Rapid harvesting of stem cell sheets by thermoresponsive bulk poly(N-isopropylacrylamide) (PNIPAAm) nanotopography.

To date, cell sheet engineering-based technologies have actualized diverse scaffold-free bio-products to revitalize unintentionally damaged tissues/organs, including cardiomyopathy, corneal defects, and periodontal damage. Although substantial interest is now centered on the practical utilization of these bio-products for patients, the long harvest period of stem cells- or other primary cell-sheets has become a huge hurdle. Here, we dramatically reduce the total harvest period of a cell sheet (from cell layer formation to cell sheet detachment) composed of human bone marrow mesenchymal stem cells (hBMSCs) down to 2 d with the help of bulk thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) substrate nanotopography, which is not achievable via the previous grafting methods using PNIPAAm. We directly replicated an isotropic 400 nm-nanopore-array pattern on a bulk PNIPAAm substrate through UV polymerization of highly concentrated NIPAAm monomers, which was achieved using a remarkably increased Young's modulus of bulk PNIPAAm that was 1500 times higher than conventional PNIPAAm. The rapid harvesting of the hBMSC sheet on the bulk PNIPAAm substrate nanotopography was not only based on the accelerated formation and maturation of the hBMSC layer, but also the easy detachment of the hBMSC sheet induced by the abrupt change in the surface roughness of the substrate below the lower critical solution temperature (LCST) owing to the enlarged surface area of the substrate. Our findings may contribute to reverse presumptions about the limitations regarding the grafting methods for the cell sheet harvest and could broaden the practical utilization of cell sheets for patients in the near future.

Compressed collagen intermixed with cornea-derived decellularized extracellular matrix providing mechanical and biochemical niches for corneal stroma analogue.

Compressed collagen is a promising scaffold for corneal stroma analogue due to its facile incorporation of keratocytes while mimicking the mechanical niche of a native cornea with dense collagen fibrillar structures. However, it does not offer the sufficient biochemical niche crucial for in vivo-like quiescent keratocyte phenotype. In this study, we engineered a scaffold for a corneal stroma analogue that mimics both the mechanical and biochemical niches of the corneal stroma by introducing cornea-derived decellularized extracellular matrix (Co-dECM) to the collagen compression process. The compressed collagen intermixed with Co-dECM (COLEM; Co-dECM content, <50 wt%) maintained a uniform structure and showed an elastic modulus and tensile strength on the order of 100 kPa, which is comparable with that of conventional compressed collagen. The COLEM with the 50 wt% Co-dECM content was found to possess 2-fold higher amount of the glycosaminoglycans as compared to the compressed collagen. The biochemical components of Co-dECM in the COLEM were verified to significantly promote the expression of quiescent keratocyte-specific genes, i.e., KERA and ALDH3A1, while improving the optical transmittance of the COLEM by reducing the diameter of collagen fibrils. The ability of the COLEM to construct multicellular in vitro corneal tissue was demonstrated by an additional corneal epithelial cell culture. The results support the hypothesis that COLEM has strong potential use in the development of corneal equivalent for in vitro models and tissue transplantation.

Bulk poly(N-isopropylacrylamide) (PNIPAAm) thermoresponsive cell culture platform: toward a new horizon in cell sheet engineering.

Although a conventional method of utilizing thermoresponsive grafted poly(N-isopropylacrylamide) (PNIPAAm) enables the harvest of a healthy confluent cell-to-cell junction preserved cell sheet while limiting the use of the trypsin enzyme, the absolute necessity in the delicate control of a sensitive nm-scale PNIPAAm chain length inevitably decelerates the advancement of cell sheet engineering. In this study, we demonstrate, for the first time, a thermoresponsive cell culture platform composed only of a 'bulk' form of a PNIPAAm hydrogel with the Young's modulus being increased up to the MPa scale. The surface roughness of the bulk PNIPAAm hydrogel initially modulated by the cross-linker concentration was altered from the nm- to µm-scale in response to a change in temperature above/below the low critical solution temperature (LCST) of 32 °C. The appropriate control of the surface roughness allowed the stable attachment (above the LCST) and easy detachment (below the LCST) of diverse cells and enabled the harvest of cell sheets composed of cell lines (C2C12 and NIH3T3) or even primary cells (human umbilical vein endothelial cells and keratinocytes). During their incubation at 37 °C, the cell lines were able to be attached on every surface of the prepared PNIPAAm cell culture platforms, whereas the primary cells were found to be only attached on a surface having a roughness below ∼30 nm. Furthermore, in the aspect of cell sheet detachment at the incubation temperature of 20 °C, the cell sheets composed of cell lines were fully detached from the surface of the platform having a roughness of ∼10 µm or higher, while the cell sheets composed of primary cells were entirely detached from the surface with a roughness of ∼19 µm or higher. Based on such behaviors of the diverse cells at a given surface roughness, this study further suggests a universal thermoresponsive cell culture platform which allows the harvest of all types of cells from cell lines to primary cells in a desired shape. Our suggested universal cell culture platform could play a powerful and versatile role in accelerating the advancement of cell sheet engineering.

Versatile Fabrication of Size- and Shape-Controllable Nanofibrous Concave Microwells for Cell Spheroid Formation.

