Sheet Engineering Research Articles

Allogeneic mesenchymal stem cell sheet therapy: A new frontier in drug delivery systems

The evolution of drug discovery exploded in the early 20th century with the advent of critical scientific advancements in organic chemistry, chemical analysis, and purification. Early drug generations focused largely on symptom control and pain management, effective targets for small-molecule drugs. Recently, the attention in drug discovery has shifted to pursuit of radical cures. Cell therapy presents the ideal attributes of a promising new drug, targeting specific tissues based on chemotactic cues and modulating secretion of instructive regenerative molecules in response to dynamic signaling from disease environments. To actuate the therapeutic potential of cell therapy toward worldwide clinical use, cell delivery methods that can effectively localize and engraft mesenchymal stem cells (MSCs) with high disease-site fidelity and enable dynamic MSC bioactive function are paramount. In this review, we discuss the evolution of cell therapies with a focus on stem cell advantages, as well as the limitations to these therapies. This review aims to introduce cell sheet technology as a breakthrough cell therapy with demonstrated therapeutic success across indications for heart, liver, and kidney tissue regeneration. Opportunities and anticipated clinical impacts of cell sheet technology using MSCs are discussed.

Hepatic cell-sheet fabrication of differentiated mesenchymal stem cells using decellularized extracellular matrix and thermoresponsive polymer

PurposeLiver tissue engineering via cell sheet technology would open new doors for treatment of patients with liver failure. Decellularized tissues could provide sufficient extracellular matrix (ECM) to support development of hepatocytes in in vivo niches. Besides, with the potential of temperature responsive polymer (pNIPAAm) as an intelligent surface for controlling the attachment/detachment of cell, we set out to generate three in vitro microenvironments models including I: pNIPAAm hydrogel (pN hydrogel), II: decellularized ECM incorporated into pNIPAAm hydrogel (dECM + pN hydrogel) and III: decellularized ECM scaffold (dECM scaffold) to investigate the structural and function cues of hepatocyte-like cells after differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) on the surface of these models. MethoddECM scaffold was obtained after decellularization of rat liver, and its efficiency was analyzed. pN hydrogel and dECM + pN hydrogel (1:3 and 2:3 ratios) of were fabricated, and scaffold architecture was characterized. Each well of culturing plates was coated separately with these three constructs and AT-MSCs were instructed to differentiate into hepatocyte-like cells (HLCs). After recellularization, patterns of differentiation, and expression of hepatogenic markers were investigated via biochemical assays and qRT-PCR at different time points. ResultsMultipotency of AT-MSCs, after their ability for osteogenesis and adipogenesis was documented. Production of dense and intact cell sheets was reported in dECM + pN hydrogel, as opposed to pN hydrogel and dECM scaffold. Also, statistically significant difference of HLCs functionality in dECM + pN hydrogel was confirmed after evaluation of the expression of hepatocyte markers including, alpha-fetoprotein, cytokeratin 18, cytochrome P450-2E1 and phosphoenolpyruvate carboxykinase. ConclusionOur results proved dECM + pN hydrogel were able to preserve hepatocyte function in cell sheets owing to the high level of albumin, urea, hepatogenic markers, and glycogenesis potential of HLCs. Accordingly, dECM incorporated in pN hydrogel could remodel microenvironments to guide the AT-MSCs into conducive differentiation and proliferation to give rise to multilayer sheets of cells in their own ECM.

Fabrication of hyaline-like cartilage constructs using mesenchymal stem cell sheets

Cell and tissue engineering approaches for articular cartilage regeneration increasingly focus on mesenchymal stem cells (MSCs) as allogeneic cell sources, based on availability and innate chondrogenic potential. Many MSCs exhibit chondrogenic potential as three-dimensional (3D) cultures (i.e. pellets and seeded biomaterial scaffolds) in vitro; however, these constructs present engraftment, biocompatibility, and cell functionality limitations in vivo. Cell sheet technology maintains cell functionality as scaffold-free constructs while enabling direct cell transplantation from in vitro culture to targeted sites in vivo. The present study aims to develop transplantable hyaline-like cartilage constructs by stimulating MSC chondrogenic differentiation as cell sheets. To achieve this goal, 3D MSC sheets are prepared, exploiting spontaneous post-detachment cell sheet contraction, and chondrogenically induced. Results support 3D MSC sheets’ chondrogenic differentiation to hyaline cartilage in vitro via post-contraction cytoskeletal reorganization and structural transformations. These 3D cell sheets’ initial thickness and cellular densities may also modulate MSC-derived chondrocyte hypertrophy in vitro. Furthermore, chondrogenically differentiated cell sheets adhere directly to cartilage surfaces via retention of adhesion molecules while maintaining the cell sheets’ characteristics. Together, these data support the utility of cell sheet technology for fabricating scaffold-free, hyaline-like cartilage constructs from MSCs for future transplantable articular cartilage regeneration therapies.

