiation. In this work, both embryonic and mesenchymal stem cells are used to evaluate the feasibility of such an application. First, "degas induced patterning" and soft UV nanoimprint lithography methods are introduced, taking into account the specificity of the cell patterning requirements. While the "degas induced patterning" allows to define easily both physical and chemical patterns, the soft UV nanoimprint lithography provides a much higher resolution with an improved pattern stability. Also single cell pattern arrays could be obtained in microfluidic devices and then cultured for a long term observation. Second, a general microfluidic device configuration, which is composed of a micro-well array with micro-channel connections, has been studied to evaluate the performance of embryonic stem cell culture and differentiation on different substrates. The results show that even though the culture of stem cells can be done with different substrates, the differentiation is more critically depending on the cell-material adhesion. Third, multi-chamber and multi-compartment microfluidic devices are also designed for culture and differentiation of mesenchymal stem cells. By adapting the existing protocols, both adipogenic and neural differentiation are realized. Then a cellular network patterning has been tested with both mesenchymal stem cells and mouse embryonic stem cells, in order to achieve a derived neuronal network formation. Finally, multi-electrode arrays have been fabricated for on chip signal transduction studies.
Microfluidics and patterning technologies are now widely used for cell biology studies. Of particular interest is to use these new technologies to control more precisely the stem cell culture and differentiation. In this work, both embryonic and mesenchymal stem cells are used to evaluate the feasibility of such an application. First, "degas induced patterning" and soft UV nanoimprint lithography methods are introduced, taking into account the specificity of the cell patterning requirements. While the "degas induced patterning" allows to define easily both physical and chemical patterns, the soft UV nanoimprint lithography provides a much higher resolution with an improved pattern stability. Also single cell pattern arrays could be obtained in microfluidic devices and then cultured for a long term observation. Second, a general microfluidic device configuration, which is composed of a micro-well array with micro-channel connections, has been studied to evaluate the performance of embryonic stem cell culture and differentiation on different substrates. The results show that even though the culture of stem cells can be done with different substrates, the differentiation is more critically depending on the cell-material adhesion. Third, multi-chamber and multi-compartment microfluidic devices are also designed for culture and differentiation of mesenchymal stem cells. By adapting the existing protocols, both adipogenic and neural differentiation are realized. Then a cellular network patterning has been tested with both mesenchymal stem cells and mouse embryonic stem cells, in order to achieve a derived neuronal network formation. Finally, multi-electrode arrays have been fabricated for on chip signal transduction studies.
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