Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro

The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabrication system for functional 3D tissues by stacking cell sheets of confluent cultured cells detached from a temperature-responsive culture dish. Here we describe the protocols for the fabrication of 3D tissues by cell sheet engineering. Three-dimensional cardiac tissues fabricated by stacking cardiac cell sheets pulsate spontaneously, synchronously and macroscopically. Via this protocol, it is also possible to fabricate other tissues, such as 3D tissue including capillary-like prevascular networks, from endothelial cells sandwiched between layered cell sheets. Cell sheet stacking technology promises to provide in vitro tissue/organ models and more effective therapies for curing tissue/organ failures.     

Application of a cell sheet–polymer film complex with temperature sensitivity for increased mechanical strength and cell alignment capability

We have succeeded in fabricating a cell sheet–polymer film complex involving a temperature‐sensitive polymer that has enough mechanical strength that can be manipulated even by forceps. The polymer film can be removed by lowering the temperature after transplantation, demonstrating its potential use in regenerative medicine. Recently, tissue engineering involving cell sheets was developed, tissues being fabricated by layering of these cell sheets. This technique promises high density cell packing, which is important for native cell functions, and successful heart therapy using cardiac cell sheets has been reported. On the other hand, the fabrication of a large tissue using cell sheets is difficult because of fragility of the cell sheets. Here, we have developed a novel method in which cells are attached to a temperature‐sensitive poly‐N‐isopropylacrylamide film mixed with laminin and collagen IV, and report that the cell sheet–polymer film complex can be manipulated with forceps. A cell sheet can be removed from the polymer film by lowering the temperature after the manipulation. We have utilized this technique for the primary myocardium and fabricated a physiologically active multi‐layered cardiac cell sheet. By applying a micropattern to this polymer film, we have succeeded in making a skeletal muscle cell sheet in which myotubes are oriented in the desired direction. Overall, we showed that this method is useful for cell sheet manipulation, morphogenesis, and transplantation. Biotechnol. Bioeng. 2009;103: 370–377. © 2009 Wiley Periodicals, Inc.     

Effects of surface substances of untransformed fibroblastic cells on the adhesion and proliferation of neighboring cells: a study using fixed confluent cell sheets

To study the effects of surface materials of cells on the behavior of other neighboring cells in a crowded culture, confluent sheets of rat 3Y1 fibroblasts were fixed and then 3Y1 cells were seeded on to them. Among confluent sheets unfixed, fixed with formalin and fixed with ethanol and an empty plastic dish surface, the substrate activity to permit cell adhesion was compared. After confluent 3Y1 cells (mainly composed of cells with a G1-DNA content) were reseeded with fresh medium on to these substrates, the capacity to initiate DNA synthesis per attached cell was also compared. The substrate activity of the ethanol-fixed cell sheet to permit cell adhesion was as high as that of the empty dish surface, whereas that of the unfixed cell sheet and that of the formalin-fixed cell sheet were low. When the ethanol-fixed cell sheet and the empty dish surface were coated with the ethanol extract of the unfixed cell sheet, the substrate activity diminished, indicating that during the fixation process with ethanol an adhesion-inhibitory factor (s) was removed. The capacity to initiate DNA synthesis of each cell that had completed adhesion and spreading on the cell sheets unfixed, fixed with formalin, and fixed with ethanol was lower compared to the cell that had adhered to the empty dish surface. We conclude that factors over the 3Y1 cell surface inhibit the overlapping cell adhesion and the proliferation of cells contacting each other, resulting in the ordered cell configuration in the confluent culture.     

