Abstract

Despite the increasing prevalence of Nonalcoholic steatohepatitis (NASH) worldwide, there is no effective treatment available for this disease. “Ballooned hepatocyte” is a characteristic finding in NASH and is correlated with disease prognosis, but their mechanisms of action are poorly understood; furthermore, neither animal nor in vitro models of NASH have been able to adequately represent ballooned hepatocytes. Herein, we engineered cell sheets to develop a new in vitro model of ballooned hepatocytes. Primary human hepatocytes (PHH) and Hepatic stellate cells (HSC) were co-cultured to produce cell sheets, which were cultured in glucose and lipid containing medium, following which histological and functional analyses were performed. Histological findings showed hepatocyte ballooning, accumulation of fat droplets, abnormal cytokeratin arrangement, and the presence of Mallory–Denk bodies and abnormal organelles. These findings are similar to those of ballooned hepatocytes in human NASH. Functional analysis showed elevated levels of TGFβ-1, SHH, and p62, but not TNF-α, IL-8. Exposure of PHH/HSC sheets to a glucolipotoxicity environment induces ballooned hepatocyte without inflammation. Moreover, fibrosis is an important mechanism underlying ballooned hepatocytes and could be the basis for the development of a new in vitro NASH model with ballooned hepatocytes.

Tissue engineering has attracted attention worldwide because of its application in regenerative medicine, drug screening, and cultured meat. Numerous biofabrication techniques for producing tissues have been developed, including various scaffold and printing methods. Here, we have proposed a novel tissue engineering method using a net metal mould without the use of a scaffold. Briefly, normal human dermal fibroblasts seeded on a dimple plate were subjected to static culture technique for several days to form spheroids. Spheroids of diameter > 200 mu m were poured into a net-shaped mould of gap <= 100 mu m and subjected to shake-cultivation for several weeks, facilitating their fusion to form a three-dimensional (3D) tissue. Through this study, we successfully constructed a scaffold-free 3D tissue having strength that can be easily manipulated, which was difficult to construct using conventional tissue engineering methods. We also investigated the viability of the 3D tissue and found that the condition of the tissues was completely different depending on the culture media used. Collectively, this method allows scaffold-free culture of 3D tissues of unprecedented thickness, and may contribute largely to next-generation tissue engineering products.

Culturing three-dimensional (3D) tissues with an appropriate microenvironment is a critical and fundamental technology in broad areas of cutting-edge bioengineering research. In addition, many technologies have engineered tissue functions. However, an effective system for transporting nutrients, waste, or oxygen to affect the functions of cell tissues has not been reported. In this study, we introduce a novel system that employs diffusion and convection to enhance transportation. To demonstrate the concept of the proposed system, three layers of normal human dermal fibroblast cell sheets are used as a model tissue, which is cultured on a general dish or porous collagen scaffold with perfusable channels for three days with and without the perfusion of culture media in the scaffold. The results show that the viability of the cell tissue was improved by the developed system. Furthermore, glucose consumption, lactate production, and oxygen transport to the tissues were increased, which might improve the viability of tissues. However, mechanical stress in the proposed system did not cause damage or unintentional functional changes in the cultured tissue. We believe that the introduced culturing system potentially suggests a novel standard for 3D cell cultures.

Three-dimensional (3D) cardiac tissue reconstruction using tissue engineering technology is a rapidly growing area of regenerative medicine and drug screening development. However, there remains an urgent need for the development of a method capable of accurately measuring the contractile force of physiologically relevant 3D myocardial tissues to facilitate the prediction of human heart tissue drug sensitivity. To this end, our laboratory has developed a novel drug screening model that measures the contractile force of cardiac cell sheets prepared using temperature-responsive culture dishes. To circumvent the difficulties that commonly arise during the stacking of cardiomyocyte sheets, we established a stacking method using centrifugal force, making it possible to measure 3D myocardial tissue. Human induced pluripotent stem cell-derived cardiomyocytes were seeded in a temperature-responsive culture dish and processed into a sheet. The cardiac cell sheets were multilayered to construct 3D cardiac tissue. Measurement of the contractile force and cross-sectional area of the multilayered 3D cardiac tissue were then obtained and used to determine the relationship between the cross-sectional area of the cardiac tissue and its contractile force. The contractile force of the 1-, 3-, and 5-layer tissues increased linearly in proportion to the cross-sectional area. A result of 6.4 mN/mm2, accounting for one-seventh of the contractile force found in adult tissue, was obtained. However, with 7-layer tissues, there was a sudden drop in the contractile force, possibly because of limited oxygen and nutrient supply. In conclusion, we established a method wherein the thickness of the cell sheets was controlled through layering, thus enabling accurate evaluation of the cardiac contractile function. This method may enable comparisons with living heart tissue while providing information applicable to regenerative medicine and drug screening models.