Novel therapies using cell sheets engineered from allogeneic mesenchymal stem/stromal cells.

Mesenchymal stem/stromal cells (MSCs) have long been recognized to help regenerate tissues, by exploiting their intrinsic potentials for differentiation and secretion of therapeutic paracrine factors together with feasibility for cell banking. These unique MSC properties are attractive to provide effective new cell-based therapies for unmet medical needs. Currently, the infusion of suspended MSCs is accepted as a promising therapy to treat systemic inflammatory diseases. However, low cell engraftment/retention in target organs and off-target entrapment using conventional cell infusion must be improved to provide reliable localized disease treatments. Cell sheet technology offers an alternative: three-dimensional (3D) tissue-like structures can be harvested from culture using mild temperature reduction, and transplanted directly onto target tissue sites without suturing, yielding stable cell engraftment and prolonged cell retention in situ without off-target losses. Engineered MSC sheets directly address two major cell therapy strategies based on their therapeutic benefits: (1) tissue replacements based on mult-ilineage differentiation capacities, focusing on cartilage regeneration in this review, and (2) enhancement of tissue recovery via paracrine signaling, employing their various secreted cytokines to promote neovascularization. MSCs also have production benefits as a promising allogeneic cell source by exploiting their reliable proliferative capacity to facilitate expansion and sustainable cell banking for off-the-shelf therapies. This article reviews the advantages of both MSCs as allogeneic cell sources in contrast with autologous cell sources, and allogeneic MSC sheets engineered on thermo-responsive cell dishes as determined in basic studies and clinical achievements, indicating promise to provide robust new cell therapies to future patients.

Sheet Type All-Solid-State Batteries with Sulfide Electrolyte

Lithium ion batteries have received great attention due to emerging electric vehicle market to suppress global climate change issues. Batteries with higher energy density is required to improve driving distance of electric vehicle. As energy density increase, safety become more important point to prevent unexpected accident. Safety issues of current lithium ion battery system mainly comes from the oxide cathode and flammable organic electrolyte. All solid state batteries with non-flammable inorganic solid electrolytes come into the limelight due to its potential for securing higher energy density and safe characteristics over current lithium ion battery system.In this study, composite electrode sheet and cell fabrication technology was examined by slurry mixing coating technique. Cathode electrode sheets were obtained by coating slurries consisting of LiNi0.8Co0.1Mn0.1O2 as the active material, an argyrodite structure Li6PS5Cl solid electrolyte, conductive carbon, butadiene-based binder with solvent on carbon coated aluminum foil as the current collector. Homogeneous mixing and coating conditions were examined by controlling composition, kinds of binder in order to achieve optimal conduction pathway of lithium ion and electron. All solid state batteries were fabricated and evaluated both half-cell and full-cell type. Electrochemical tests were investigated and the detailed results will be discussed in this presentation.

Preseeding of Mesenchymal Stem Cells Increases Integration of an iPSC-Derived CM Sheet into a Cardiac Matrix.

Cell sheet technology has demonstrated great promise in delivering a large amount of therapeutic cells for tissue repair, including in the myocardium. However, the lack of host integration remains one of the key challenges in using cell sheets for cardiac repair. Paracrine factors secreted by mesenchymal stem cells (MSCs) have been reported to facilitate tissue repair and regeneration in a variety of ways. It has been demonstrated that paracrine factors from MSCs could enhance scaffold recellularization and vascularization. In this study, we used an in vitro cardiac matrix mimic platform to examine the effects of hMSCs preseeding on the interactions between cell sheets and cardiac matrix. The fabricated human induced pluripotent stem cells-derived cardiomyocyte sheets were attached to a decellularized porcine myocardium slice with or without preseeding of hMSCs. The hMSCs preseeding significantly enhanced the interactions between cardiomyocyte sheets and cardiac matrix in terms of cell migration distance, cell distribution, and mature vascular and cardiomyocyte marker expressions in the matrix. Growth factor and matrix metalloproteinases array analysis suggested that hMSCs- induced vascularization and MMPs regulation are the two possible mechanisms that lead to the improved CMs and cardiac matrix interactions. Further examination of these two mechanisms will enable the development of new approaches to facilitate transplanted cells for tissue repair.

