Temperature-responsive Cell Culture Surfaces Research Articles

Engineered Bone Marrow Stem Cell-Sheets Alleviate Renal Damage in a Rat Chronic Glomerulonephritis Model

Although mesenchymal stem cell (MSC)-based regenerative therapy is being developed for the treatment of kidney diseases, cell delivery and engraftment still need to be improved. Cell sheet technology has been developed as a new cell delivery method, to recover cells as a sheet form retaining intrinsic cell adhesion proteins, which promotes its transplantation efficiency to the target tissue. We thus hypothesized that MSC sheets would therapeutically reduce kidney disease with high transplantation efficiency. When the chronic glomerulonephritis was induced by two injections of the anti-Thy 1.1 antibody (OX-7) in rats, the therapeutic efficacy of rat bone marrow stem cell (rBMSC) sheet transplantation was evaluated. The rBMSC-sheets were prepared using the temperature-responsive cell-culture surfaces and transplanted as patches onto the surface of two kidneys of each rat at 24 h after the first injection of OX-7. At 4 weeks, retention of the transplanted MSC-sheets was confirmed, and the animals with MSC-sheets showed significant reductions in proteinuria, glomerular staining for extracellular matrix protein, and renal production of TGFß1, PAI-1, collagen I, and fibronectin. The treatment also ameliorated podocyte and renal tubular injury, as evidenced by a reversal in the reductions of WT-1, podocin, and nephrin and by renal overexpression of KIM-1 and NGAL. Furthermore, the treatment enhanced gene expression of regenerative factors, and IL-10, Bcl-2, and HO-1 mRNA levels, but reduced TSP-1 levels, NF-kB, and NAPDH oxidase production in the kidney. These results strongly support our hypothesis that MSC-sheets facilitated MSC transplantation and function, and effectively retarded progressive renal fibrosis via paracrine actions on anti-cellular inflammation, oxidative stress, and apoptosis and promoted regeneration.

Influence of poly(N-isopropylacrylamide) (PIPAAm) graft density on properties of PIPAAm grafted poly(dimethylsiloxane) surfaces and their stability

A previous report shows that poly(N-isopropylacrylamide) (PIPAAm) gel grafted onto poly(dimethylsiloxane) (PDMS) (PI-PDMS) surfaces with large PIPAAm graft density (Lar-PI-PDMS), is prepared by using electron beam irradiation, demonstrating that applied mechanical stretching affects properties of the Lar-PI-PDMS surface. However, the influence of PIPAAm graft density on the properties of PI-PDMS surfaces and their stability are not understood. To provide insight into these points, the properties of PI-PDMS surfaces with low PIPAAm graft density (Low-PI-PDMS) surfaces with stretched (stretch ratio = 20%) and unstretched states were examined as stretchable temperature-responsive cell culture surface using contact angle measurement and cell attachment/detachment assays, compared to those with Lar-PI-PDMS, as previously reported. Long-term contact angle measurements (61 days) for unstretched Low-PI-PDMS and Lar-PI-PDMS surfaces indicated that the cross-linked structure of the grafted PIPAAm gel suppressed hydrophobic recovery of the basal PDMS surface. The cell attachment assay revealed that the stretched Low-PI-PDMS surface was less cell adhesive than that of the unstretched Low-PI-PDMS surface despite of a larger amount of adsorbed fibronectin (FN). The lower cell adhesiveness was possibly explained by denaturation of adsorbed FN, which was induced by the strong hydrophobic property of the stretched Low-PI-PDMS surface. The cell detachment assay revealed that dual stimuli, low temperature treatment and mechanical shrinking stress applied to the stretched Low-PI-PDMS surface promoted cell detachment compared to a single stimulus, low temperature treatment or mechanical shrinking stress. These results suggested that the PIPAAm gelgrafted PDMS surface was chemically stable and did not suffer from hydrophobic recovery. External mechanical stretching stress not only strongly dehydrated grafted PIPAAm chains, but also denatured the adsorbed FN when the grafted PIPAAm layer was extremely thin, as in Low-PI-PDMS surfaces. Thus, PI-PDMS may be utilized as a stretchable temperature-responsive cell culture surface without significant hydrophobic recovery.

Design of Temperature-Responsive Cell Culture Surfaces for Cell Sheet Engineering.

