Isolation And Characterization Of Tumor Cells With Different Motility Based On A Paper-based 3D Culture System

Malignant tumors are one of the most important diseases that endanger human health.In particular,the recurrence and metastasis of malignant tumors after treatment have caused the greatest obstacle to improving the anti-tumor efficacy and improving the survival rate of patients.According to statistics,more than 90%of cancer patients’death causes are related to tumor metastasis and its complications.Tumor metastasis has undoubtedly become an important direction of cancer research.Isolation of tumor cells with different migration abilities can further analyze the genetic characteristics of cells with different migration abilities,which is of great significance for the study of tumor invasion and cancer treatment.Paper is one of the oldest important inventions of mankind.In recent years,because of liquid infiltration and diffusion in the paper fiber substrate,paper-based microfluidic analysis system（Microfluidic paper-based analytical devices,μPADs）was developed.By mixing the cells in the hydrogel sol and then added dropwise to the filter paper to establish the"cells-in-gels-in-paper,CiGiP"cell culture.A large number of tiny pores like tissue gaps exist in the paper substrate,allowing substances to penetrate and provide nutrients for the cells.More importantly,the construction of 3D structures can be easily achieved through multi-layer stacking,and the structural transformation from 3D to 2D can be easily achieved by disassembling the multi-layer paper base,which is convenient for observation and analysis of the cell morphology and function inside the chip.Based on the analysis of the status quo of the above research fields,in this thesis,the paper-based three-dimensional cell culture chip:CiGiP（cells-in-gel-in-paper）chip was constructed by means of lens encapsulation paper and Parafilm^？.A multi-layer CiGiP paper-based chip is superimposed to construct a cell migration platform,which is used to study the migration of tumor cells between different layers of paper-based cell chip.The multi-layer CiGiP can be superimposed-disassembled to quantify cell movement and sort out cells with different migration abilities,and further analyze the genetic characteristics of cells with different migration abilities.Finally,an integrated RT-LAMP（RT:reverse transcription;LAMP:loop-mediated isothermal amplification）rapid detection device was constructed,which can use to detect tumor markers of multi-layer CiGiP sorted tumor cells to verify the cells of the multi-layer CiGiP cell migration platform sorting performance.The main research contents of this thesis are as follows:1.Fabrication of CiGiP cell culture chip based on lens paper and thermosensitive flexible Parafilm^？Paper is a new type of cell culture material and has important application value in cell culture.In order to develop a simple and low-cost paper-based three-dimensional cell culture protocol,this study compared the physical and chemical properties of Whatman^？1#filter paper,printing paper,and Whatman^？105#lens paper.It was found that compared with Whatman^？1#filter paper and printing paper,Whatman^？105#lens paper has better flexibility,stability and permeability.CiGiP paper-based three-dimensional cell culture chip was obtained by combining lens paper and Parafilm^？through thermoplastic sealing method.To establish cell culture chip,the gel concentration was optimized by comparing the gel spreading and cell growth on the gel-embeded paper chip.By detecting the proliferation rate of cells in the CiGiP paper-based cell culture chip,it was found that the cells in the CiGiP paper-based chip maintained a good proliferation rate,and the cells tended to aggregate and grow in the paper-based chip.Comparing with other methods of constructing paper-based cell chips,the method designed in this research has the advantages of low cost,simple fabrication precedure.2.Separation and characterization of cancer cell subtype according to their motility using a multi-layer CiGiP cultureCancer cell metastasis is considered to be a sign of malignant tumor progression.Therefore,measuring the movement of cancer cells and exploring the relationship between tumor cell migration ability and cell invasion will help to further understand the mechanism of tumor metastasis.In order to study the genetic characteristics of cells with different migration ability,this study constructed a multi-layer CiGiP three-dimensional cell chip as a tumor cell migration platform.First,the number of superimposed layers of multi-layer CiGiP three-dimensional cell migration platform and the the culture time of cells in the chip were optimized.By disassembling the multi-layer paper-based chip,it was found that only a small part of cells could successfully invade from the cell seeding layer to the top and bottom layers of the multi-layer cell chip,which has stronger migration ability.Further detection of cells in each layer by q PCR showed that the cells with higher migration ability had significantly increased expression of cancer stem cell（CSC）biomarkers ALDH1A1,SOX2,NANOG and OCT4.Finally,the tumor cells that migrated to different layers were collected for chemotherapeutic drug sensitivity testing.The results showed that the highly invasive tumor cells sorted by the multi-layer CiGiP chip were more resistant to adriamycin.This research is the first to study the multi-layer CiGiP three-dimensional cell chip to sort tumor cells with different invasive capabilities,and further explores the genetic characteristics of tumor cells with high invasive capabilities,revealing the relationship between tumor invasive capabilities and cancer stem cells.3.Fabrication of paper-based RT-LAMP chip for rapid detection of cancer cells sorted by CiGiP three-dimensional cell chipIn order to detect cancer cells sorted by CiGiP three-dimensional paper-based cell chip,this work designed a paper-based RT-LAMP chip to detect prostate cancer cell-specific biomarker prostate cancer antigen 3（PCA3）.LAMP is a new type of nucleic acid amplification method without temperature cycle process,so it has great application prospects in the field of biological detection.The detection device includes paper-based RT-LAMP reaction chip,temperature control module and imaging module.The RT-LAMP reaction chip is composed of a nucleic acid amplification area loaded with RT-LAMP reagent and a colorimetric detection area loaded with of calcein and manganese chloride,amplification area and colorimetric detection area are connected by a channel constructed by 3D printing.The separation design on RT-LAMP chip avoids the influence of the chromogenic dye on the efficiency of nucleic acid amplification.The temperature control module can be powered by batteries,which can improve the portability of the detection equipment.By squeezing the sponge pad located in the amplification area,the nucleic acid amplification product is squeezed into the colorimetric detection area to perform colorimetric reaction.By optimizing the RT-LAMP reaction time and temperature,the detection sensitivity is improved.Finally,successfully detected the cancer marker PCA3 of different invasive cells isolated from the multilayer CiGiP chip.Furthermore,it can be proved that the separation ability of multilayer CiGiP cell chip.