Although the microfabrication techniques for microwells enabled to guide physiologically relevant three-dimensional cell spheroid formation, there have been substantial interests to more closely mimic nano/microtopographies of in vivo cellular microenvironment. Here, we developed a versatile fabrication process for nanofibrous concave microwells (NCMs) with a controllable size and shape. The key to the fabrication process was the use of an array of hemispherical convex electrolyte solution drops as the grounded collector for electrospinning, which greatly improved the degree of freedom of the size, shape, and curvature of an NCM. A polymer substrate with through-holes was prepared for the electrolyte solution to come out through the hole and to naturally form a convex shape because of surface tension. Subsequent electrolyte-assisted electrospinning process enabled to achieve various arrays of NCMs of triangular, rectangular, and circular shapes with sizes ranging from 1000 µm down to 250 µm. As one example of biomedical applications, the formation of human hepatoma cell line (HepG2) spheroids was demonstrated on the NCMs. The results indicated that the NCM enabled uniform, size-controllable spheroid formation of HepG2 cells, resulting in 1.5 times higher secretion of albumin from HepG2 cells on the NCM on day 14 compared with those on a nanofibrous flat microwell as a control.

Aquatic flower-inspired cell culture platform with simplified medium exchange process for facilitating cell-surface interaction studies.

Establishing fundamentals for regulating cell behavior with engineered physical environments, such as topography and stiffness, requires a large number of cell culture experiments. However, cell culture experiments in cell-surface interaction studies are generally labor-intensive and time-consuming due to many experimental tasks, such as multiple fabrication processes in sample preparation and repetitive medium exchange in cell culture. In this work, a novel aquatic flower-inspired cell culture platform (AFIP) is presented. AFIP aims to facilitate the experiments on the cell-surface interaction studies, especially the medium exchange process. AFIP was devised to capture and dispense cell culture medium based on interactions between an elastic polymer substrate and a liquid medium. Thus, the medium exchange can be performed easily and without the need of other instruments, such as a vacuum suction and pipette. An appropriate design window of AFIP, based on scaling analysis, was identified to provide a criterion for achieving stability in medium exchange as well as various surface characteristics of the petal substrates. The developed AFIP, with physically engineered petal substrates, was also verified to exchange medium reliably and repeatedly. A closed structure capturing the medium was sustained stably during cell culture experiments. NIH3T3 proliferation results also demonstrated that AFIP can be applied to the cell-surface interaction studies as an alternative to the conventional method.

The effect of adenovirus-IkappaBalpha transduction on the chemosensitivity of lung cancer cell line with resistance to cis-diamminedichloroplatinum(II)(cisplatin) and doxorubicin(adriamycin).

Resistance to chemotherapeutic agents is the major reason for treatment failure of cancer chemotherapy. Some chemotherapeutic drugs induce the activation of NF-kappaB in cancer cells that results in their resistance to anticancer drugs. But the role of NF-kappaB in acquired resistance has not been well investigated. In this study, we transferred the "super-repressor" form of the NF-kappaB inhibitor by adenoviral vector (ad-IkappaBalpha) to human lung cancer cell lines with resistant to cisplatin (PC-14-DDP) and adriamycin (PC-14-ADR), and observed the sensitivity change. Electrophoretic mobility shift assay showed that ad-IkappaBalpha blocked the activation of NF-kappaB induced by cisplatin and adriamycin. Transduction with ad-IkappaBalpha restored the sensitivity of cisplatin and adriamycin resistant lung cancer cell lines (PC-14-DDP and PC-14-ADR) to a level compatible to the parental cell lines. Annexin-V analysis suggested that the enhancement of chemosensitivity was probably a result of the induction of apoptosis. These data demonstrated that ad-IkappaBalpha blockade of chemotherapeutic induced NF-kappaB activation increased apoptosis induction and the chemosensitivity of lung cancer cell lines with acquired resistance to cisplatin and adriamycin. Therefore, gene transfer of IkappaBalpha-SR seems to represent a new therapeutic strategy for the solution of low sensitivity and lung cancer resistance to anticancer drugs.

Inhibitory effect of adenovirus-uteroglobin transduction on the growth of lung cancer cell lines.

Uteroglobin is a secretory protein synthesized by most epithelia, including the respiratory tract. It has strong anti-inflammatory properties that appear to be related to the inhibition of phospholipase A2. Recent experimental evidence indicates that uteroglobin has an inhibitory effect on the proliferation and invasion of cancer cells. We investigated the effects of the adenovirus-uteroglobin (ad-UG) transduction on the growth of lung cancer cell lines, which did not express the uteroglobin gene. Upon transduction of ad-UG, the rate of cell growth and the ability to produce colonies in soft agar were evaluated. Cell cycle analysis, Western blot for cell cycle-related proteins and annexin V staining for apoptosis were carried out to see if they were associated with the changes in cell growth. All the tested lung cancer cell lines did not express the uteroglobin gene. The growth rates, and colony-forming ability of transformed cells, were significantly inhibited by the induction of uteroglobin gene expression. The DNA histogram showed that the cell fraction of the G2/M phase was increased, and this G2/M phase arrest was related to a decrease of cdk1 and cyclin A. However, a fraction of apoptotic cells were same as the control. From these results, uteroglobin is thought to have an inhibitory effect on the growth of lung cancer cells. This suggests a potential role for uteroglobin in gene therapy for lung cancer.