Temperature-responsive cell culture surfaces, which modulate cell attachment/detachment characteristics with temperature, have been used to fabricate cell sheets. Extensive study on fabrication of cell sheet with the temperature-responsive cell culture surface, manipulation, and transplantation of the cell sheet has established the interdisciplinary field of cell sheet engineering, in which engineering, biological, and medical fields closely collaborate. Such collaboration has pioneered cell sheet engineering, making it a promising and attractive technology in tissue engineering and regenerative medicine. This review introduces concepts of cell sheet engineering, followed by designs for the fabrication of various types of temperature-responsive cell culture surfaces and technologies for cell sheet manipulation. The development of various methods for the fabrication of temperature-responsive cell culture surfaces was also summarized. The availability of cell sheet engineering for the treatment and regeneration of damaged human tissue has also been described, providing examples of the clinical application of cell sheet transplantation in humans.

Phenotypic traits of mesenchymal stem cell sheets fabricated by temperature-responsive cell culture plate: structural characteristics of MSC sheets

BackgroundIn most stem cell therapy strategies reported to date, stem cells are introduced to damaged tissue sites to repair and regenerate the original tissue structure and function. MSC therapeutic efficacies are inconsistent, largely attributed to transplanted MSC difficulties both in engrafting at tissue sites and in retaining their therapeutic functions from suspension formulations. MSC functional components, including cell adhesion and cell–cell junction proteins, and ECM that contribute to essential cellular therapeutic effects, are damaged or removed by proteolytic enzymes used in stem cell harvesting strategies from culture. To overcome these limitations, methods to harvest and transplant cells without disrupting critical stem cell functions are required. Cell sheet technology, exploiting temperature-responsive cell culture surfaces, permits cell harvest without cell protein damage. This study is focused on phenotypic traits of MSC sheets structurally and functionally to understand therapeutic benefits of cell sheets.Methods/resultsThis study verified cleaved cellular proteins (vinculin, fibronectin, laminin, integrin β-1, and connexin 43) and increased apoptotic cell death produced under standard trypsin harvesting treatment in a time-dependent manner. However, MSC sheets produced without trypsin using only temperature-controlled sheet harvest from culture plastic exhibited intact cellular structures. Also, MSCs harvested using enzymatic treatment (i.e., chemical disruption) showed higher pYAP expression compared to MSC sheets.ConclusionRetention of cellular structures such as ECM, cell–cell junctions, and cell–ECM junctions is correlated with human umbilical cord mesenchymal stem cell (hUC-MSC) survival after detachment from cell culture surfaces. Retaining these proteins intact in MSC cultures using cell sheet technology is proposed to enhance stem cell survival and their function in stem cell-based therapy.

Cell sheet tissue engineering: Cell sheet preparation, harvesting/manipulation, and transplantation.

Cell sheet tissue engineering is a concept for creating transplantable two-dimensional (2D) and three-dimensional (3D) tissues and organs. This review describes three elements of cell sheet tissue engineering in terms of the chemical and physical effects of material surfaces and the interfacial properties of cell sheets: preparation, harvesting/manipulation and transplantation of cell sheets. An essential technology for the preparation of cell sheets is the use of a temperature-responsive cell culture surface, where the surface of tissue culture polystyrene (TCPS) dish is modified with thin layer of temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). PIPAAm-immobilized TCPS allows cultured cells to be harvested as a contiguous cell sheet with extracellular matrices (ECMs) by reducing the temperature, while chemical and physical disruption impair ECMs, cell-cell junction, and membrane proteins. Ligand-immobilized and porous hydrophilic PIPAAm-grafted surfaces are able to accelerate cell sheet preparation and harvesting, respectively. In addition, the manipulation of harvested cell sheets with the aid of cell sheet manipulator facilitates the formation of 3D tissues. Cell sheet-based tissues and their transplantation are in seven clinical settings such as heart, cornea, esophagus, periodontal, middle chamber of ear, knee cartilage, and lung. In order to create thick and large 3D tissues and organs, large production of differentiated parenchymal cells from induced pluripotent stem (iPS) cells and vascularization within 3D tissues are key issues. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 955-967, 2019.

