Cell Sheet Detachment Research Articles

Photothermal dual-responsive polymers for trypsin-free cell sheet harvesting

Cell sheet engineering has emerged to tackle cell therapy's bottlenecks and increase the likelihood of life-saving potentials of tissue engineering. In this study, Poly(N-isopropylacrylamide-co-spiropyran) (P(NIPAAm-co-Sp)) copolymer and Poly(N-isopropylacrylamide-co-styrene-co-spiropyran) (P(NIPAAm-co-St-co-Sp)) terpolymer were synthesized by free radical polymerization (FRP). Fourier transform infrared (FTIR) was utilized to characterize the chemical structure of the resultant polymers. The molecular weight and molecular weight distribution were measured by gel permeation chromatography (GPC). The reversible photosensitivity of the synthesized polymers was proved by ultraviolet–visible (UV–Vis) spectroscopy. Thin layers of polymers were spin-coated onto ultraviolet/ozone (UV-O3) and silane-modified glass slides. To confirm the coating process, the attenuated total reflection FTIR spectroscopy (ATR-FTIR) was used. The cross-sectional images of the spin-coated glass slides, obtained using field emission scanning electron microscopy (FESEM), showed thin layers about 300 to 400 nm thick. The roughness of the coated layers was examined by atomic force microscopy which were below 1 nm. The water contact angle (WCA) measurements showed that changes in the surface wettability of thin layers occurred in response to temperature alteration and light illumination. The spin-coated glass slides were used to culture the mouse fibroblast cell line L929. The cell culture results showed that the thickness of the thin layers was adequate for forming a confluent and integrated sheet of L929 cells. By reducing the temperature from 37 °C to 20 °C and illuminating the glass slide, fragments of the cell sheet were harvested. The use of light besides temperature reduction decreased the time needed for cell sheet detachment from 2 h to 1 h.

Temperature responsive poly(N-isopropylacrylamide-co-styrene) nanofilms for non-enzymatic cell sheet harvesting

In this study, temperature-responsive cell culture substrates with non-enzymatic cell sheet detachment attributes in response to temperature reduction were prepared. Poly(N-isopropylacrylamide) (PNIPAAm) with a physiologically proximal lower critical solution temperature (LCST) induces a temperature responsivity to the substrate, and polystyrene was used to regulate the hydrophobicity of the substrate. Different compositions of poly(NIPAAm-co-styrene) were synthesized by free radical polymerization. The resulting copolymers were characterized using Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1HNMR) spectroscopy. The copolymer composition was calculated from 1HNMR results. The molecular weight and molecular weight distribution were measured by gel permeation chromatography (GPC). The nanofilms of the prepared copolymers were physically coated by spin-coating technique on tetramethyl orthosilicate (TMOS) pre-coated glass slides. Attenuated total reflection-FTIR (ATR-FTIR) and field emission scanning electron microscopy (FESEM) analyses showed that the spin coating procedure was performed successfully, and the change in the water contact angle (WCA) in response to temperature alteration indicated the temperature-responsive behavior of the polymer layers. L929 mouse fibroblast cell line was cultured on nanofilm-coated glass slides until a confluent cell sheet covered the entire surface. An almost integrated cell sheet was harvested by temperature reduction from 37 °C to 20 °C. The results revealed that copolymer composition is vital in harvesting contiguous cell sheets. This fabrication method, compared to others such as plasma polymerization and surface-initiated atom transfer radical polymerization (SI-ATRP), is very cost-effective and applicable for large-scale cell culture investigations.

