Research Of Microfluidic And Single Cell Sorting Technology In Biomedical Applications

Objective: In recent years, with the development of microfluidic technology and second- generation sequencing technology, applications of microfluidic chip in the biomedical field are more and more widespread, while the single-cell analysis also has attracted more and more attention. Firstly, we developed a biomimetic microfluidic device for in vitro antihypertensive drug evaluation; secondly, by the lack of existing single-cell separation technology, we established a single cell identification, enrichment, and sorting system.Methods:(1) ① A PDMS-glass microfluidic chip with a channel of 4 cm length, 2 mm width, and 100 μ m height was fabricated using a standard soft pattern photolithography technique. Chip was coated with FN in 37℃ for 1 h,then the HUVECs were cultured in the chip. ② The system consists of a syringe pump, a microfluidics chip, a digital manometer, and several joints and tubes. The flow rate was regulated by the syringe pump; the pressure was shown by the digital manometer, then the appropriate pressure was applied to HUVECs in the microfluidic chip. ③ Hydralazine hydrochloride was then added to the medium at one of four different concentrations(0, 50, 250, or 500 μ mol/L) and infused into the channel using the syringe pump under the pressure of 0 KPa, 12 KPa, and 18 KPa. Two experiments was set: for the first one, the drug was added to the medium during and after the pressure was applied; for the other one, the drug was added only after the pressure was applied. The chip was cultured inside the incubator, with other components of the system staying outside. ④ The medium during and after applying the pressure was collected for ET-1 assay, and cells in chip were then stained with anti-CD62 P antibody, Alexa Fluor 546 goat anti mouse IgG antibody, Alexa Fluor 488 phalloidin and DAPI. The cells were then imaged under a fluorescence microscope.(2) ① The computer was used as the control center of the whole system, a three-axis electric cylinder was used as the liquid distribution of 384 well plate, a micromanipulator was used as the controller of the capillary glass needle positioning, a microinjectior was used as the controller of cell’s picking- up and blowing-out manipulation, and the microscope was used as displaying the real-time imaging of the samples. ② C++ programing language was used to control every components of the system, generating the graphical user interface of 384-well plate and micro-well. ③ For efficient enrichment of a single cell, two steps are used to achieve enrichment and selection function: for the first step, the 384-well plates were used for sample preliminary screening. The samples were pretreated into cell suspension, dispensed into the well plate, the wells in the plate were imaged one by one, the target cells were determined using software, and all the cells of target well were collected; for the second step, the cells collected from the first step were dropped onto micro-well glass slide, forming a large droplet. This cell droplet was sectioned and imaged for each section under the microscope. The target cell location within the droplet was determined using the software. Finally the target cells were picked up one by one and blown out with the micromanipulator and microinjection pump. ④ To test the efficiency of the cell picker system, the breast cancer cell line MDA-MB-231 transfected with RFP spiked into breast cancer cell line MCF-7 were used as the test sample. Five sets of experiments, in which 1, 2, 5, 10 and 20 MDA-MB-231-RFP cells were spiked into 1 million MCF-7 cells, were repeated 5 times. The mouse liver tumor tissue was used as a sample. After tissue digestion into single cell suspension, single cell was picked out and amplified, libraried using the whole genome, transcriptome and methylation method, and sequenced by Illumina Hiseq 2500 high-throughput sequencing platform.Results:(1) ① We have established a microfluidic system to study the responses of HUVECs to different concentrations of hydralazine hydrochloride under pressures of 0 KPa, 12 KPa, and 18 KPa. ② Results of cell staining of Alexa Fluor 488 phalloidin and DAPI indicated that with an increased pressure cytoskeletons collapsed, and when an increased concentration of hydralazine hydrochloride was added to the medium, a more prominent network of actin filaments occupied the cytoplasm. ③ Results of cell staining of anti CD62 P antibody, Alexa Fluor 546 goat anti mouse IgG antibody and DAPI indicated that pressure could result in cell injury and then stimulate the secretion of P-selectin. Hydralazine hydrochloride could restrain the secretion of P-selectin. ④ ET-1 release increased with increasing pressure and obviously decreased with addition of the drug.(2) ① The system we established can identify, enrich, and pick single cell with the help of GUI. ② The GUI of 384-well plate and micro well plate can identify the image, save the data and assistantly display the process in real time. ③ Spike- in experiments indicated that the average efficiency of the cell picker system can be as high as 60%. ④ Mouse liver tumor sample experiments showed that the system could pick single tumor cell, and the cell could be used for single cell genome, transcriptome, and methylation analysis.Conclusion:(1) In this study, we have established an easily fabricated and assembled, low-cost microfluidic system to successfully study the response of endothelial cells to different concentrations of an antihypertensive drug while under pressure.(2) We also have proposed a new method of identifying, enriching, and sorting single, established a semi-automatic system to achieve this purpose, and successfully picked up single cell using this system.