Temperature-responsive culture surfaces for insect cell sheets to fabricate a bioactuator

ABSTRACTFor the first time, we have fabricated insect-derived cell sheets by using temperature-responsive culture surfaces having a phase-transition temperature below 25°C. We prepared the temperature-responsive cell culture surfaces (tissue culture polystyrene, TCPS) by grafting a copolymeric gel consisting of hydrophobic N-tert-butylacrylamide (tBAAm) and N-isopropylacrylamide (IPAAm) units. First, to characterize the hydrophilic and hydrophobic properties of the copolymeric gel-grafted surfaces, static water contact angles of each surface were measured at various temperatures. By increasing the amount of tBAAm in the grafted copolymeric gel, the transition temperature of the gel was shifted to lower temperatures. At 25°C, the grafted copolymeric gel was dehydrated, and the insect-derived cells (AeAl2 cells) adhered on all the copolymeric gel-grafted surfaces. At 20°C, AeAl2 cells cannot adhere on the P(IPAAm-1.62tBAAm)-TCPS surface (the initial molar ratio of IPAAm and tBAAm (tBAAm = 1.62 mol%)) better than on other surfaces (TCPS and tBAAm = 4.88, 8.17 mol%). These two findings implied that the lower critical solution temperature of the copolymeric gel-grafted-TCPS existed from 20°C to 25°C. The laminin-coated P(IPAAm-1.62tBAAm)-TCPS surface showed temperature-dependent cell attachment and detachment properties, while AeAl2 cells were not detached from the extracellular matrix uncoated P(IPAAm-1.62tBAAm)-TCPS surface. AeAl2 cells and insect muscle cells were harvested as the respective sheets.

Poly( N-isopropylacrylamide)-Grafted Polydimethylsiloxane Substrate for Controlling Cell Adhesion and Detachment by Dual Stimulation of Temperature and Mechanical Stress.

Stretchable temperature-responsive cell culture surfaces composed of poly( N-isopropylacrylamide) (PIPAAm) gel-grafted polydimethylsiloxane (PIPAAm-PDMS) were prepared to demonstrate that dual stimulation of temperature and mechanical stress extensively altered graft polymer thickness, surface wettability, and cell detachment behavior. The PIPAAm-PDMS surface was hydrophilic and hydrophobic below and above the lower critical solution temperature, respectively, which was ascribed to the phase transition of PIPAAm chains. When uniaxial stretching was applied, the grafted PIPAAm gel surface was modulated to be more hydrophobic as shown by an increase in the contact angle. Atomic force microscopy observation revealed that uniaxial stretching made the grafted gel layer thinner and deformed the nanoscale aggregates of the grafted PIPAAm gel, implying extension of the PIPAAm chains. The stretched PIPAAm-PDMS became more cell adhesive than the unstretched PIPAAm-PDMS at 37 °C. Furthermore, dual stimulation, shrinking the already stretched PIPAAm-PDMS and decreasing the temperature, induced more rapid cell detachment than only a change in temperature did. Similarly, upon comparison with a single stimulation of a change in temperature or mechanical stress, dual stimulation accelerated cell sheet detachment and harvesting. This new stretchable and temperature-responsive culture surface can easily adjust the surface property to a different cell adhesiveness by appropriately combining each stimulus and enable the fabrication of cell sheets of various species.

Temperature-Responsive Cell Culture Surface for Cell-Sheet Tissue Engineering and Its Design to Express Temperature-Dependent Cell Attachment/Detachment Character

Temperature-Responsive Cell Culture Surface for Cell-Sheet Tissue Engineering and Its Design to Express Temperature-Dependent Cell Attachment/Detachment Character

Design of Temperature-Responsive Cell Culture Surfaces for Cell Sheet-Based Regenerative Therapy and 3D Tissue Fabrication.

This chapter describes the concept of "cell sheet engineering" for the creation of transplantable cellular tissues and organs. In contrast to scaffold-based tissue engineering, cell sheet engineering facilitates the reconstruction of scaffold-free, cell-dense tissues. Cell sheets were harvested by changing the temperature of thermoresponsive cell culture surfaces modified with poly(N-isopropylacrylamide) (PIPAAm) with a thickness on the nanometer scale. The transplantation of 2D cell sheet tissues has been used in clinical settings. Although 3D tissues were formed simply by layering 2D cell sheets, issues related to vascularization within 3D tissues and the large-scale production of cells must be addressed to create thick and large 3D tissues and organs.