Magnetic Rotating Flow of a Hybrid Nano-Materials Ag-MoS2 and Go-MoS2 in C2H6O2-H2O Hybrid Base Fluid over an Extending Surface Involving Activation Energy: FE Simulation

Numeric simulations are performed for a comparative study of magnetohydrodynamic (MHD) rotational flow of hybrid nanofluids (MoS2-Ag/ethyleneglycol-water (50–50%) and MoS2-Go/ethyleneglycol-water (50–50%)) over a horizontally elongated plane sheet. The principal objective is concerned with the enhancement of thermal transportation. The three-dimensional formulation governing the conservation of mass, momentum, energy, and concentration is transmuted into two-dimensional partial differentiation by employing similarity transforms. The resulting set of equations (PDEs) is then solved by variational finite element procedure coded in Matlab script. An intensive computational run is carried out for suitable ranges of the particular quantities of influence. The primary velocity component decreases monotonically and the magnitude of secondary velocity component diminishes significantly when magnetic parameter, rotational parameter, and unsteadiness parameter are incremented. Both the primary and secondary velocities are smaller in values for the hybrid phase Ag-MoS2 than that of hybrid phase Go-MoS2 but the nanoparticle concentration and temperature are higher for hybrid phase Ag-MoS2. The increased values of parameters for thermophoresis, Brownian motion, shape factor, and volume fraction of ϕ2 made significant improvement in the temperature of the two phases of nano liquids. Results are also computed for the coefficients of skin friction(x, y-directions), Nusselt number, and Sherwood number. The present findings manifest reasonable comparison to their existing counterparts. Some of the practical engineering applications of the present analysis may be found in high-temperature nanomaterial processing technology, crystal growing, extrusion processes, manufacturing and rolling of polymer sheets, academic research, lubrication processes, and polymer industry.

Novel pre-vascularized tissue-engineered dermis based on stem cell sheet technique used for dermis-defect healing.

Insufficient donor dermis and the shortage of three-dimensional vascular networks are the main limitations in the tissue-engineered dermis (TED). To solve these problems, we initially constructed pre-vascularized bone marrow mesenchymal stem cell sheet (PBMCS) and pre-vascularized fibroblasts cell sheet (PFCS) by cell sheet technology, and then superimposed or folded them together to construct a pre-vascularized TED (PTED), aiming to mimic the real dermis structure. The constructed PTED was implanted in nude mice dorsal dermis-defect wound and the wound-healing effect was quantified at Days 1, 7 and 14 via the methods of histochemistry and immunohistochemistry. The results showed that PTED could rapidly promote the wound closure, especially at Day 14, and the wound-healing rate of three-layer PTED could reach 97.2% (P < 0.01), which was faster than the blank control group (89.1%), PBMCS (92.4%), PFCS (93.8%) and six-layer PTED (92.3%). In addition, the vessel density in the PTED group was higher than the other groups on the 14th day. Taken together, it is proved that the PTED, especially three-layer PTED, is more conducive to the full-thickness dermis-defect repair and the construction of the three-dimensional vascular networks, indicating its potential application in dermis-defect repair.

Test Method for Friction Characterization of Rivets

Due to continuously increasing requirements on lightweight sheet metal constructions, new processes and technologies are necessary to face current and future challenges. A trend in lightweight construction is the multi-material mix with sheets of different geometric and mechanical properties. To manufacture these so-called composite structures, new joining technologies and methods are required. One possibility is the further development of the self-pierce riveting joining process. For the simulation and evaluation of new and adapted processes of self-pierce riveting, numerical models are used to investigate process parameters and constraints. Besides precise material models for the numerical simulation of this joining process, the identification of friction parameters between the individual sheets and rivet elements is necessary to achieve exact results. Thus, a method is necessary to identify the friction coefficients between rivet elements and sheets. Such a method is presented and evaluated in this work. Different process parameters like the relative speed are varied for the experimental investigations and an analysis of the respective influence on the resulting friction coefficients is conducted. For the use of the test set-up, coefficients of friction are determined for rivet elements “RIVSET® C 5.3x5.5” (Böllhoff GmbH) coated with Almac® in combination with two different sheet materials HCT590X+Z and EN‑AW 6014.

Construction and Evaluation of Osteochondral-Like Tissue Using Chondrocyte Sheet and Cancellous Bone.