Fabrication of 3D cylindrical thermosensitive hydrogels as supports for cell culture and detachment of tubular cell sheets

Pseudo interpenetrating vinyl-caprolactam (VCL) based thermosensitive tubular hydrogels with a volume phase transition temperature, VPTT, around 35 °C, have been prepared by combining two different crosslinkers, a di-methacrylate (C1) and a di-vinyl urea (C2). The molar ratio between the two crosslinkers (for a global crosslinker molar percentage of 1.9) has shown to play a key role on the properties of the hydrogel. Increasing the amount of di-vinyl urea, leads to transparent but rather fragile materials and to a lower extent of thermosensitivity, that is, to a lower variation in the hydrogel swelling upon temperature change. However, tubes prepared with a selected crosslinker molar ratio C1/C2 of 65/35 provided a compromise between transparency, thermosensitivity and maneuverability and were, thus, evaluated as supports for cell culture using premyoblastic cells. These hydrogels, used as supports, allow for surface adhesion and cell proliferation until confluence, and eventually an efficient monolayer detachment (and transplant to a 3D-printed polylactic acid (PLA) support) through a controlled drop in temperature. As a result, this method permits to obtain tubular tissue constructs with potential applications in tissue engineering such as in the elaboration of vascular grafts.

"Fuzzy hair" promotes cell sheet detachment from thermoresponsive brushes already above their volume phase transition temperature.

Thermoresponsive poly(glycidyl ether) (PGE) brushes have shown to be viable substrates for the culture and temperature-triggered detachment of confluent cell sheets. Surface-tethered PGEs with a cloud point temperature (TCP) around ~30°C exhibit phase transitions well-centered within the physiological range (20-37°C), which makes them ideal candidates for cell sheet fabrication. However, PGEs with TCPs at ~20°C also afford the detachment of various types of cell sheets, even at room temperature (20-23°C), i.e., above the polymers' TCPs. In this study, we investigate the phase transition of PGE brushes tethered to polystyrene (PS) culture substrates with varying grafting density and TCP to arrive at a mechanistic understanding of their functionality in cell sheet fabrication. Using quartz crystal microbalance with dissipation (QCM-D) monitoring, we demonstrate that brushes fabricated from PGEs with TCPs at ~20°C display volume phase transition temperatures (VPTTs) well below room temperature. Although the investigated coatings obviously do not exhibit marked thermal switching in terms of brush hydration and layer thickness, their physical properties at the brush-water interface, as ascertained by QCM-D and AFM measurements, undergo subtle changes upon cooling from 37°C to room temperature which is sufficient to promote cell sheet detachment. Thus, it appears that discreet rehydration of the outmost brush layer, resembling "fuzzy hair" at the brush-water interface, renders the surfaces less protein- and cell-adhesive at room temperature. This minor structural change of the interface allows for the reliable detachment of human dermal fibroblast sheets already at 20°C well above the VPTT of the brushes.

Photo- and thermo-responsive extracellular matrix mimicking nano-coatings prepared from poly(N-isopropylacrylamide)-spiropyran copolymer for effective cell sheet harvesting

Poly(N-isopropylacrylamide)-spiropyran (PNIPAAm-SP) copolymers were tethered onto the glass substrate using surface-initiated atom transfer radical polymerization (SI-ATRP). The substrate was previously activated by ultra-violet ozone (UVO) irradiation and the pre-synthesized initiator was immobilized on it. A block of PNIPAAm was grafted onto the glass substrates, followed by a second block of mixed NIPAAm and spiropyran acrylate (SPA). The effects of UVO irradiation time and polymerization time of spiropyran containing block on cell sheet formation and detachment characteristics were evaluated. The photo-responsiveness of SPA was evaluated by ultra-violet visible spectroscopy (UV–vis). The presence and thickness of the grafted polymer layers were evidenced by attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and field emission scanning electron microscopy (FE-SEM). The results of water contact angle (WCA) analysis showed that the modified surfaces became hydrophilic with decreasing temperature from 37 to 20 °C as well as with light irradiation. L929 mouse fibroblast cell culture results indicated a complete cell sheet formation. By applying temperature or light stimuli, the cell sheets were detached in a trypsin-free procedure.

Detachment of bovine corneal endothelial cell sheets by cooling-induced surface hydration of poly[(R)-3-hydroxybutyrate]-based thermoresponsive copolymer coating.