Time Course of Cell Sheet Adhesion to Porcine Heart Tissue after Transplantation

Multilayered cell sheets have been produced from bone marrow-derived mesenchymal stem cells (MSCs) for investigating their adhesion properties onto native porcine heart tissue. Once MSCs reached confluence after a 7-day culture on a temperature-responsive culture dish, a MSCs monolayer spontaneously detached itself from the dish, when the culture temperature was reduced from 37 to 20°C. The basal extracellular matrix (ECM) proteins of the single cell sheet are preserved, because this technique requires no proteolytic enzymes for harvesting cell sheet, which become a basic building block for assembling a multilayer cell sheet. The thickness of multilayered cell sheets made from three MSC sheets was found to be approximately 60 μm. For investigating the adhesion properties of the basal and apical sides, the multilayered cell sheets were transplanted onto the surface of the heart’s left ventricle. Multilayered cell sheets were histological investigated at 15, 30, 45 and 60 minutes after transplantation by hematoxylin eosin (HE) and azan dyes to determine required time for the adhesion of the multilayered sheets following cell-sheet transplantation. The results showed that only the basal side of multilayered cell sheets significantly enhanced the sheets adhesion onto the surface of heart 30 minutes after transplantation. This study concluded that (1) cell sheets had to be transplanted with its basal side onto the surface of heart tissue and (2) at least 30 minutes were necessary for obtaining the histological adhesion of the sheets to the heart tissue. This study provided clinical evidence and parameters for the successful application of MSC sheets to the myocardium and allowed cell sheet technology to be adapted clinical cell-therapy for myocardial diseases.     

The effect of surface charge property on Escherichia coli initial adhesion and subsequent biofilm formation

Polyethylene (PE) sheets were modified by radiation-induced graft polymerization (RIGP) of an epoxy-group containing monomer glycidyl methacrylate (GMA). The epoxy group of GMA was opened by introducing sodium sulfite (SS) and diethylamine (DEA) as representatives of negatively and positively charged functional groups, respectively. These modified surfaces by RIGP, termed GMA, SS, and DEA sheets, were investigated to elucidate their effects on initial adhesion and subsequent biofilm formation of Escherichia coli. Initial adhesion test revealed that E. coli density and viability were governed by sheet surface electrostatic property: E. coli cell density on the DEA sheet was 23 times higher than that on the SS sheet after 8 h incubation. The viability of E. coli cells dramatically decreased after contact with the DEA sheet, but remained high on the SS sheet. E. coli biofilm structure on the DEA sheet was dense, homogeneous, and uniform, with biomass higher than that of the GMA and SS sheets by factors of 14.0 and 37.5, respectively. On the contrary, biofilm structure on the SS sheet was sparse, heterogeneous, and mushroom-shaped. More than 40% of E. coli biofilm on the DEA sheet was retained under a high liquid shear force condition (5,000 s(-1)), whereas 97% and 100% of biofilms on the GMA and SS sheets were sloughed, indicating that E. coli biofilm robustness depends on surface charge property of the substratum. This suggests that substratum surface fabrication by RIGP may enhance or suppress biofilm formation, a finding with potentially important practical implications.     

Aligning cells in arbitrary directions on a membrane sheet using locally formed microwrinkles

Sheets of cells can be used for tissue regenerative medicine. Cell alignment within the sheet is now a key factor in the next generation of this technology. Anisotropic cell sheets without random cell orientations have been conventionally produced with photolithographically, microfabricated substrates using special facilities and equipment. Here we demonstrate a more accessible approach to the fabrication of anisotropic substrates. We locally deformed part of an elastic membrane and simultaneously oxidized the surface to create microwrinkles as well as to enable adhesion to the extracellular matrix. The approach with the local loading made it possible to orient cells in controlled directions within a single membrane sheet depending on the strains determined by the controllable deformation. This technique potentially enables a versatile design of microwrinkles for target-compatible cell alignments.     

Noninvasive cross-sectional observation of three-dimensional cell sheet-tissue-fabrication by optical coherence tomography

Cell sheet engineering allows investigators/clinicians to prepare cell-dense three-dimensional (3-D) tissues, and various clinical trials with these fabricated tissues have already been performed for regenerating damaged tissues. Cell sheets are easily manipulated and 3-D tissues can be rapidly fabricated by layering the cell sheets. This study used optical coherence tomography (OCT) to noninvasively analyze the following processes: (1) adhesions between layered cell sheets, and (2) the beating and functional interaction of cardiac cell sheet-tissues for fabricating functional thicker 3-D tissues. The tight adhesions and functional couplings between layered cell sheets could be observed cross-sectionally and in real time. Importantly, the noninvasive and cross-sectional analyses of OCT make possible to fabricate 3-D tissues by confirming the adherence and functional couplings between layered cell sheets. OCT technology would contribute to cell sheet engineering and regenerative medicine.     