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.

In this report, we describe a microfluidic vascular-bed (micro-VB) device providing a platform for 3D tissue engineering with vascular network formation. The micro-VB device allows functional connections between endothelial capillaries of heterogeneous sections (5-100 μm in diameter) and artificial plastic tubes or reservoirs (1-10 mm in diameter). Moreover, the micro-VB device can be installed in a standard 100 mm-diameter Petri dish. Endothelial networks in 3D engineered tissues were obtained by cellular self-assembly on the device, after co-culturing of human umbilical vein endothelial cells (HUVECs) and normal human dermal fibroblasts (NHDFs) in fibrin gel. Endothelial capillary connection between vascularized tissues and microfluidic channels, mimicking arteries and veins, was confirmed by perfusion of fluorescent microspheres. The micro-VB devices were compatible with the use of commercially available culture dishes and did not require the involvement of additional equipment. Thus, these micro-VB devices are expected to substantially improve the routine application of 3D tissue engineering to regenerative medicine.

Ballooned hepatocytes (BH) are enlarged, abnormal hepatocytes, which are usually involved in liver diseases, in particular, nonalcoholic steatohepatitis (NASH). However, formation of BHs in vitro has been seldom reported. This study reported an in vitro strategy to produce human BHs in a cell sheet-based three-dimensional (3D) model where primary human hepatocytes were cocultured with normal human dermal fibroblasts. Enlargement of hepatocytes (2.3 times larger than normal, p < 0.01), loss of cytoplasmic keratin, appearance of Mallory-Denk bodies (MDBs), and abundant fat droplets accumulation were observed after only a few days culture. Additionally, ultrastructural characteristic findings of BHs in human NASH, including enlarged mitochondria with crystalline inclusions, dilated endoplasmic reticulum, and MDBs formation were also observed in the 3D model. Furthermore, pathophysiological features of human NASH, such as increased secretion of sonic hedgehog ligands and myofibroblast activation were found. This study reports in vitro production of human BHs by using a cell sheet-based 3D model. Similar histological, ultrastructural, and pathophysiological features to human NASH are discovered in this model. This model may facilitate study of BHs and increase our knowledge of the pathogenesis of human liver diseases. Impact Statement Human ballooned hepatocytes (BH), which are present in nonalcoholic steatohepatitis (NASH) are mainly studied based on human liver biopsies and animal models. In this study, human BHs can be successfully reproduced in a cell sheet-based in vitro model, which, as far as we know, is the first in vitro model that recapitulates so many histological and ultrastructural hallmarks of BHs found in human NASH. Additionally, this study also demonstrated presence of some NASH pathophysiological features. This model may facilitate the study of hepatocellular ballooning and prove beneficial in translational preclinical drug discovery in NASH.

Pluripotent stem cell including induced pluripotent stem cells (iPSC) are promising cell sources for regenerative medicine and for three-dimensional suspension culture technologies which may enable the generation of robust numbers of desired cells through cell aggregation. Although manual procedure is widely used for dissociating cell aggregates, the development of non-manual procedures using devices will contribute to efficient cell manufacturing. In the present study, we developed novel cell aggregate dissociation devices with a rotating cylinder inside based on taylor couette flow-mediated shear stress. The shear stress can be increased according to an increase in the size of the rotating cylinder inside the devices and the rotation rate. Adequate device size and suitable rotation rate efficiently dissociated cell aggregates after the undifferentiated expansion and the cardiac differentiation of human iPSC. These finding suggest that non-manual device procedure might be useful for harvesting single cells from human iPSC-derived cell aggregates.

The purpose of this study was to fabricate pulsatile tubular cardiac tissue using cell sheet based-tissue engineering. First, we fabricated human induced pluripotent stem cell (hiPSc)-derived cardiomyocyte sheets and normal human dermal fibroblast (NHDF) sheets which are harvested from temperature responsive culture dishes only by lowering the temperature. Then tubular cardiac tissues are formed by wrapping one hiPSc-derived cardiomyocyte sheet and three NHDF sheets around an octagonal column, and both ends of the tubular tissue were covered with fibrin and collagen gel. The octagonal column with the tubular tissue was connected to an in vitro circulation system in a culture box. After four-day culture, the cardiac tissue survived and pulsated spontaneously in the circulation system. Furthermore, the analysis with a Millar catheter inserted into the cardiac tubes revealed significant inner pressure changes generated by their beating. In addition, the tubular cardiac tissue pulsated in response to the electrical stimulation. Although histological analyses demonstrated that cardiac troponin T-positive cells stratified the inner surface of the tubular tissues, gene expression analyses showed an immature state of these cardiomyocytes. Thus, cell sheet-based tissue engineering realized human pulsatile tubular cardiac tissue fabrication and we believe that these tubular cardiac tissues should contribute to future drug screening and regenerative therapy for heart diseases.