A Facile Method for Preparing Temperature‐Responsive Cell Culture Surfaces by Using a Thioxanthone Photoinitiator Immobilized on a Polystyrene Surface

AbstractWe developed a new facile method for the preparation of temperature‐responsive cell culture surfaces (TRCS) by photo‐polymerization of N‐isopropylacrylamide using a surface‐immobilized photoinitiator. In the first step, thioxanthone groups, which served as the photoinitiator, were directly formed on polystyrene culture dish surfaces (TX‐PSt). Second, poly(N‐isopropylacrylamide) (PIPAAm) was grafted onto the TX‐PSt surfaces (PIPAAm‐TX‐PSt) by using the visible light or light‐emitting diode irradiation‐induced surface‐initiated polymerization method. By optimizing the amount of grafted PIPAAm, the resulting PIPAAm‐TX‐PSt successfully exhibited characteristics of a TRCS. In addition, a striped micropatterned polyacrylamide area was successively formed on the photoirradiated PIPAAm‐TX‐PSt surface by using a chromium photomask. These results show that temperature‐responsive cell culture surfaces can be prepared through this new method.

Mechanical stretching induced alternation of cell attachment and detachment character of poly(N-isopropylacrylamide) gel grafted PDMS surface

Event Abstract Back to Event Mechanical stretching induced alternation of cell attachment and detachment character of poly(N-isopropylacrylamide) gel grafted PDMS surface Yoshikatsu Akiyama1, Miki Matsuyama2, Naoya Takeda2, Masayuki Yamato1 and Teruo Okano1 1 Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Japan 2 Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University, Japan Introduction: Temperature-responsive cell culture surface, poly(N-isopropylacrylamide) (PIPAAm) hydrogel grafted tissue culture polystyrene (PIPAAm-TCPS), has been utilized for cell-sheet based tissue-engineering. PIPAAm-TCPS shows temperature-dependent cell attachment and detachment character, which is one of characteristics of PIPAAm-TCPS[1]. Based on a hypothesis that thickness of graft PIPAAm hydrogel layer were altered by applying mechanical stress to PIPAAm gel grafted substrate (Fig. 1), we modified PDMS surfaces with ultra-thin PIPAAm hydrogel (PIPAAm-PDMS) and characterized stretched and unstretched PIPAAm-PDMS surfaces as a new stretchable temperature-responsive cell culture surfaces. Experimental Methods: PIPAAm-PDMS was prepared referring to a previous report[2]. IPAAm monomer dissolved in 2-propanol (monomer concentration, 40 wt%) was spread onto 4-well PDMS cell culture surfaces (each chamber size was 16 mm square), which was purchased from STREX, Inc., followed by O2 plasma and (3-aminoproyl)trimethoxysilane (APTMS) treatments. And then, the surface was subjected to EB irradiation. During EB irradiation, polymerization and grafting polymer on PDMS surface occurred simultaneously. PIPAAm-PDMS was characterized by FT-IR / ATR, XPS analysis, contact angle measurement and cell detachment / attachment assay. For the cell attachment and detachment assay, PIPAA-PDMS with about 10.0 ug/cm2 of PIPAAm graft density was used. Uniaxial mechanical-stress was applied to the PIPAAm-PDMS surfaces with a special device (purchased from STEX, Inc.). In this experiment, 20% of stretch ratio was used for the characterization of stretched PIPAAm-PDMS surface. Results: To prove a hypothesis, PDMS was selected as a model elastic substrate in this experiment. Uniaxially stretched and unstretched PIPAAm-PDMS surfaces were evaluated by a variety of surface analysis methods. By modifying PDMS with PIPAAm hydrogel, contact angle decreased from 110º (unstretched PDMS) to 78º (unstretched PIPAAm-PDMS) at 37ºC (Fig. 2). The stretched and unstrethced PIPAAm-PDMS surfaces showed temperature-dependent contact angles alternation. However, by applying 20% of uniaxial mechanical stretching stress to PIPAAm-PDMS, the contact angles increased form 78º (unstretched PIPAAm-PDMS) to 83º (stretched PIPAAm-PDMS). AFM image of PIPAAm-PDMS surface showed PIPAAm nano-particles, which uniformly covered PDMS surface. The thickness of the graft PIPAAm hydrogel layer decreased from 700 nm to 480 nm (Fig. 3). Considering that temperature-responsive cell culture surfaces with thinner PIPAAm hydrogel layer shows more hydrophobic property, that PIPAAm-PDMS surface properties were altered by the applied mechanical stretching stress. Cell attachment assay also suggested that the stretched PIPAAm-PDMS was more cells adhesive. These results possibly suggest that PIPAAm-PDMS surface properties are controllable by the degree of applied mechanical stress. Grant-in-Aid for Scientific Research (No. 23106009) in Innovative Areas "Bio Assembler" from MEXT

