Living Cell Sheets Research Articles

Tailoring cell sheets for biomedical applications

AbstractCell sheet technology has emerged as a novel scaffold‐free approach for cell‐based therapies in regenerative medicine. Techniques for harvesting cell sheets are essential to preserve the integrity of living cell sheets. This review provides an overview of fundamental technologies to fabricate cell sheets and recent advances in cell sheet‐based tissue engineering. In addition to the commonly used temperature‐responsive systems, we introduce alternative approaches, such as ROS‐induced, magnetic‐controlled, and light‐induced cell sheet technologies. Moreover, we discuss the modification of the cell sheet to improve its function, including stacking, genetic modification, and vascularization. With the significant advances in cell sheet technology, cell sheets have been widely applied in various tissues and organs, including but not limited to the lung, cornea, cartilage, periodontium, heart, and liver. This review further describes both the preclinical and clinical applications of cell sheets. We believe that the progress in cell sheet technology would further propel its biomedical applications.

Tug-of-war between stretching and bending in living cell sheets.

The balance between stretching and bending deformations characterizes shape transitions of thin elastic sheets. While stretching dominates the mechanical response in tension, bending dominates in compression after an abrupt buckling transition. Recently, experimental results in suspended living epithelial monolayers have shown that, due to the asymmetry in surface stresses generated by molecular motors across the thickness e of the epithelium, the free edges of such tissues spontaneously curl out-of-plane, stretching the sheet in-plane as a result. This suggests that a competition between bending and stretching sets the morphology of the tissue margin. In this paper, we use the framework of non-Euclidean plates to incorporate active pre-strain and spontaneous curvature to the theory of thin elastic shells. We show that, when the spontaneous curvature of the sheet scales like 1/e, stretching and bending energies have the same scaling in the limit of a vanishingly small thickness and therefore both compete, in a way that is continuously altered by an external tension, to define the three-dimensional shape of the tissue.

Harvesting of Living Cell Sheets by the Dynamic Generation of Diffractive Photothermal Pattern on PEDOT

Near infrared (NIR) photothermal pattern on conductive polymer film enables unique approaches to harvest large‐area cell sheets with various patterns without the use of patterned culture dish. The NIR photothermal pattern is generated from a patterned optical lens (POL), which creates a dynamic near IR light pattern and the corresponding photothermal pattern (PTP) on the polymer film. The POL is prepared from transparent polydimethylsiloxane designed to generate various light patterns. The PTPs allow a noninvasive harvest of cultured cells as an intact living cell sheet with a high harvesting efficiency (ηcell > 100). Various PTPs are generated by the diffraction of NIR light through POLs having different micropatterns, which afford cell sheets with a desired pattern without changing the original cell morphology at cultured state. Furthermore, a large‐area living cell sheet is obtained with a detached area larger than 19 cm2, which is the largest living cell sheet up to date. Further optical engineering of the harvesting system allows multiple productions of cell sheets with one dose of light. It is possible to harvest cell sheets not only from human fibroblast cells but also from human adipose‐derived stem cells, indicating that the method can be applied to engineer various cells.

Cell-Derived Extracellular Matrix: Basic Characteristics and Current Applications in Orthopedic Tissue Engineering.

The extracellular matrix (ECM) is a dynamic and intricate microenvironment with excellent biophysical, biomechanical, and biochemical properties, which can directly or indirectly regulate cell proliferation, adhesion, migration, and differentiation, as well as plays key roles in homeostasis and regeneration of tissues and organs. The ECM has attracted a great deal of attention with the rapid development of tissue engineering in the field of regenerative medicine. Tissue-derived ECM scaffolds (also referred to as decellularized tissues and whole organs) are considered a promising therapy for the repair of musculoskeletal defects, including those that are widely used in orthopedics, although there are a few shortcomings. Similar to tissue-derived ECM scaffolds, cell-derived ECM scaffolds also have highly advantageous biophysical and biochemical properties, in particular their ability to be produced in vitro from a number of different cell types. Furthermore, cell-derived ECM scaffolds more closely resemble native ECM microenvironments. The products of cell-derived ECM have a wide range of biomedical applications; these include reagents for cell culture substrates and biomaterials for scaffolds, hybrid scaffolds, and living cell sheet coculture systems. Although cell-derived ECM has only just begun to be investigated, it has great potential as a novel approach for cell-based tissue repair in orthopedic tissue engineering. This review summarizes and analyzes the various types of cell-derived ECM products applied in cartilage, bone, and nerve tissue engineering in vitro or in vivo and discusses future directions for investigation of cell-derived ECM.

Fat Prospects for Healing Cutaneous Wounds: New Activities Under the Sheets.