Gelatin is useful for biofabrication, because it can be used for cell scaffolds and it has unique properties. Therefore, we attempted to fabricate biodevices of gelatin utilizing micro 3D printer which is able to print with high precision. However, it has been difficult to fabricate 3D structure of gelatin utilizing 3D printer, because a printed gelatin droplet on the metal plate electrode would spread before solidification. To clear this problem, we developed a new experimental set-up with a peltier device that can control temperature of the impact point. At an impact point temperature of 80 °C, the spreading of printed gelatin droplets was prevented. Therefore, we were able to print a ball gelatin. In addition, we were able to print a narrower gelatin line than at an impact point temperature of 20 °C.

Background
Hepatocellular carcinoma (HCC) is considered the 3rd leading cause of death by cancer worldwide with the majority of patients were diagnosed in the late stages. Currently, there is no effective therapy. The selection of an animal model that mimics human cancer is essential for the identification of prognostic/predictive markers, candidate genes underlying cancer induction and the examination of factors that may influence the response of cancers to therapeutic agents and regimens. In this study, we developed a HCC nude rat models using cell sheet and examined the effect of human stromal cells (SCs) on the development of the HCC model and on different liver parameters such as albumin and urea.
Methods
Transplanted cell sheet for HCC rat models was fabricated using thermo-responsive culture dishes. The effect of human umbilical cord mesenchymal stromal cells (UC-MSCs) and human bone marrow mesenchymal stromal cells (BM-MSCs) on the developed tumour was tested. Furthermore, development of tumour and detection of the liver parameter was studied. Additionally, angiogenesis assay was performed using Matrigel.
Results
HepG2 cells requires five days to form a complete cell sheet while HepG2 co-cultured with UC-MSCs or BM-MSCs took only three days. The tumour developed within 4 weeks after transplantation of the HCC sheet on the liver of nude rats. Both UC-MSCs and BM-MSCs improved the secretion of liver parameters by increasing the secretion of albumin and urea. Comparatively, the UC-MSCs were more effective than BM-MSCs, but unlike BM-MSCs, UC-MSCs prevented liver tumour formation and the tube formation of HCC.
Conclusions
Since this is a novel study to induce liver tumour in rats using hepatocellular carcinoma sheet and stromal cells, the data obtained suggest that cell sheet is a fast and easy technique to develop HCC models as well as UC-MSCs have therapeutic potential for liver diseases. Additionally, the data procured indicates that stromal cells enhanced the fabrication of HepG2 cell sheets. This provides the foundation for future research using stromal cells in preclinical and clinical investigations.

Biotechnology has drastically been advanced by the development of iPS and ES cells, which are representative forms induced pluripotent stem cells. In the micro/nano bio field, the development of cells and Taylor-made medicine for a potential treatment of incurable diseases has been a center of attention. The melting point of gelatin is between 25 and 33 °C, and the sol–gel transition occurs in low temperature. This makes the deformation of this useful biomaterial easy. The examples of gelatin fiber applications are suture threads, blood vessel prosthesis, cell-growth-based materials, filter materials, and many others. Because the cell size differs depending on the species and applications, it is essential to fabricate gelatin fibers of different diameters. In this paper, we have developed a fabrication method for gelatin fibers the coacervation method. We fabricated narrow gelatin fibers having a diameter over 10 μm.