Measurement of the dynamic behavior of thin poly(N-isopropylacrylamide) hydrogels and their phase transition temperatures measured using reflectometric interference spectroscopy

Temperature-responsive cell culture surfaces prepared by modifying tissue-culture polystyrene with nanoscale poly(N-isopropylacrylamide) (PIPAAm) hydrogels are widely used as intelligent surfaces for the fabrication of various cell sheets that change with temperature. In this work, the characteristics of nanoscale PIPAAm hydrogels were phenomenologically elucidated on the basis of time-dependent surface evaluations under conditions of changing temperature. Because the dynamic characteristics of the nanoscale hydrogel did not exhibit good performance, the nanoscale PIPAAm hydrogel was analyzed by monitoring its temperature-dependent dynamic swelling/deswelling changes using reflectometric interference spectroscopy (RIfS) on an instrument equipped with a microfluidic system. RIfS measurements under ambient atmosphere provided the precise physical thickness of the dry PIPAAm hydrogel (6.7 nm), which agreed with the atomic force microscopy results (6.6 nm). Simulations of the reflectance spectra revealed that changes in the wavelength of the minimum reflectance (Δλ) were attributable to the changes in the refractive index of the thin PIPAAm hydrogel induced by a temperature-dependent volume phase transition. The temperature-dependent Δλ change was used to monitor the swelling/deswelling behavior of the nanoscale PIPAAm hydrogel. In addition, the phase transition temperature of the thin PIPAAm hydrogel under aqueous conditions was also determined to be the inflection point of the plot of the change in Δλ as a function of temperature. The dynamic behavior of a thin PIPAAm hydrogel chemically deposited on a surface was readily analyzed using a new analytical system with RIfS and microfluidic devices.

Recent development of temperature-responsive surfaces and their application for cell sheet engineering

Cell sheet engineering, which fabricates sheet-like tissues without biodegradable scaffolds, has been proposed as a novel approach for tissue engineering. Cells have been cultured and proliferate to confluence on a temperature-responsive cell culture surface at 37°C. By decreasing temperature to 20°C, an intact cell sheet can be harvested from the culture surface without enzymatic treatment. This new approach enables cells to keep their cell–cell junction, cell surface proteins and extracellular matrix. Therefore, recovered cell sheet can be easily not only transplanted to host tissue, but also constructed a three-dimensional (3D) tissue by layering cell sheets. Moreover, cell sheet manipulation technology and bioreactor have been combined with the cell sheet technology to fabricate a complex and functional 3D tissue in vitro. So far, cell sheet technology has been applied in regenerative medicine for several tissues, and a number of clinical studies have been performed. In this review, recent advances in the preparation of temperature-responsive cell culture surface, the fabrication of organ-like tissue and the clinical application of cell sheet engineering are summarized and discussed.

Evidence of the Survival of Ectopically Transplanted Oral Mucosal Epithelial Stem Cells After Repeated Wounding of Cornea

Tissue engineering has become an essential tool in the development of regenerative medicine. We have developed cell sheet-based techniques for use in regenerative medicine that have already been successfully used in clinical applications. Native corneal epithelium is produced from limbal stem cells located in the transition zone between the cornea and the bulbar conjunctiva. Limbal stem cell deficiency (LSCD) is a severe defect of the limbal stem cells leading to vision loss due to conjunctival epithelial invasion and neovascularization. Rabbit LSCD models were treated with transplantable autologous oral mucosal epithelial cell (OEC) sheets fabricated on temperature-responsive cell culture surfaces, after which, the ocular surfaces were clear and smooth with no observable defects. The central part of the reconstructed ocular surface was scraped and wounded, after which proliferating epithelial cells covered the scraped area within a few days. The ocular surfaces were clear and smooth even after repeated scrapings and consisted of only OECs or heterogeneously mixed with corneal epithelial cells. This study demonstrates that transplanted cell sheets containing oral mucosal epithelial stem cells could reconstruct the ocular surface to maintain cornea homeostasis; moreover, they provide an ideal microenvironment to support the proliferation of remaining native limbal stem cells.