The manipulation of human chondrocyte sheets in target areas frequently results in their tearing because they are thin and fragile. In this study, human cancellous bones were used as a supporting material to create chondrocyte sheet-cancellous bone tissues, and their properties were evaluated. Using cell sheet technology, human chondrocytes were constructed into triple-layered chondrocyte sheets that displayed chondrogenic properties. After transferring the chondrocyte sheets onto cancellous bones, the top area of the chondrocyte sheet-cancellous bone tissues exhibited a smooth surface topography without cell sheet floating within 7 days of culture. The immunofluorescence staining of collagen type II (COL2A1) and fibronectin (FN1) was also performed and examined. Using the shotgun proteomic analysis, the proteins associated with cell adhesion, extracellular matrix (ECM) organization, cell-substrate junction assembly, and cell adhesion mediated by integrin were observed in the chondrocyte sheets, cancellous bones, and chondrocyte sheet-cancellous bone tissues. Three integrin members, including integrin β4 (ITGB4), ITGB6, and ITGB8, were found in the chondrocyte sheets. Only ITGB8 was found in the chondrocyte sheets and chondrocyte sheet-cancellous bone tissues. During 48 h, the mean velocity of the individual cell migration was low, which did not affect the structure and chondrogenic properties of the chondrocyte sheets. Staining of the filamentous actin (F-actin) cytoskeleton in the migratory cells also provided a better understanding of the dynamic communication between the cell cytoskeleton and adhesion molecules through ITGB8, which may play a key role in the attachment of the chondrocyte sheets and the synthesis of the cartilage ECM. Therefore, we suggest that cancellous bone could be used as a supporting material to construct chondrocyte sheet-cancellous bone tissues for potential treatment of osteochondral lesions. Impact Statement We proposed a method to construct an osteochondral-like tissue by placing human chondrocyte sheets onto cancellous bone. The stationary chondrocyte sheets and the low mean velocity of the individual cell migration on the cancellous bone with the expression of COL2A1 indicated that the cancellous bone served as an appropriate supporting material. Moreover, the cellular mechanism for the adhesion of the chondrocyte sheets on the cancellous bone based on ITGB8-mediated adhesion through the rearrangement of filamentous actin provided a better understanding to improve the construction of osteochondral-like tissues, and to predict the repair mechanism in osteoarthritis therapy.

Recent Advances in Light-Induced Cell Sheet Technology

Various stimuli have been applied to harvest complete cell sheets, including temperature, magnetic, pH, and electrical stimuli. Cell sheet technology is definitely a convenient and efficient means of providing beneficial effects for tissue regeneration and cell therapy. Light of different wavelengths, such as ultraviolet (UV), visible light, and near infrared ray (NIR) light, were also confirmed to aid in fabricating a cell sheet. The alteration of wettability, potential, or water content of the culturing surfaces occurs under light illumination, inducing conformational changes of adhesive proteins or collagens, which then leads to cell sheets detachment. However, several limitations exist in current investigations, as few applications and researches based on genes have been reported. Studies in the future should aim at generating new materials available for culturing and releasing cell sheets rapidly and effectively.

Evaluation of Multi-Layered Pancreatic Islets and Adipose-Derived Stem Cell Sheets Transplanted on Various Sites for Diabetes Treatment.

Islet cell transplantation is considered an ideal treatment for insulin-deficient diabetes, but implantation sites are limited and show low graft survival. Cell sheet technology and adipose-derived stem cells (ADSCs) can be useful tools for improving islet cell transplantation outcomes since both can increase implantation efficacy and graft survival. Herein, the optimal transplantation site in diabetic mice was investigated using islets and stem cell sheets. We constructed multi-layered cell sheets using rat/human islets and human ADSCs. Cell sheets were fabricated using temperature-responsive culture dishes. Islet/ADSC sheet (AI sheet) group showed higher viability and glucose-stimulated insulin secretion than islet-only group. Compared to islet transplantation alone, subcutaneous AI sheet transplantation showed better blood glucose control and CD31+ vascular traits. Because of the adhesive properties of cell sheets, AI sheets were easily applied on liver and peritoneal surfaces. Liver or peritoneal surface grafts showed better glucose control, weight gain, and intraperitoneal glucose tolerance test (IPGTT) profiles than subcutaneous site grafts using both rat and human islets. Stem cell sheets increased the therapeutic efficacy of islets in vivo because mesenchymal stem cells enhance islet function and induce neovascularization around transplanted islets. The liver and peritoneal surface can be used more effectively than the subcutaneous site in future clinical applications.