Cell sheet technology (CST) is a fascinating scaffoldless tissue engineering technique to generate a physiologically representative tissue replacement from autologous sources. As compared to conventional enzymatic cell harvesting methods, CST enables the preservation of important cell-to-cell junctions and extracellular matrix (ECM) components. However, covalent grafting methods are often employed for CST. In this study, a series of triblock copolymers with a hydrophobic and biocompatible poly[(R)-3-hydroxybutyrate] (PHB) central block flanked by varying lengths of terminal poly(N-isopropylacrylamide) (PNIPAAm) blocks (PNIPAAm-PHB-PNIPAAm) was synthesized via atom transfer radical polymerization of NIPAAm. The thermoresponsive triblock copolymers were explored as a non-covalent surface coating for culturing and detaching bovine corneal endothelial cell (BCEC) sheets. Aqueous solutions of the triblock copolymers produced thermosensitive micelles which can be drop-casted on glass substrates, resulting in a temperature-responsive surface. Importantly, incorporating a central hydrophobic PHB block enabled the anchoring of the coating to the bare substrate and enhanced the proliferation rate of the BCECs studied. Effective detachment of an intact cell sheet was also demonstrated via a cooling treatment at 4 °C for 20 min, and the viability of the detached cell sheet was found to be unaffected by the cooling. This work may potentially inspire more studies involving the non-covalent thermoresponsive polymer coatings for corneal tissue engineering applications.

Thin Thermoresponsive Polymer Films for Cell Culture: Elucidating an Unexpected Thermal Phase Behavior by Atomic Force Microscopy.

Application of poly-N-isopropylacrylamide (PNIPAM) and its more hydrophobic copolymers with N-tert-butylacrylamide (NtBA) as supports for cell sheets has been validated in numerous studies. The binary systems of these polymers with water are characterized by a lower critical solution temperature (LCST) in a physiologically favorable region. Upon lowering the temperature below the LCST, PNIPAM chains undergo a globule-to-coil transition, causing the film dissolution and cell sheet detachment. The character of the PNIPAM-water miscibility behavior is rather complex and not completely understood. Here, we applied atomic force microscopy to track the phase transition in thin films of linear thermoresponsive (co)polymers (PNIPAM and PNIPAM-co-NtBA) prepared by spin-coating. We studied the films' Young's modulus, roughness, and thickness in air and in distilled water in a full thermal cycle. In dry films, in the absence of water, all the measured parameters remained invariant. The swollen films in water above the LCST were softer by 2-3 orders of magnitude and about 10 times rougher than the corresponding dry films. Upon lowering the temperature to the LCST, the films passed through the phase transition observed as a drastic drop of Young's modulus (about an order of magnitude) and decrease in roughness in both polymers in a narrow temperature range. However, the films did not lose their integrity and demonstrated almost fully reversible changes in the mechanical properties and roughness. The thermal dependence of the films' thickness confirmed that they dissolved only partially and required an external force to induce the complete destruction. The reversible thermal behavior which is generally not expected from non-cross-linked polymers is a key finding, especially with respect to their practical application in cell culture. Both the thermodynamic and kinetic factors, as well as the confinement effect, may be responsible for this peculiar film robustness, which requires overcooling and the aid of an external force to destroy the film.

On the foundation of thermal “Switching”: The culture substrate governs the phase transition mechanism of thermoresponsive brushes and their performance in cell sheet fabrication