Accelerated cell-sheet recovery from a surface successively grafted with polyacrylamide and poly(N-isopropylacrylamide)

A double polymeric nanolayer consisting of poly(N-isopropylacrylamide) (PIPAAm) and hydrophilic polyacrylamide (PAAm) was deposited on tissue culture polystyrene (TCPS) surfaces using electron beam irradiation to form a new temperature-responsive cell culture surface in which the basal hydrophilic PAAm component in the double polymeric layer promotes the hydration of the upper PIPAAm layer and induces rapid cell detachment compared to a conventional temperature-responsive cell culture surface, PIPAAm-grafted TCPS (PIPAAm-TCPS). Take-off angle-dependent X-ray photoelectron spectroscopy spectral analysis demonstrated that the grafted PIPAAm and PAAm components were located in the upper and basal regions of the double polymeric layer, respectively, suggesting that the double polymeric layer forms an inter-penetrating-network-like structure with PAAm at the basal portion of the PIPAAm grafted chains. The wettability of the temperature-responsive cell culture surfaces with the double polymeric layer tended to be more hydrophilic, with an increase in the basal PAAm graft density at a constant PIPAAm graft density. However, when the graft densities of the upper PIPAAm and basal PAAm were optimized, the resulting temperature-responsive cell culture surface with the double polymeric layer exhibited rapid cell detachment while maintaining cell adhesive character comparable to that of PIPAAm-TCPS. The cell adhesive character was altered from cell-adhesive to cell-repellent with increasing PAAm or PIPAAm graft density. The cell adhesive character of the temperature-responsive cell culture surfaces was relatively consistent with their contact angles. These results strongly suggest that the basal PAAm surface properties affect the degree of hydration and dehydration of the subsequently grafted PIPAAm. In addition, the roles of the hydrophilic component in accelerating cell detachment are further discussed in terms of the mobility of the grafted PIPAAm chains. Applications of this insight might be useful for designing temperature-responsive cell culture surfaces for achieving efficient cell culture and quick target cell detachment.

Cell sheets engineering for esophageal regenerative medicine.

Recently, cell-based therapies, regenerative medicine, and tissue engineering have been progressing rapidly. We have developed a novel strategy for regenerative medicine to recover tissue functions using temperature-responsive cell culture surfaces. To overcome of conventional methods such as the usage of single-cell suspension injection, we have applied transplantable cell sheets fabricated with temperature-responsive culture surfaces for cell delivery. In the field of gastroenterology, transplantable cell sheets from autologous oral mucosal epithelial cells can prevent esophageal stricture following extensive endoscopic mucosal resection.

Preparation of Poly(N-isopropylacrylamide) Grafted Polydimethylsiloxane by Using Electron Beam Irradiation

A poly(N-isopropylacrylamide) (PIPAAm) grafted poly(dimethylsiloxane) (PDMS) surface was prepared as a temperature-responsive cell culture surface by using electron beam (EB) irradiation. Different chemical treatments to modify the bare PDMS surface were investigated for subsequent grafting of PIPAAm, and treatment conditions were optimized to prepare the temperature-responsive cell culture surface. The PDMS surface was initially activated to form silanol groups with conventional O2 plasma or hydrochloric acid (HCl) treatment. Activated PDMS surfaces were individually immobilized with three different conventional silane compounds, i.e., 3-mercaptopropyltrimethoxysilane (MerTMS), 3-methacryloxypropyltrimethoxysilane (MetTMS), and 3-aminopropyltrimethoxysilane (AmiTMS). O2 plasma treatment made PDMS more hydrophilic. In contrast, PDMS surfaces activated with HCl treatment were relatively hydrophobic. Observation of the activated PDMS surface modified with MerTMS, MetTMS, and AmiTMS indicated that these silane compounds had been favorably immobilized on plasma-treated PDMS surfaces. FT-IR/ATR analysis demonstrated that immobilized silane compounds enabled PIPAAm grafting on the PDMS surface. Cell attachment and detachment analysis also suggested that the PDMS surface sequentially treated with O2 plasma and AmiTMS compound was a substrate appropriate for preparing a temperature-responsive cell culture surface by EB irradiation-induced PIPAAm grafting method. The intelligent surface may further be applied to mechanically stretchable temperature-responsive cell culture surfaces.

Hormone supplying renal cell sheet in vivo produced by tissue engineering technology.