Rapid Fabricating Technique for Multi-Layered Human Hepatic Cell Sheets by Forceful Contraction of the Fibroblast Monolayer

Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell) sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells) as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions.     

Responsive systems for cell sheet detachment

Cell sheet engineering has been progressing rapidly during the past few years and has emerged as a novel approach for cell based therapy. Cell sheet harvest technology enables fabrication of viable, transplantable cell sheets for various tissue engineering applications. Currently, the majority of cell sheet studies use thermo-responsive systems for cell sheet detachment. However, other responsive systems began showing their potentials for cell sheet harvest. This review provides an overview of current techniques in creating cell sheets using different types of responsive systems including thermo-responsive, electro-responsive, photo-responsive, pH-responsive and magnetic systems. Their mechanism, approach, as well as applications for cell detachment have been introduced. Further development of these responsive systems will allow efficient cell sheet harvesting and patterning of cells to reconstruct complex tissue for broad clinical applications.     

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.     

MECHANICAL PROPERTIES OF THE CELL SURFACE IN STARFISH EGGS

The mechanical properties of the cell surface of the starfish egg at various stages of maturation have been investigated using the cell elastimeter. When constant negative pressure was applied to a part of the cell with a micropipette closely in contact with it, it bulged out, and the bulge rapidly increased at first and then gradually reached a steady value within one min. The relation between the deformation of the cell surface (i.e. degree of bulging) and applied negative pressure was almost linear in both oocytes at the germinal vesicle stage and mature eggs. The surface force and the elasticity value: i.e., the product of the elastic modulus of the surface membrane (layer) and its thickness, were determined from the relation between the deformation and the negative pressure. The elasticity value was about 5 times the surface force in both oocytes at the germinal vesicle stage and mature eggs. When maturation of the oocyte was induced by 1-methyladenine, the stiffness of the cell surface decreased shortly before the breakdown of the germinal vesicle. The stiffness transiently increased at the time of formation of the first and second polar-bodies.     

Gravitropic bending of cress roots without contact between amyloplasts and complexes of endoplasmic reticulum

The polar arrangement of cell organelles in Lepidium root statocytes is persistently converted to a physical stratification during lateral centrifugation (the centrifugal force acts perpendicular to the root long axis) or by apically directed centrifugation combined with cytochalasin-treatment. Lateral centrifugation (10 min, 60 min at 10\g or 50\g) causes displacement of amylplasts to the centrifugal anticlinal cell wall and shifting of the endoplasmic reticulum (ER) complex to the centripetal distal cell edge. After 60 min of lateral centrifugation at 10\g or 50\g all roots show a clear gravitropic curvature. The average angle of curvature is about 40° and corresponds to that of roots stimulated gravitropically in the horizontal position at 1\g in spite of the fact that the gravistimulus is 10-or 50-fold higher. Apically directed centrifugation combined with cytochalasin B (25 g\ml^-1) or cytochalasin D (2.5 g\ml^-1) incubation yields statocytes with the amyloplasts sedimented close to the centrifugal periclinal cell wall and ER cisternae accumulated at the proximal cell pole. Gravitropic stimulation for 30 min in the horizontal position at 1\g and additional 3 h rotation on a clinostat result in gravicurvature of cytochalasin B-treated centrifuged (1 h at 50\g) roots, but because of retarded root growth the angle of curvature is lower than in control roots. Cytochalasin D-treatment during centrifugation (20 min at 50\g) does not affect either root growth or gravicurvature during 3 h horizontal exposure to 1\g relative to untreated roots. As lateral centrifugation enables only short-term contact between the amyloplasts and the distal ER complex at the onset of centrifugation and apically directed centrifugation combined with cytochalasin-treatment even exclude any contact the integrity of the distal cell pole need not necessarily be a prerequisite for graviperception in Lepidium root statocytes.Abbreviations CB cytochalasin B - CD cytochalasin D - ER endoplasmic reticulum - g gravitational acceleration     

