Microfluidic systems for separation, counting, sorting, culture and differentiation of stem cells

• Main Authors: Huei-WenWu, 吳慧紋
• Other Authors: Gow-Bin Lee
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/21188844130002589661

博士 === 國立成功大學 === 工程科學系碩博士班 === 100 === Microfluidic techniques have been recently developed for cell-based assays. In microfluidic systems, the objective is for these microenvironments to mimic in-vivo surroundings. With advantageous characteristics such as optical transparency and the capability for automating protocols, different types of cells can be cultured, screened and monitored in real time to systematically investigate their morphology and functions under well-controlled microenvironments in response to various stimuli. Recently, the study of stem cells using microfluidic platforms has attracted considerable interest. Even though stem cells have been studied extensively using bench-top systems, an understanding of their behavior in in-vivo-like microenvironments which stimulate stem cell proliferation and differentiation is still lacking. In this paper, several stem cell studies using microfluidic systems are purposed. The various miniature systems for stem cell separation/isolation, sorting, isolation, culture, differentiation, and stimulation, are then systematically introduced. Compared with conventional cell culture protocols, the microfluidic techniques provide versatile approaches to mimic more in vivo-like extracellular conditions for more realistic cell-based assay research. There still exist several inherent advantages including low biosamples/reagents consumption, a single integrated chip with multiple functions, and ability to run the array assays simultaneously. In this study, it was presented several new microfluidic devices fabricated based on SU-8 lithography process, a computer numerical controlled (CNC) milling for molds, and polydimethylsiloxane (PDMS) replica molding processes for stem cells researches. First, a passive separation chip with louver-like structures in the microchannel is proposed as a filter to separate mesenchymal stem cells (MSCs) from amniotic fluid. Buffer solution is used to squeeze the sample flow by using the syringe pumps to form a narrow stream so that the sample flows close to the louver-like structures to obtain a higher separating efficiency. The device can alleviate the clogging problem and avoid the use of the external force such that cells will not be damaged during the separation process. Preliminary results show that the developed microfluidic device can perform a good separation of 86% (beads). It also shows that that the developed microfluidic device can perform a good separation of 82.8 % for MSCs. Furthermore, the separation process can be repeated to improve the separation efficiency to 97.1 %. Another magnetic-bead technology integrated with the microfluidic system was purposed to develop a platform capable of isolating, counting, and sorting the hematopoietic stem cells. Since there is only an extremely small amount of stem cells existing in the umbilical cord blood, it is crucial to isolate and count the cell sample. In this research, the processes including mixing, transporting, counting and sorting can be completed automatically using the microfluidic control module. The target stem cells will be first captured by the antibody coated onto the magnetic beads, and then be successfully counted and sorted by a detection system. In addition, a continuous microfluidic device capable of automating culturing and differentiating the MSCs was proposed. Microfluidic-based pneumatic trumpet-like micropump activated by two electromagnetic valves (EMVs) with three air chambers plus an elusive side-channel was used to suck the culture medium so that the medium in the culture area can be continuously supplied. Moreover, the waste can be moved through the elusive side-channel without contamination. The results represented that MSCs can be cultured and differentiated into different kinds of phenotypes stably for a long time. The stem cell culture chip not only can provide stable and well-defined microenvironments, but also features in low consumption of research resource. Finally, an integrated microfluidic system capable of fine-tuning the insulin concentration automatically and applying different levels of shear stresses simultaneously was developed to investigate the effects of chemical and mechanical stresses on adipogenic differentiation of MSCs. It is comprised of a dilution device which can automatically fine-tune the concentrations of insulin for chemical stimulation on stem cells and three different levels of shear stresses produced by deflecting the PDMS membranes used to induce stem cells at the same time. The experimental results showed that an optimum insulin concentration of 10 μg/ml for differentiation of adipocytes can be determined. Moreover, the adipogenic differentiation can be suppressed by applying stronger shear stress and higher pulsation frequency of mechanical stimulation. In summary, we have demonstrated several microfluidic based platforms of separation/isolation, counting, sorting, culture, differentiation and stimulation for the stem cell which may provide a promising development in the this new medical field.