Tissues engineered utilizing biofabrication techniques have recently been the focus of much attention, because these bioengineered tissues have great potential to improve the quality of life of patients with various hard-to-treat diseases. Most tissues contain micro-tubular structures including blood vessels, lymphatic vessels, and bile canaliculus. Therefore, we bioengineered a micro diameter tube using alginate gel to coat the core gelatin gel. Micro-gelatin fibers were fabricated by the coacervation method and then coated with a very thin alginate gel layer by dipping. A micro diameter alginate tube was produced by dissolving the core gelatin gel. Consequently, these procedures led to the formation of micro-alginate gel tubes of various shapes and sizes. This biofabrication technique should contribute to tissue engineering research fields. (C) 2017 The Japan Society of Applied Physics

Optical coherence tomography (OCT) is a valuable tool in the cross-sectional observation/analysis of three-dimensional (3-D) biological tissues, and that histological observation is important clinically. However, the resolution of the technology is approximately 10-20 m. In this study, optical coherence microscopy (OCM), a tomographic system combining OCT technology with a microscopic technique, was constructed for observing cells individually with a resolution at the submicrometer level. Cells and 3-D tissues fabricated by cell sheet technology were observed by OCM. Importantly, the cell nuclei and cytoplasm could be clearly distinguished, and the time-dependent dynamics of cell-sheet tissues could be observed in detail. Additionally, the 3-D migration of cells in the bioengineered tissue was also detected using OCM and metal-labeled cells. Bovine aortic endothelial cells, but not NIH3T3 murine embryonic skin fibroblasts, actively migrated within the 3-D tissues. This study showed that the OCM system would be a valuable tool in the fields of cell biology, tissue engineering, and regenerative medicine. (c) 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 481-488, 2017.

In this paper, we report an in vitro co-culture system that combines mammalian cells and algae, Chlorococcum littorale, to create a three-dimensional (3-D) tissue. While the C2C12 mouse myoblasts and rat cardiac cells consumed oxygen actively, intense oxygen production was accounted for by the algae even in the co-culture system. Although cell metabolism within thicker cardiac celllayered tissues showed anaerobic respiration, the introduction of innovative co-cultivation partially changed the metabolism to aerobic respiration. Moreover, the amount of glucose consumption and lactate production in the cardiac tissues and the amount of ammonia in the culture media decreased significantly when co-cultivated with algae. In the cardiac tissues devoid of algae, delamination was observed histologically, and the release of creatine kinase (CK) from the tissues showed severe cardiac cell damage. On the other hand, the layered cell tissues with algae were observed to be in a good histological condition, with less than one-fifth decline in CK release. The co-cultivation with algae improved the culture condition of the thicker tissues, resulting in the formation of 160 mu m-thick cardiac tissues. Thus, the present study proposes the possibility of creating an in vitro "symbiotic recycling system" composed of mammalian cells and algae.

Gelatin has unique properties. The form is changed by temperature. In addition, the melting point is around 37 degree C. For these reasons, gelatin is useful for biofabrication. We are able to fabricate biodevices of biomaterials with cave utilizing gelatin. In this study, we spun gelatin fibers utilizing three methods. We were able to fabricate micro gelatin fibers which diameter was 20~200 μm. Each methods have merits and demerits. Therefore, utilizing three methods, we were able to fabricate complex structures of micro gelatin fibers.

Cellular self-assembly based on cell-to-cell communication is a well-known tissue organizing process in living bodies. Hence, integrating cellular self-assembly processes into tissue engineering is a promising approach to fabricate well-organized functional tissues. In this research, we investigated the capability of endothelial cells (ECs) to control shape and position of vascular formation using arbitral-assembling techniques in three-dimensional engineered tissues. To quantify the degree of migration of ECs in endothelial network formation, image correlation analysis was conducted. Positive correlation between the original positions of arbitrarily assembled ECs and the positions of formed endothelial networks indicated the potential for controlling shape and position of vascular formations in engineered tissues. To demonstrate the feasibility of controlling vascular formations, engineered tissues with vascular networks in triangle and circle patterns were made. The technique reported here employs cellular self-assembly for tissue engineering and is expected to provide fundamental beneficial methods to supply various functional tissues for drug screening and regenerative medicine.

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 degrees 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 mu m. 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.

Construction of three-dimensional (3D) tissues with pre-isolated cells is a promising achievement for novel medicine and drug-discovery research. Our laboratory constructs 3D tissues with an innovative and unique method for layering multiple cell sheets. Cell sheets maintain a high-efficiently regenerating function, because of the higher cell density and higher transplantation efficiency, compared to other cell-delivery methods. Cell sheets have already been applied in clinical applications for regenerative medicine in treating patients with various diseases. Therefore, in our search to develop a more efficient treatment with cell sheets, we are constructing 3D tissues by layering cell sheets. Native animal tissues and organs have an abundance of capillaries to supply oxygen and nutrients, and to remove waste molecules. In our investigation of vascularized cardiac cell sheets, we have found that endothelial cells within cell sheets spontaneously form blood vessel networks as in vivo capillaries. To construct even thicker 3D tissues by layering multiple cell sheets, it is critical to have a medium or blood flow within the vascular networks of the cell sheets. Therefore, to perfuse medium or blood in the cell sheet vascular network to maintain the viability of all cells, we developed two types of vascular beds; (1) a femoral muscle-based vascular bed, and (2) a synthetic collagen gel-based vascular bed. Both vascular beds successfully provide the critical flow of culture medium, which allows 12-layer cell sheets to survive. Such bioreactor systems, when combined with cell sheet engineering techniques, have produced functional vascularized 3D tissues. Here we explain and discuss the various processes to obtain vascular networks by properly connecting cell sheets and the engineering of 3D tissues. (C) 2014 Elsevier B.V. All rights reserved.