Collagen-Supplemented Incubation Rapidly Augments Mechanical Property of Fibroblast Cell Sheets.

Cell sheet technology using UpCell™ (Thermo Fisher Scientific, Roskilde, Denmark) plates is a modern tool that enables the rapid creation of single-layered cells without using extracellular matrix (ECM) enzymatic digestion. Although this technique has the advantage of maintaining a sheet of cells without needing artificial scaffolds, these cell sheets remain extremely fragile. Collagen, the most abundant ECM component, is an attractive candidate for modulating tissue mechanical properties given its tunable property. In this study, we demonstrated rapid mechanical property augmentation of human dermal fibroblast cell sheets after incubation with bovine type I collagen for 24 h on UpCell plates. We showed that treatment with collagen resulted in increased collagen I incorporation within the cell sheet without affecting cell morphology, cell type, or cell sheet quality. Atomic force microscopy measurements for controls, and cell sheets that received 50 and 100 μg/mL collagen I treatments revealed an average Young's modulus of their respective intercellular regions: 6.6 ± 1.0, 14.4 ± 6.6, and 19.8 ± 3.8 kPa during the loading condition, and 10.3 ± 4.7, 11.7 ± 2.2, and 18.1 ± 3.4 kPa during the unloading condition. This methodology of rapid mechanical property augmentation of a cell sheet has a potential impact on cell sheet technology by improving the ease of construct manipulation, enabling new translational tissue engineering applications.

An effective light activated TiO2 nanodot platform for gene delivery within cell sheets to enhance osseointegration

Cell sheets play a vital role in gene therapies. Nonetheless, gene delivery platforms based on cell sheet technology are restricted to small genes. Here, we produced a rapid and efficient light activated gene delivery nanodot platform for large genes. Due to its large gene sequence, low-density lipoprotein receptor-related protein 5 (LRP5) is rarely used for gene therapy in vivo. Our study successfully constructed a nanodot platform using light and adenoviruses to harvest LRP5-overexpressing bone marrow mesenchymal stem cell (BMSC) sheets with a nearly 2-fold increase in efficiency and a 40% reduction in time compared with a traditional gene delivery platform. Overexpression of LRP5 upregulated the expression levels of alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP2), and runt-related transcription factor-2 (Runx2). Further, in vivo results suggested that LRP5 enhanced BMSC sheet-mediated osseointegration. This light activated gene delivery nanodot platform may serve as a potentially powerful tool for introducing various genes and for increasing the scope of application for cell sheet-based gene therapies.

Printing support hydrogels for creating vascular-like structures in stacked cell sheets

Vascular structures are essential for the survival of thick artificial three-dimensional (3D) tissues. However, it is difficult to create high-cell-density artificial tissue with vascular structures of a few hundred micrometers in diameter. Bioprinting technology can create artificial 3D tissues with vascular structures of a few hundred micrometers in diameter, but the cell density of bio-printed artificial 3D tissues is low. On the other hand, cell sheet technology can create high-cell-density artificial 3D tissues by stacking, but it is not possible to set small vascular structures at any place. In this study, we successfully demonstrated high-cell-density artificial 3D tissues with vascular-like structures by stacking cell sheets combined with bioprinting technology.

Bending technology of high strength steel sheets and tools for its realization

The article proposes and theoretically justifies a method for bending high-strength steels with compression. The technology allows to get parts from high-strength steels without cracks along the bending line. In the case of bending according to the proposed method, the deformation is carried out by the combined action of longitudinal and lateral forces. Equitations are derived showing the stress distribution over the cross section in the deformation zone. The article proposes and theoretically justifies a method for bending high-strength steels with compression. The technology allows you to get parts from high-strength steels without cracks along the bending line. In the case of bending according to the proposed method, the deformation is carried out by the action of longitudinal and transverse forces. Equitations were derived for finding the longitudinal compressive force at which the neutral layer in the workpiece moves to the outer surface based on the equations of stress distribution. Equitations are derived for finding the moment created by the longitudinal compressive force, as well as the moment formed by the action of compressive stresses, which made it possible to derive the equation for the external bending moment during bending with compression. Equitation is derived for finding the shoulder of the force action creating an external bending moment. Equitation is derived for finding the force of the punch for bending with compression.

Stable cell adhesion affects mesenchymal stem cell sheet fabrication: Effects of fetal bovine serum and human platelet lysate.

