신개념 거칠기 전환 폴리 (N-이소프로필아크릴아미드)의 개발 및 이를 통한 줄기세포 시트 치료 활용

Abstract

Although a conventional method of utilizing a thermoresponsiveness of grafted poly (N-isopropylacrylamide) (PNIPAAm) enables a harvest of a healthy cell-to-cell junction preserved cell sheet, the absolute necessity in the control of the nm-scale PNIPAAm chain length decelerate the advancement of cell sheet engineering. In this thesis currently known Young’s modulus of PNIPAAm, which is in kPa-scale, was increased up to MPa-scale and successful fabrication of PNIPAAm cell culture platform was firstly achieved in a ‘bulk’ form. An initial surface roughness of the PNIPAAm cell culture platform was controlled by altering the cross-linker (CS) concentration and the change in the temperature above/below the lower critical solution temperature (LCST) swtiched the surface roughness of the bulk PNIPAAm from nm to μm-scale. After the examination of the cell lines (mouse myoblasts, C2C12, and mouse embryo fibroblasts, NIH3T3) and the primary cells (human umbilical vein endothelial cells, HUVEC, and human epidermal cells, keratinocytes) on the fabrication bulk PNIPAAm, a universal thermoresponsive cell culture platform enabling the attachment/detachment of all types of the cells were found to have initial surface roughness below ~ 30 nm and final surface roughness above 15 μm. Our suggested universal cell culture platform would function as a powerful and versatile tool in accelerating the forthcoming advancement of cell sheeting engineering. At the current state, the practical utilization of cell sheet based technology is hugely hampered due to an inevitable long course of time required for individual cell sheet harvest. Widely, the harvest period of a single cell sheet is generalized to an average of 10 days; and depending on the severity/thickness of the target wound, multiple cell sheets are certainly needed. Under such circumstances, we herein for the first time in the history of cell sheet engineering report a method for ultrafast cell sheet harvest based on a unique nano-replication technology of bulk PNIPAAm substrate possessing 1,500 times higher level of Young’s modulus compared with the conventional PNIPAAm. We successfully replicated various isotropic nanopore patterns on the surface of the bulk PNIPAAm substrates with different pore diameters of 200, 300, and 400 nm and fixed interpore distance of 480 nm to investigate the effect of nanotopographical cues on the fibroblast cell sheet harvest. Interestingly, the nanopore-patterened bulk PNIPAAm substrate with pore diameter of 400 nm not only enlarged the spreading area of individual cells by 2.2-fold but also promoted the cell proliferation with induced extracellular matrix secretion by 2.5-fold; thereby, shortened the cell sheet harvest period from 10 days to 2 days. Lastly, the therapeutic effect of the stem cell sheets which were harvested from the bulk PNIPAam was examined on the myocardial infract (MI) model. While the therapeutic effect (paracrine effect) of the stem cell sheet is known to be last only a week, the stacked cell sheet was observed to preserve its paracrine effect for 4 weeks of the examination period in vitro and further prolonged the cardiac deterioration in vivo during the examination period of 8 weeks. In addition, while the realization of various methods of adopting PNIPAAm on cell culture platform is continuously being pursued to attain in vivo-like heterogeneous or vascularized cell sheets, there are several issues that hamper maximizing the potential of cell sheet engineering. It is still arduous to achieve stable adhesion behavior of the cells on the fabricated cell culture platform and the inherently constrained geometry of the harvested cell sheet limits the range of its utilization. We tailored the polymer network of conventional PNIPAAm through the modification of its substance composition and for the first time introduce a multi-responsive dynamic PNIPAAm cell culture platform which can harvest different types of cell sheets in the desired shape. Stable adhesion behavior of different cell types was observed in varying ambient conditions and the shape memory effect driven by the hygroscopic-responsive behavior of the modified PNIPAAm was confirmed. We believe this novel cell culture platform could ignite a spark to the further expansion of cell sheet engineering.