In vitro scaling up of bioengineered tissues is known to be limited by diffusion issues, specifically a lack of vasculature. Here, we report a new strategy for preserving cell viability in three-dimensional tissues using cell sheet technology and a perfusion bioreactor having collagen-based microchannels. When triple-layer cardiac cell sheets are incubated within this bioreactor, endothelial cells in the cell sheets migrate to vascularize in the collagen gel, and finally connect with the microchannels. Medium readily flows into the cell sheets through the microchannels and the newly developed capillaries, while the cardiac construct shows simultaneous beating. When additional triple-layer cell sheets are repeatedly layered, new multi-layer construct spontaneously integrates and the resulting construct becomes a vascularized thick tissue. These results confirmed our method to fabricate in vitro vascularized tissue surrogates that overcomes engineered-tissue thickness limitations. The surrogates promise new therapies for damaged organs as well as new in vitro tissue models.

In vitro fabrication of functional vascularized three-dimensional tissues has been a long-standing objective in the field of tissue engineering. Here we report a technique to engineer cardiac tissues with perfusable blood vessels in vitro. Using resected tissue with a connectable artery and vein as a vascular bed, we overlay triple-layer cardiac cell sheets produced from coculture with endothelial cells, and support the tissue construct with media perfused in a bioreactor. We show that endothelial cells connect to capillaries in the vascular bed and form tubular lumens, creating in vitro perfusable blood vessels in the cardiac cell sheets. Thicker engineered tissues can be produced in vitro by overlaying additional triple-layer cell sheets. The vascularized cardiac tissues beat and can be transplanted with blood vessel anastomoses. This technique may create new opportunities for in vitro tissue engineering and has potential therapeutic applications.

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.

Cell therapy is expected to a new tool to treat refractory diseases. In heart regeneration, it has been firstly conducted with needle injection of cell suspensions. Recently, cell sheet engineering emerged as another method of cell therapy. Cell sheet is prepared with a temperature responsive dish by temperature reduction. It is a thin-patch-like tissue construct and its thickness is several tens of micrometers. It is composed of cells and intrinsic extra cellular matrix only. The transplantation of the cell sheet has been already conducted in animal experiments and even in clinical trials. The cell sheet is transplanted at the surface of the heart, but it is difficult to transplant the cell sheet under the beating heart. To overcome this difficulty, we designed a device that was composed of two thin polymer films that have different friction. The films were made of polyurethane, polyethylene, or polypropylene. The cell sheet was set up on the device by sandwiching it with the less frictional film and the more frictional film. In this paper, using two different films having the different friction, the cell sheet was successfully transplanted to the static round polymer surface, the harvested heart, and even the beating heart of pig by removing the films step by step using the difference in friction. Also, surface properties such as friction, adhesion force and roughness of the films were studied by an atomic force microscopy (AFM). From the results of the study, the friction of the film was found to be likely proportional to the adhesion force and the inverse of roughness. © 2011 IEEE.

Recently, researchers have challenged to create three-dimensional (3-D) tissues with tissue engineering technology in order to establish in vitro models and new therapy for damaged organ. Most popular approach of tissue engineering is using 3-D biodegradable scaffolds as alternatives of extracellular matrix. By contrast, we have bioengineered pulsatile myocardial tissues by stacking cardiomyocyte sheets, which were harvested from temperature-responsive culture dishes only by lowering temperature. However, the shortage of oxygen and nutrition limits the final tissue thickness. In this study, we tried to fabricate thicker myocardial tissues by promoting oxygen and nutrition permeation using a novel perfusion bioreactor. Triple-layer neonatal rat cardiomyocyte sheets were attached in the culture chamber. Chambers were perfused with 2.4mL/h culture media and 15mmHg pressure gradient was applied to perfuse the culture media through the construct. After 5-days culture, the construct in the bioreactor was thicker and cell-denser than in static condition. These results indicate that the perfusion bioreactor should contribute to myocardial tissue engineering in vitro.