Cell sheet technology exploits temperature responsive cell culture dishes (TRCDs) as versatile cell harvesting methods to yield contiguous cell monolayers robustly held together by cell-cell junctions, receptors, and endogenous extracellular matrix. More than 15 years of clinical data using autologous-sourced cell sheets demonstrate enhanced therapeutic properties through increased cell retention at target tissue sites. Recently, several preclinical studies have also been reported using mesenchymal stem cell (MSC) sheets in wound healing, cardiac ischemia therapies, and pancreatic regeneration. However, optimized MSC sheet fabrication conditions have not yet been reported. In this study, we identified specific conditions for reliable human MSC sheet fabrication by comparing cell growth media supplements (fetal bovine serum [FBS] and human platelet lysate [hPL]). Human umbilical cord-derived MSCs cultured in FBS and hPL exhibit different actin cytoskeletal structures related to their cell morphologies and adhesion. MSCs cultured in FBS media showed stable cell adhesion on TRCDs with flattened cell shapes and aligned actin cytoskeletal structure. This stable cell adhesion enables production of consistent MSC cell sheets, with controlled cell sheet detachment. Conversely, cell sheet fabrication in hPL media exhibits poor reproducibility being more sensitive to temperature- and culture time-induced release due to weak cell adhesion. These findings suggest that stable MSC adhesion to TRCDs is important to reliable MSC sheet fabrication methods and that MSC growth media supplementation directly affects cell adhesion during culture.

The soft wheat flour usage in the wafer sheet and sugar wafers technology

The soft wheat flour usage in the wafer sheet and sugar wafers technology

Perfection of the technology of corrosion-resistant sheet rolled stock production at JSC “Ural Steel”

Manufacturing of field oil pipelines require mainly pipes of 09ГСФ and 13ХФА steels, which correspond to the K52 class of strength. The pipe products and connection details of field oil- and oil products pipelines must meet increased requirements to their resistance against aggressive media, containing water and hydrogen sulfide. At the JSC “Ural Steel”, a unified comprehensive technology of production sheet rolled stock of 09ГСФ and 13ХФА steels in a wide range of assortment. The steels ensure the requirements to hydrogen fracturing as per NACE TM0284 and sulfide corrosion fracturing under stress method A of NACE TM0177 standard. The regimes of controlled sheet rolling with accelerated after-deformation cooling following by a high tempering elaborated. The technology regimes ensure necessary level of mechanical characteristics, corresponding to K52 class of strength. To ensure a satisfactory flatness of sheets, variants of the steels chemical composition depending on finished sheet thickness recommended. The elaborated production regimes ensure metal high resistance at low temperatures and actual value of the impact toughness KCV –40 exceeding considerably the standard requirements. It was shown, that decrease of relation Ca/S in the metal down to ~0,6–1,0 at Sulphur content less than 0.002% does not result in corrosion resistance decreasing and also minimizes the steel liquation inhomogeneity and contamination by non-metallic inclusions. The technology elaborated at JSC “Ural Steel” ensures the possibility of industrial production of corrosion-resistant rolled stock of 09ГСФ and 13ХФА steels in a wide range of assortment.

A Surface-Tailoring Method for Rapid Non-Thermosensitive Cell-Sheet Engineering via Functional Polymer Coatings.

Cell sheet engineering, a technique utilizing a monolayer cell sheet, has recently emerged as a promising technology for scaffold-free tissue engineering. In contrast to conventional tissue-engineering approaches, the cell sheet technology allows cell harvest as a continuous cell sheet with intact extracellular matrix proteins and cell-cell junction, which facilitates cell transplantation without any other artificial biomaterials. A facile, non-thermoresponsive method is demonstrated for a rapid but highly reliable platform for cell-sheet engineering. The developed method exploits the precise modulation of cell-substrate interactions by controlling the surface energy of the substrate via a series of functional polymer coatings to enable prompt cell sheet harvesting within 100 s. The engineered surface can trigger an intrinsic cellular response upon the depletion of divalent cations, leading to spontaneous cell sheet detachment under physiological conditions (pH 7.4 and 37 °C) in a non-thermoresponsive manner. Additionally, the therapeutic potential of the cell sheet is successfully demonstrated by the transplantation of multilayered cell sheets into mouse models of diabetic wounds and ischemia. These findings highlight the ability of the developed surface for non-thermoresponsive cell sheet engineering to serve as a robust platform for regenerative medicine and provide significant breakthroughs in cell sheet technology.