In this issue of Diabetes , Kato et al. (1) describe new results for dermal wound healing in full-thickness skin defects in Zucker diabetic fatty (ZDF) rats. The team fabricated and applied transplanted living cell sheets comprising allogeneic adipose-derived stem cells (ASCs) harvested from the inguinal fat pads of normal rats. They attempt to address the current challenges with abnormal wound healing in increasing numbers of patients with diabetes who present with difficult cutaneous wound healing problems (2,3). This deficiency is often clinically characterized by persistent inflammation, hypergranulation, excessive tissue bed exudates, and chronic inability to resolve, resulting in enhanced bacterial burdens, infections, ulcerations, and more serious complications. Delayed cutaneous healing in these patients is attributed to combinations of predisposing factors associated with chronic diabetes pathology, including oxidative imbalance, vascular abnormalities, neuropathy, infection, and local metabolic deficiencies intrinsic to diabetes. Pathological disruption of normal regenerative and degenerative extracellular matrix processing alters the dermal matrix composition, resulting in reduced amounts and quality of tissue matrix (2,3). Natural balances between proinflammatory cytokines (e.g., interleukin [IL]-1α and -1β and tumor necrosis factor-α) stimulating endogenous protease activities and prohealing cytokines (e.g., IL-4, IL-8, IL-10, IL-11, IL-13, granulocyte macrophage colony-stimulating factor, MCP-1, and macrophage inflammatory protein 2) apparently do not exist in diabetic tissues. Additionally, reduced levels of potent growth factors platelet-derived growth factor, basic fibroblast growth factor, epidermal growth factor, and transforming growth …

Real-time, noninvasive optical coherence tomography of cross-sectional living cell-sheets in vitro and in vivo.

Cell sheet technology has a history of application in regenerating various tissues, having successfully completed several clinical trials using autologous cell sheets. Tomographic analysis of living cell sheets is an important tool in the field of cell sheet-based regenerative medicine and tissue engineering to analyze the inner structure of layered living cells. Optical coherence tomography (OCT) is commonly used in ophthalmology to noninvasively analyze cross-sections of target tissues at high resolution. This study used OCT to conduct real-time, noninvasive analysis of living cell sheet cross sections. OCT showed the internal structure of cell sheets in tomographic images synthesized with backscatter signals from inside the living cell sheet without invasion or damage. OCT observations were used to analyze the static and dynamic behaviors of living cell sheets in vitro and in vivo including (1) the harvesting process of a C2C12 mouse skeletal myoblast sheet from a temperature-responsive culture surface; (2) cell-sheet adhesion onto various surfaces including a culture surface, a synthetic rubber glove, and the dorsal subcutaneous tissue of rats; and (3) the real-time propagation of beating rat cardiac cells within cardiac cell sheets. This study showed that OCT technology is a powerful tool in the field of cell sheet-based regenerative medicine and tissue engineering.

A device for the rapid transfer/transplantation of living cell sheets with the absence of cell damage

In this study, we developed a device that could easily, rapidly, and completely transfer cell sheets from one material to another or transplant cell sheets onto the dorsal subcutaneous tissues of rats without leaving residual cells. Because the manipulation is as simple as pipetting, technical expertise is not required to transfer cell sheets very rapidly (the transfer time was 3.7 ± 1.6 s) using the device compared with that of a conventional method using a pipette (430 ± 180 s). After transfer by the device, C2C12 skeletal myoblast sheets showed active cell metabolism, cell viability, and very high production of vascular endothelial growth factor and stromal-derived factor-1α, indicating transfer without cell damage. Cardiac cell sheets after transfer showed spontaneous and synchronous beating, indicating intact cell–cell junctions and ion channel proteins on the cell surface. In addition, the device allowed us to transfer C2C12 cell sheets onto soft, rugged and curved surfaces such as human hands. Furthermore, cardiac cell sheets adhered rapidly and tightly onto the dorsal subcutaneous tissues of rats. This transfer/transplantation device may be a powerful tool in cell sheet-based tissue engineering and regenerative medicine.

HARVESTING EPITHELIAL CELL SHEET BASED ON THERMO- SENSITIVE HYDROGEL

Thermo-sensitive hydrogels were prepared by graft copolymerization of chitosan and NIsopropylacrylamide via gamma radiation. Characterization of hydrogels such as 13 C-NMR, DSC analysis and swelling test and cell assessments for harvesting living cell sheet were investigated. 13 CNMR and DSC analysis showed chitosan and NIPAAm monomer were grafted via gamma radiation successfully. Swelling ratio and curves results administrated hydrophilicity / hydrophobicity of hydrogel that this property is due to presence of PNIPAAm in different temperatures. The hydrogel was tested for harvesting epithelial cells after carrying out cell culture at 37 °C and incubating the confluent cells at 4°C for spontaneous detachment of cell sheet from hydrogel surface without enzyme treatment. Cell viability assay results and microscopic observations demonstrated that cells could attach to the hydrogel surface and maintain high viability and proliferation ability. Cell detachment efficiency from the hydrogel was high. These unique properties of the hydrogel would make it a promising support for epithelial cell grafting especially cornea regeneration.