Thermally “switchable” poly(glycidyl ether) (PGE) brushes constitute effective coatings for the temperature-triggered harvest of confluent cell sheets. Based on a simple “grafting-to” approach, such coatings can be tethered to various applied plastic culture substrate materials. Herein, we elucidate the self-assembly of PGE brushes with tunable grafting densities up to 0.12 and 0.22 chains nm−2 on polystyrene (PS) and tissue culture PS (TCPS), respectively. In terms of temperature-dependent wettability and protein adsorption, we found that brushes exhibit distinct grafting density-dependent properties which correlate with their cell sheet fabrication performance. In addition, temperature-ramped quartz-crystal microbalance with dissipation (QCM-D) measurements revealed marked substrate-specific PGE phase transitions which allowed us to deduce comprehensive switching mechanisms. Thus, we demonstrate that brushes tethered to hydrophilic TCPS (contact angle (CA) ∼ 60°) undergo a “cushioned” transition comprising a non-switchable, hydrated basal layer as well as a switchable top layer which regulates cell sheet detachment. In contrast, PGE brushes tethered to PS undergo a “grounded” transition which is substantially influenced by the dehydrating effect of the less hydrophilic PS substrate (CA ∼ 90°). These divergent phase transition mechanisms give rise to a broad scope in cell sheet fabrication performance, yielding staggered detachment times within a 30 min to 3 h range. Hence, we emphasize the importance of a detailed knowledge on the effect of applied culture substrates on the thermal switchability and phase transition characteristics of thermoresponsive brush coatings to accomplish an optimized design for functional cell culture dishes. Statement of significanceAs the first comparative study of its kind, we elucidate the substrate-dependent thermal switchability of thermoresponsive brush coatings and evaluate their grafting density-dependent phase transition mechanism and its effect on cell sheet fabrication performance.

Efficient fabrication of stretching hydrogels with programmable strain gradients as cell sheet delivery vehicles

Fabricating functional cell sheets with excellent mechanical strength for tissue regeneration remains challenging. Therefore, we devised a novel 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide/N-hydroxy-succinimide crosslinked hydrogel carrier composed of gelatin (Ge) and beta-cyclodextrin (β-CD) that promoted the adhesion and proliferation of keratinocytes (Kcs) compared with those cultured on a Ge hydrogel due to significantly higher pore size, porosity, and stiffness, as confirmed using field emission scanning electron microscopy (FE-SEM) and shear wave elastography (SWE). Upon exposure to a programmable gradient microenvironment, cells displayed a stress/strain-dependent spatial-temporal distribution of extended cellular phenotypes and cytoskeletons. The promoted proliferation of Kcs and the increased retention of the undifferentiated cell phenotype on Ge-β-CD composite hydrogels under a 15% strain led to the accelerated detachment of cell sheets with retained cell-cell junctions. Moreover, the stretch-triggered upregulated expression of phosphorylated yes-associated protein (YAP) 1 suggested that this effect might be associated with the mechanical stimulation-induced activation of the YAP pathway.

Cruciate Ligament Cell Sheets Can Be Rapidly Produced on Thermoresponsive poly(glycidyl ether) Coating and Successfully Used for Colonization of Embroidered Scaffolds.

Anterior cruciate ligament (ACL) cell sheets combined with biomechanically competent scaffolds might facilitate ACL tissue engineering. Since thermoresponsive polymers allow a rapid enzyme-free detachment of cell sheets, we evaluated the applicability of a thermoresponsive poly(glycidyl ether) (PGE) coating for cruciate ligamentocyte sheet formation and its influence on ligamentocyte phenotype during sheet-mediated colonization of embroidered scaffolds. Ligamentocytes were seeded on surfaces either coated with PGE or without coating. Detached ligamentocyte sheets were cultured separately or wrapped around an embroidered scaffold made of polylactide acid (PLA) and poly(lactic-co-ε-caprolactone) (P(LA-CL)) threads functionalized by gas-phase fluorination and with collagen foam. Ligamentocyte viability, protein and gene expression were determined in sheets detached from surfaces with or without PGE coating, scaffolds seeded with sheets from PGE-coated plates and the respective monolayers. Stable and vital ligamentocyte sheets could be produced within 24 h with both surfaces, but more rapidly with PGE coating. PGE did not affect ligamentocyte phenotype. Scaffolds could be colonized with sheets associated with high cell survival, stable gene expression of ligament-related type I collagen, decorin, tenascin C and Mohawk after 14 d and extracellular matrix (ECM) deposition. PGE coating facilitates ligamentocyte sheet formation, and sheets colonizing the scaffolds displayed a ligament-related phenotype.