Reconstruction of functional endometrium-like tissue in vitro and in vivo using cell sheet engineering

Uterus is a female specific reproductive organ and plays critical roles in allowing embryo to grow. Therefore, the endometrial disorders lead to female infertility. Hence, the regeneration of endometrium allowing fertilized ovum to implant might be valuable in the field of fertility treatment. Recently, cell sheet engineering using a temperature-responsive culture dish has advanced in regenerative medicine. With this technology, endometrial cells were harvested as a contiguous cell sheet by reducing temperature. Firstly, mouse endometrial cell sheets were re-cultured for 3 days to evaluate the function. Histological analyses revealed that endometrial epithelial cell-specific cytokeratin 18 and female-specific hormone receptors, estrogen receptor β and progesterone receptor, were expressed. Furthermore, endometrial epithelial cells constructed epithelial layer at the apical side. Then, endometrial cell sheets from green-fluorescent-protein rat cells were transplanted onto the buttock muscle of nude rat for evaluating the function in vivo. Histological analyses showed that endometrial cell sheets reconstructed endometrium-like tissue, which was found to form uterus-specific endometrial glands having hormonal receptor to estrogen. In this study, endometrial cell sheets were speculated to contribute to the regeneration of functional endometrium as a new therapy.     

Micropatterned thermoresponsive polymer brush surfaces for fabricating cell sheets with well-controlled orientational structures

Newly developed fabrication technique of thermoresponsive surface using RAFT-mediated block copolymerization and photolithography achieved stripe-like micropatterning of poly(N-isopropylacrylamide) (PIPAAm) brush domains and poly(N-isopropylacrylamide)-b-poly(N-acryloylmorpholine) domains. Normal human dermal fibroblasts were aligned on the physicochemically patterned surfaces simply by one-pot cell seeding. Fluorescence images showed the well-controlled orientation of actin fibers and fibronectin in the confluent cell layers with associated extracellular matrix (ECM) on the surfaces. Furthermore, the aligned cells were harvested as a tissue-like cellular monolayer, called "cell sheet" only by reducing temperature below PIPAAm's lower critical solution temperature (LCST) to 20 °C. The cell sheet harvested from the micropatterned surface possessed a different shrinking rate between vertical and parallel sides of the cell alignment (approximately 3:1 of aspect ratio). This indicates that the cell sheet maintains the alignment of cells and related ECM proteins, promising to show the mechanical and biological aspects of cell sheets harvested from the functionalized thermoresponsive surfaces.     

MECHANICAL PROPERTIES OF SEA URCHIN EGGS III. VISCO-ELASTICITY OF THE CELL SURFACE

When a sea urchin egg was compressed between two parallel plates, the force required to keep the distance between the plates constant gradually decreased with time. The contours of the compressed egg were different from the contours expected from the assumption that the surface forces are uniform over the entire surface. The surface forces of the egg without deformation computed from the area of the cell surface in contact with the substratum, the density of the egg and its size were 0.02–0.04 dynes/cm in Hemicentrotus pulcherrimus. Larger values were obtained in eggs during compression. Surface forces, which were computed from measurements of the form of the egg and the applied force when the egg was deformed by a rod and a plate supporting the egg, increased as the deformation increased.
From these results, it was concluded that the cell surface is visco-elastic in sea urchin eggs.     

Centrifugal dewatering of acid casein curd: effect of casein manufacturing and centrifugation variables on curd compression in a laboratory centrifuge

Data relevant to curd compression in a horizontal, solid bowl decanter centrifuge have been obtained by studying the dewatering of acid casein curd in a batch laboratory centrifuge. Analysis of curd compression under centrifugal force predicts a moisture content gradient in the dewatered curd from a maximum at the curd-liquid interface to a minimum at the centrifuge bowl wall. This moisture content gradient was also measured experimentally, and its practical implications are discussed. Increases in centrifugal force, centrifugation time, and centrifugation temperature all caused a marked de crease in dewatered curd moisture content, whereas in creases in precipitation pH and maximum washing temperature caused a smaller decrease in dewatered curd moisture content.     