Temperature-responsive intelligent interfaces for biomolecular separation and cell sheet engineering

Temperature-responsive intelligent surfaces, prepared by the modification of an interface with poly(N-isopropylacrylamide) and its derivatives, have been used for biomedical applications. Such surfaces exhibit temperature-responsive hydrophilic/hydrophobic alterations with external temperature changes, which, in turn, result in thermally modulated interactions with biomolecules and cells. In this review, we focus on the application of these intelligent surfaces to chromatographic separation and cell cultures. Chromatographic separations using several types of intelligent surfaces are mentioned briefly, and various effects related to the separation of bioactive compounds are discussed, including wettability, copolymer composition and graft polymer architecture. Similarly, we also summarize temperature-responsive cell culture substrates that allow the recovery of confluent cell monolayers as contiguous living cell sheets for tissue-engineering applications. The key factors in temperature-dependent cell adhesion/detachment control are discussed from the viewpoint of grafting temperature-responsive polymers, and new methodologies for effective cell sheet culturing and the construction of thick tissues are summarized.

Applications of chitosan-based thermo-sensitive copolymers for harvesting living cell sheet

A thermo-sensitive chitosan-based copolymer hydrogel was used for harvesting living cell sheets. The hydrogel was tested for harvesting 3T3 cells after carrying out cell culture at 37 °C and incubating the confluent cells at 20 °C for spontaneous detachment of cell sheets from hydrogel surface without enzyme treatment. Results from cell viability assay and microscopy observations demonstrated that cells could attach to the hydrogel surface and maintain high viability and proliferation ability. Cell detachment efficiency from the hydrogel was about 80%. The detached cell sheet retained high viability and could proliferate again after transferred to a new culture surface.

Novel Living Cell Sheet Harvest System Composed of Thermoreversible Methylcellulose Hydrogels

In this study, a novel yet simple method, using a thermoreversible hydrogel system coated on tissue culture polystyrene (TCPS) dishes, was developed for harvesting living cell sheets. The hydrogel system was prepared by simply pouring aqueous methylcellulose (MC) solutions blended with distinct salts on TCPS dishes at 20 degrees C. For the applications to cell culture, only those aqueous MC compositions that may form a gel at 37 degrees C were chosen for the study. It was found that the hydrogel coating composed of 8% MC blended with 10 g/L PBS (phosphate buffered saline) (the MC/PBS hydrogel, with a gelation temperature of approximately 25 degrees C) stayed intact throughout the entire course of cell culture. To improve cell attachments, the MC/PBS hydrogel at 37 degrees C was evenly spread with a neutral aqueous collagen at 4 degrees C. The spread aqueous collagen gradually reconstituted with time and thus formed a thin layer of collagen (the MC/PBS/collagen hydrogel). After cells reached confluence, a continuous monolayer cell sheet formed on the surface of the MC/PBS/collagen hydrogel. When the grown cell sheet was placed outside of the incubator at 20 degrees C, it detached gradually from the surface of the thermoreversible hydrogel spontaneously, without treating with any enzymes. The results obtained in the MTT assay demonstrated that the cells cultured on the surface of the MC/PBS/collagen hydrogel had an even better activity than those cultured on an uncoated TCPS dish. After harvesting the detached cell sheet, the remaining viscous hydrogel system is reusable. Additionally, the developed hydrogel system can be used for culturing a multilayer cell sheet. The obtained living cell sheets may be used for tissue reconstructions.

Nanostructured designs of biomedical materials: applications of cell sheet engineering to functional regenerative tissues and organs

Biomaterials surface design is critical for control of cell–materials interactions. Materials surface characteristics important to cell–materials interactions are the following: (a) nonfouling surfaces where cells cannot interact; (b) surfaces that interact with cells but do not alter cell morphology or metabolism (passive adhesion processes); and (c) surfaces that strongly interact with cells and cell–surface receptors to alter cell shape after metabolic interactions (active adhesion). In this paper, we briefly discuss the relationship between materials surface characteristics and cells for biomaterials designs in these categories. We have extensively investigated the thermoresponsive polymer, poly( N-isopropylacrylamide) (PIPAAm), as grafted surfaces allowing recovery of confluent cell monolayers as contiguous living cell sheets for tissue engineering applications. Cellular interactions with PIPAAm-grafted surfaces can be regulated vertically using the thickness of the PIPAAm-grafted layers in nanometer-scale levels, as well as laterally (spatially) using nano-patterned PIPAAm chemistry on various other surface chemistries. PIPAAm-grafted surfaces with 15–20-nm thick layers exhibit temperature-dependent cell adhesion/detachment control, while surfaces with PIPAAm layer thicknesses of more than 30 nm do not support cell adhesion. These changes in cell adhesion are explained by the limited mobility of the surface grafted polymer chains as a function of grafting, hydration, and temperature.

Intracellular phase of existence of lymphycotes during remission of acute lymphatic leukaemia.

THAT lymphocytes may be found inside other larger cells is now well established1,2. In these laboratories this phenomenon has been observed in material examined by tissue culture methods from many patients suffering from Hodgkin's disease and the other malignant reticuloses. Further work3 has shown that in certain cases of acute leukaemia, the leukaemic cells may be difficult to maintain alive by themselves but can be maintained for long periods in association with established living cell sheets, for example, thyroid cells or embryonic tissues.