Thermoresponsive surfaces designed for the proliferation and differentiation of human pluripotent stem cells

Thermoresponsive surfaces enable the detachment of cells or cell sheets by decreasing the temperature of the surface when harvesting the cells. However, human pluripotent stem cells (hPSCs), such as embryonic stem cells and induced pluripotent stem cells, cannot be directly cultured on a thermoresponsive surface; hPSCs need a specific extracellular matrix to bind to the integrin receptors on their surfaces. We prepared a thermoresponsive surface by using poly(N-isopropylacrylamide-co-butylacrylate) and recombinant vitronectin to provide an optimal coating concentration for the hPSC culture. hPSCs can be cultured on the same thermoresponsive surface for 5 passages by partial detachment of the cells from the surface by decreasing the temperature for 30 min; then, the remaining hPSCs were subsequently cultured on the same dishes following the addition of new cultivation media. The detached cells, even after continual culture for five passages, showed high pluripotency, the ability to differentiate into cells derived from the 3 germ layers and the ability to undergo cardiac differentiation.

Adhesion, proliferation, and detachment of cells on poly(N-isopropyl acrylamide) brushes tethered on polystyrene using surface-initiated atom transfer radical polymerization

The preparation of thermo-responsive cell culture dishes through grafting poly(N-isopropyl acrylamide) (PNIPAAm) brushes is described. The effect of different treatments, including ultraviolet-ozone (UVO) and ozone exposure on hydroxyl functionalization of the tissue culture polystyrene (TCPS) dishes, was investigated. The surface of TCPS dishes was activated by UVO irradiation, and the synthesized initiator was immobilized on the surface. PNIPAAm brushes were yielded by surface-initiated atom transfer radical polymerization (SI-ATRP). Surface composition and properties of grafted PNIPAAm on polystyrene surface were studied by X-ray photoelectron spectroscopy (XPS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), energy-dispersive X-ray (EDX) spectroscopy, and water contact angle (WCA). The results show that PNIPAAm brushes were successfully grafted on the TCPS, and the WCA of the surface was raised from 33 to 64° by increasing the temperature from 20 to 40 °C, which is suitable for cell sheet detachment. Different cell types, including MG, endothelial, and fibroblast cells, were cultured on the PNIPAAm-grafted TCPS. The cell culture results show that the attachment to the surface and proliferation were successfully carried out. The formed cell sheet was also detached by decreasing the temperature from 37 to 20 °C. Therefore, the grafted PNIPAAm brushes are useful for the preparation of cell sheets from different cell types.

Detachment of cell sheets from clinically ubiquitous cell culture vessels by ultrasonic vibration

Proteinases that digest the extracellular matrix are usually used to harvest cells from culture vessels in a general culture process, which lowers the initial adhesion rate in regenerative medicine. Cell sheet engineering is one of the most important technologies in this field, especially for transplantation, because fabricated cell sheets have rich extracellular matrixes providing strong initial adhesion. Current cell sheet fabrication relies on temperature-responsive polymer-coated dishes. Cells are cultured on such specialized dishes and subjected to low temperature. Thus, we developed a simple but versatile cell sheet fabrication method using ubiquitous culture dishes/flasks without any coating or temperature modulation. Confluent mouse myoblasts (C2C12 cell line) were exposed to ultrasonic vibration from underneath and detached as cell sheets from entire culture surfaces. Because of the absence of low temperature, cell metabolism was statically increased compared with the conventional method. Furthermore, viability, morphology, protein expression, and mRNA expression were normal. These analyses indicated no side effects of ultrasonic vibration exposure. Therefore, this novel method may become the standard for cell sheet fabrication. Our method can be easily conducted following a general culture procedure with a typical dish/flask, making cell sheets more accessible to medical experts.

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.

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.