Regenerative medicine is a new medical field and is expected to have a profoundly positive effect in curing difficult-to-treat diseases. Cell sheet fabrication is an important tissue engineering technology used in regenerative medicine. This study investigated the creation of a hormone-releasing tissue using cell sheet technology, which could be utilized in future therapy for chronic renal disease. Renal cell sheets were fabricated on a temperature-responsive cell culture surface with primary renal cells from adult porcine kidney. These sheets contained various kinds of renal cells that showed cyst-like formation. An important renal function is the synthesis of 1,25-dihydroxyvitamin D3, and this was confirmed in the cell sheets in vitro. Erythropoietin (EPO) production is another important renal function. This ability was also observed in the renal cell sheets in vitro, and then again after transplantation in a nude rat. In particular, the relative expression of EPO mRNA increased more under cell sheet culture conditions compared with exponential cell growth conditions. Histological analysis of the implanted renal cell sheets showed them to be Dolichos biflorus agglutinin-positive and to have regenerated renal tubular-like morphology. These results indicated that both functional and morphological regenerative renal tissues were fabricated by cell sheet technology. This study introduces a hormone-supplying treatment for renal dysfunctional diseases using engineered renal tissues. Moreover, since our renal cell sheets developed renal tubular-like structures in vivo, it holds promise for fabricating artificially engineered true renal tissue in the future.

Shear stress-dependent cell detachment from temperature-responsive cell culture surfaces in a microfluidic device

A new approach to quantitatively estimate the interaction between cells and material has been proposed by using a microfluidic system, which was made of poly(dimethylsiloxane) (PDMS) chip bonding on a temperature-responsive cell culture surface consisted of poly(N-isopropylacrylamide) (PIPAAm) grafted tissue culture polystyrene (TCPS) (PIPAAm-TCPS) having five parallel test channels for cell culture. This construction allows concurrently generating five different shear forces to apply to cells in individual microchannels having various resistance of each channel and simultaneously gives an identical cell incubation condition to all test channels. NIH/3T3 mouse fibroblast cells (MFCs) and bovine aortic endothelial cells (BAECs) were well adhered and spread on all channels of PIPAAm-TCPS at 37 °C. In our previous study, reducing culture temperature below the lower critical solution temperature (LCST) of PIPAAm (32 °C), cells detach themselves from hydrated PIPAAm grafted surfaces spontaneously. In this study, cell detachment process from hydrated PIPAAm-TCPS was promoted by shear forces applied to cells in microchannels. Shear stress-dependent cell detachment process from PIPAAm-TCPS was evaluated at various shear stresses. Either MFCs or BAECs in the microchannel with the strongest shear stress were found to be detached from the substrate more quickly than those in other microchannels. A cell transformation rate constant Ct and an intrinsic cell detachment rate constant k0 were obtained through studying the effect of shear stress on cell detachment with a peeling model. The proposed device and quantitative analysis could be used to assess the possible interaction between cells and PIPAAm layer with a potential application to design a cell sheet culture surface for tissue engineering.

Prevention of Esophageal Stricture After Endoscopic Submucosal Dissection Using Tissue-Engineered Cell Sheets

The use of esophageal endoscopic submucosal dissection (ESD) to remove superficial esophageal neoplasms is gradually becoming more common in Japan. However, large-scale esophageal ESD often requires subsequent multiple balloon dilations to prevent postoperative esophageal stricture. We investigated the safety and efficacy of endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in preventing formation of strictures after ESD. We performed an open-label, single-arm, single-institute study. We collected specimens of oral mucosal tissue from 9 patients with superficial esophageal neoplasms. Epithelial cell sheets were fabricated ex vivo by culturing isolated cells for 16 days on temperature-responsive cell culture surfaces. After a reduction in temperature, these sheets were endoscopically transplanted directly to the ulcer surfaces of patients who had just undergone ESD. All patients were monitored by endoscopy once a week until epithelialization was complete. Autologous cell sheets were successfully transplanted to ulcer surfaces using an endoscope. Complete re-epithelialization occurred within a median time of 3.5 weeks. No patients experienced dysphagia, stricture, or other complications following the procedure, except for one patient who had a full circumferential ulceration that expanded to the esophagogastric junction. Sutureless, endoscopic transplantation of carrier-free cell sheets composed of autologous oral mucosal epithelial cells safely and effectively promotes re-epithelialization of the esophagus after ESD. Patients in this study did not experience any serious complications. This procedure might be used to prevent stricture formation following ESD and improve patients' quality of life. Further study will be needed to show that stricture formation can be prevented.