| The goal of micro total analysis systems (μTAS, also termed Lab on a Chip) is to integrate various such operation steps of chemical or biochemical analysis as sampling, sample pretreatment, separation, and detection into automatic, portable and miniaturized analytical instruments. Microfluidic chips, the core ofμTAS, have been rapidly growing in recent years. While the concept ofμTAS is being populated in the field of analytical chemistry, the idea of "lab on a chip" is spreading in the field of life sciences including the biomimics. The success ofμ-TAS mainly depends on the development of novel microfluidic chips. Fabrication of novel microfluidic chips relies not only on the micro fabrication technology but also on the materials used for fabrication of the chips.Polydimethylsiloxane (PDMS) is one of the most widely used polymer materials for fabricating microfluidic chips. The most attractive advantage of PDMS lies in its biological compatibility and excellent gas-permeability, making it suitable for biological applications. Thus, the microfluidic chips used for research in cell biology and biochemical analysis based on cells are almost fabricated with PDMS. However, PDMS does have some disadvantages. The most well-known limitation is its highly hydrophobic surface that is difficult to wet with aqueous solutions and prone to be fouled by hydrophobic molecules or proteins. Thus, various methods have been developed to modify the PDMS surface.The present work is aimed to develop a simple method for preparation of the thermo-responsive PDMS surfaces grafted with poly(N-isopropylacrylamide) (PNIPAAm), to characterize the properties of the smart surfaces, and to demonstrate the application of the PNIPAAm-grafted-PDMS (PNIPAAm-g-PDMS) surfaces for cell culture, harvest and transport operations on microfluidic chips. The thesis is composed of four parts:In chapter 1, the recent advances in both the preparation of thermo-responsive PNIPAAm surfaces and its application to cell culture were reviewed. The surface grafting polymerization techniques used for preparing PNIPAAm surfaces mainly include four types:solution radical grafting polymerization, electron beam or high-energy rays induced grafting polymerization, plasma induced grafting polymerization and UV light induced grafting polymerization. The applications of PNIPAAm surfaces in cell culture mainly include three fields:temperature-modulated, nonenzymatic harvest, patterned co-culture and tissue engineering. The current trends of the research works on the PNIPAAm-g-PDMS surfaces are also presented in detail.In chapter 2, the thermo-responsive PNIPAAm-g-PDMS surfaces were prepared by using the benzophenone-initiated photopolymerization. The chemical and physical properties of the grafted surfaces were also characterized. It was observed that thick (> 1 mm) PDMS substrates were much more difficult to be grafted with PNIPAAm than the thin ones. Systematic investigations revealed that the shortage of benzophenone molecules diffused into the surface region of the thick PDMS might be the reason for the problem. By prolonging the time used for treating the PDMS substrate with a benzophenone solution, PNIPAAm could be successfully grafted onto thick PDMS substrates. The PNIPAAm-g-PDMS surface was highly thermo-responsive. The contact angle on grafted surface increased from 38°to 91°in response to the temperature rising from 20℃to 38℃. An electroosmotic flow (EOF) mobility of 5×10"4 cm2/V·s was supported by PNIPAAm-g-PDMS channel at 50℃, whereas negligible EOF was observed in the channel at 20℃. Anticancer drug of doxorubicin (DX) could be adsorbed by the grafted surface at 40℃, and most of the adsorbed DX could be quickly released from the surface to a stripping solution at 5℃. Osteoblast cells adhered onto the PNIPAAm-g-PDMS surface and proliferate therein at 37℃, while the cell sheet detached from the surface by lowering the temperature to 25℃without using any enzymatic agent. In chapter 3, using COS7 (african green monkey kidney fibroblast cell line) as a model, the performances of the PNIPAAm-g-PDMS surfaces in cell culture were investigated. In addition, the proliferation and multi-differentiation behaviors of human mesenchymal stem cells (hMSCs) on the PNIPAAm-g-PDMS surface were also examined. The results showed that the performances of PNIPAAm-g-PDMS surfaces in cell culture could be improved by coating of gelatin and modulating the PNIPAAm-grafting yields. The mean survival rate of COS7 which harvested by treatment with trypsin from PDMS surface is 80.6%, and that of COS7 harvested from the PNIPAAm-g-PDMS surface by lowing temperature was 89.1%. The viability of COS7 from the PNIPAAm-g-PDMS surfaces was significantly higher than that from the PDMS surfaces (P=0.016). When used to harvest hMSCs, the developed method was more superior to the conventional method. The mean survival rate of hMSCs harvested by using traditional treatment with trypsin was 75.7%, and that of hMSCs harvested by using a temperature shift was 97.2%. The results also showed that the PNIPAAm-g-PDMS surface could keep the potentials of multi-differentiation of hMSCs.In chapter 4, the culture, harvest and passage of COS7 in the PNIPAAm-g-PDMS microfluidic channels were studied. Using a native PDMS chip as model, the conditions of cell culture in microchannels were optimized against such conditions as the configuration of channels, the methods of seeding and changing the culture medium. Under the optimized conditions, the long-term cell-culture could be achieved in PNIPAAm-g-PDMS microchannels. And the cells reached 90% confluence in three days. With simply lowing of temperature, cells could detach from the PNIPAAm-g-PDMS surfaces, and could be transported by a fluid flow to the downstream chips, wherein the harvested cell could be sub-cultured. The integration of the cell culture, harvest and transport operations on microfluidic chips was initially implemented. The main novelties of the present work are summarized as:1. It is revealed that the thickness of PDMS substrate was a critical factor for grafting of PNIPAAm onto PDMS surface with the benzophenone-initiated and UV induced grafting polymerization technique. It is suggested that thickness of the PDMS substrate affect the concentration of the benzophenone on the surface of the PDMS substrate, subsequently, the yields of the PNIPAAm-grafting. Based on this suggestion, a reliable method for preparation of the PNIPAAm-g-PDMS surface was established for the PDMS substrates of varied thickness.2. A simple method of thermal-switchable cell culture on and cell harvest from the PNIPAAm-g-PDMS surfaces was established. The superiority of this novel method over the traditional trypsin-based harvest method lies in the higher viability of the harvested cells. Furthermore, on-chip operations of the cell culture, harvest, transport and passage were realized in the microfluidic channels with the PNIPAAm-g-PDMS surfaces.3. It is discovered that the PNIPAAm-g-PDMS surfaces does not inhibit the potentials of multi-differentiation of hMSCs.
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