Synthesis of temperature and light sensitive mixed polymer brushes via combination of surface-initiated PET–ATRP and interface-mediated RAFT polymerization for cell sheet application

In this study, a series of temperature and light sensitive mixed polymer brushes consisting of poly(2-(2-methoxyethoxy) ethyl methacrylate) [poly(MEO2MA)] and poly(N-[4-[(4-nitrophenyl) azo] phenyl] acrylamide) [poly(NPAPAA)] units were grown on silicon wafer via the combination of surface-initiated photoinduced electron transfer-atom transfer radical polymerization (PET-ATRP) and interface-mediated reversible addition-fragmentation chain transfer (RAFT) polymerization. This process was realized in five consecutive steps: (i) coupling of previously synthesized (3-aminopropyl) triethoxysilane-2-bromoisobutyryl bromide (APTES-BIBB) and (3-aminopropyl) triethoxysilane-t-butoxycarbonyl (APTES-t-BOC) mixtures to the Si-OH surface under self-assembly monolayer conditions (ii) synthesis of poly(MEO2MA) units via surface-initiated PET-ATRP (iii) replacement of terminal-Br atoms at poly(MEO2MA) units with biocompatible maleimide molecules (iv) removal of the t-BOC group from APTES-t-BOC and bonding of the RAFT agent to the APTES, and (v) synthesis of poly(NPAPAA) units via the interface-mediated RAFT polymerization. The surface compositions of APTES-BIBB and APTES-t-BOC molecules were determined by the water contact angle data analyzed with the Israelachvili-Gee model and by the X-ray photoelectron spectroscopy measurements. The grafting density (σ, chains nm−2) and the average distance between grafting points (D, nm) of the mixed polymer brushes are about 0.56 chains nm−2 and 1.52 nm, respectively, indicating high grafting density. The lower critical solution temperatures (LCSTs), trans-cis and cis-trans transition times were in the range of 24.6–29.2 °C, 10–25 min and 8–20 min, respectively, depending on the surface composition of the polymer brushes. Cell culture studies revealed successful cell detachment either via the temperature reduction or UV irradiation methods. The cell recovery ratios were recorded as 99.05 ± 0.45% and 83.95% ± 0.58%, respectively. These experimental findings provided basic information at the molecular level in the preparation of temperature and light- sensitive bioactive surfaces for cell sheet preparation and detachment state.

Fabrication of Functional High Density Cell Sheets for Tissue Engineering Using Spin-Coated poly(N-isopropylacrylamide)Thin Films

The current goal of cell sheet engineering technology is to provide an improved approach for more predictive and effective generation of viable tissue-like constructs. Success requires validation and characterization of cell sheet growth and detachment. In this study, we perform qualitative and quantitative assessments of cell sheets lifted from thin spin-coated poly(N-isopropylacrylamide)films.

Recent Advances in ROS-Responsive Cell Sheet Techniques for Tissue Engineering.

Cell sheet engineering has evolved rapidly in recent years as a new approach for cell-based therapy. Cell sheet harvest technology is important for producing viable, transplantable cell sheets and applying them to tissue engineering. To date, most cell sheet studies use thermo-responsive systems to detach cell sheets. However, other approaches have been reported. This review provides the progress in cell sheet detachment techniques, particularly reactive oxygen species (ROS)-responsive strategies. Therefore, we present a comprehensive introduction to ROS, their application in regenerative medicine, and considerations on how to use ROS in cell detachment. The review also discusses current limitations and challenges for clarifying the mechanism of the ROS-responsive cell sheet detachment.

Gradient Photothermal Field for Precisely Directing Cell Sheet Detachment.

Due to the remaining intact intercellular interactions, cell sheets play increasing roles in biomedical fields. Recently, spatiotemporal control of cell sheets has become an urgent requirement in the context of precise surgery. Here, the development of a gradient photothermal biointerface for precisely directing cell sheet detachment through quantitatively regulating the dissociation rate of collagen is reported. This novel and facile method shows great potential for application in precise regenerative medicine.

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.