Environmental and centrifugal factors influencing the visco-elastic properties of oral biofilms in vitro

Centrifugal compaction causes changes in the surface properties of bacterial cells. It has been shown previously that the surface properties of planktonic cells change with increasing centrifugal compaction. This study aimed to analyze the influences of centrifugal compaction and environmental conditions on the visco-elastic properties of oral biofilms. Biofilms were grown out of a layer of initially adhering streptococci, actinomyces or a combination of these. Different uni-axial deformations were induced on the biofilms and the load relaxations were measured over time. Linear-Regression-Analysis demonstrated that both the centrifugation coefficient for streptococci and induced deformation influenced the percentage relaxation. Centrifugal compaction significantly influenced relaxation only upon compression of the outermost 20% of the biofilm (p < 0.05), whereas biofilm composition became influential when 50% deformation was induced, invoking re-arrangement of the bacteria in deeper biofilm structures. In summary, the effects of centrifugal compaction of initially adhering, centrifuged bacteria extend to the visco-elastic properties of biofilms, indicating that the initial bacterial layer influences the structure of the entire biofilm.     

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10⁷ cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.     

An ultra scale‐down analysis of the recovery by dead‐end centrifugation of human cells for therapy

An ultra scale‐down method is described to determine the response of cells to recovery by dead‐end (batch) centrifugation under commercially defined manufacturing conditions. The key variables studied are the cell suspension hold time prior to centrifugation, the relative centrifugal force (RCF), time of centrifugation, cell pellet resuspension velocities, and number of resuspension passes. The cell critical quality attributes studied are the cell membrane integrity and the presence of selected surface markers. Greater hold times and higher RCF values for longer spin times all led to the increased loss of cell membrane integrity. However, this loss was found to occur during intense cell resuspension rather than the preceding centrifugation stage. Controlled resuspension at low stress conditions below a possible critical stress point led to essentially complete cell recovery even at conditions of extreme centrifugation (e.g., RCF of 10000 g for 30 mins) and long (~2 h) holding times before centrifugation. The susceptibility to cell loss during resuspension under conditions of high stress depended on cell type and the age of cells before centrifugation and the level of matrix crosslinking within the cell pellet as determined by the presence of detachment enzymes or possibly the nature of the resuspension medium. Changes in cell surface markers were significant in some cases but to a lower extent than loss of cell membrane integrity. Biotechnol. Bioeng. 2015;112: 997–1011. © 2014 Wiley Periodicals, Inc.     

Preparation of bead-tailed actin filaments: estimation of the torque produced by the sliding force in an in vitro motility assay.

By coating covalently the surface of a polystyrene bead (diameter = 1 micron) with gelsolin, we have succeeded in attaching the bead selectively at the barbed end of an actin filament and forming a 1:1 bead-actin filament complex. On a layer of heavy meromyosin on a nitrocellulose-coated coverglass, this bead-actin filament complex slid smoothly, trailing the bead at its end. Therefore we called this preparation "bead-tailed" actin filaments. The sliding velocity was indistinguishable from that of nonbeaded filaments. With use of this system, we tried to detect the axial rotation (rotation around the filament axis) in a sliding actin filament. Although a single bead at the tail end did not serve as the marker for the axial rotation, we occasionally found another bead bound to the tail bead. In this case, the orientation of the bead-aggregate could be followed continuously with a video monitor while the filament was sliding over heavy meromyosin. We observed that actin filaments slid over distances of many tens of micrometers without showing a complete turn of the bead-aggregates. On the basis of the calculation of rotational friction drag on the bead-aggregate, we estimate that the rotational component of the sliding force and the torque produced on a sliding actin filament (length < or = 10 microns) did not accumulate > 1 pN and 5 pN.nm, respectively. In the present system of randomly oriented heavy meromyosin lying on a nitrocellulose film without an external load.