Exploration of Preservation Methods for Utilizing Porcine Fetal-Organ-Derived Cells in Regenerative Medicine Research.

Human pluripotent stem cells have been employed in generating organoids, yet their immaturity compared to fetal organs and the limited induction of all constituent cell types remain challenges. Porcine fetal progenitor cells have emerged as promising candidates for co-culturing with human progenitor cells in regeneration and xenotransplantation research. This study focused on identifying proper preservation methods for porcine fetal kidneys, hearts, and livers, aiming to optimize their potential as cell sources. Extracted from fetal microminiature pigs, these organs were dissociated before and after cryopreservation-thawing, with subsequent cell quality evaluations. Kidney cells, dissociated and aggregated after vitrification in a whole-organ form, were successfully differentiated into glomeruli and tubules in vivo. In contrast, freezing hearts and livers before dissociation yielded suboptimal results. Heart cells, frozen after dissociation, exhibited pulsating heart muscle cells similar to non-frozen hearts. As for liver cells, we developed a direct tissue perfusion technique and successfully obtained highly viable liver parenchymal cells. Freezing dissociated liver cells, although inferior to their non-frozen counterparts, maintained the ability for colony formation. The findings of this study provide valuable insights into suitable preservation methods for porcine fetal cells from kidneys, hearts, and livers, contributing to the advancement of regeneration and xenotransplantation research.

Successful reconstruction of the rat ureter by a syngeneic collagen tube with a cardiomyocyte sheet.

Introduction: Ureteral injuries require surgical intervention as they lead to loss of renal function. The current reconstructive techniques for long ureteral defects are problematic. Consequently, this study aimed to reconstruct the ureter in a rat model using subcutaneously prepared autologous collagen tubes (Biotubes). Methods: The lower ureter of LEW/SsNSlc rats was ligated to dilate the ureter to make anastomosis easier, and reconstruction was performed six days later by anastomosing the dilated ureter and bladder with a Biotube that was prepared subcutaneously in syngeneic rats. Some rats underwent left nephrectomy and ureter reconstruction simultaneously as negative controls to evaluate the effects of urine flow on patency. The other rats were divided into three groups as follows: a group in which the ureter was reconstructed with the Biotube alone, a group in which cardiomyocyte sheets made from the neonatal hearts of syngeneic rats were wrapped around the Biotube, and a group in which an adipose-derived stem cell sheets made from the inguinal fat of adult syngeneic rats were wrapped. Contrast-enhanced computed tomography and pathological evaluations were performed two weeks after reconstruction. Result: In the Biotube alone group, all tubes were occluded and hydronephrosis developed, whereas the urothelium regenerated beyond the anastomosis when the left kidney was not removed, suggesting that urothelial epithelial spread occurred with urinary flow. The patency of the ureteral lumen was obtained in some rats in the cardiomyocyte sheet covered group, whereas stricture or obstruction of the reconstructed ureter was observed in all rats in the other groups. Pathological evaluation revealed a layered urothelial structure in the cardiomyocyte sheet covered group, although only a small amount of cardiomyocyte sheets remained. Conclusion: Urinary flow may support the epithelial spread of the urothelium into the reconstructed ureter. Neonatal rat cardiomyocyte sheets supported the patency of the regenerated ureter with a layered urothelium.

The bioengineering of perfusable endocrine tissue with anastomosable blood vessels.

Organ transplantation is a definitive treatment for endocrine disorders, but donor shortages limit the use of this technique. The development of regenerative therapies would revolutionize the treatment of endocrine disorders. As is the case for harvested organs, the ideal bioengineered graft would comprise vascularized endocrine tissue, contain blood vessels that could be anastomosed to host vessels, have stable blood flow, and be suitable for transplantation into various sites. Here, we describe a transplantable endocrine tissue graft that was fabricated byex vivoperfusion of tricultured cell sheets (isletß-cells, vascular endothelial cells (vECs), and mesenchymal stem cells (MSCs)) on a vascularized tissue flap ofin vivoorigin. The present study has three key findings. First, mild hypothermic conditions enhanced the success ofex vivoperfusion culture. Specifically, graft construction failed at 37 °C but succeeded at 32 °C (mild hypothermia), and endocrine tissue fabricated under mild hypothermia contained aggregations of isletß-cells surrounded by dense vascular networks. Second, the construction of transplantable endocrine tissue byex vivoperfusion culture was better achieved using a vascular flap (VF) than a muscle flap. Third, the endocrine tissue construct generated using a VF could be transplanted into the rat by anastomosis of the graft artery and vein to host blood vessels, and the graft secreted insulin into the host's circulatory system for at least two weeks after transplantation. Endocrine tissues bioengineered using these techniques potentially could be used as novel endocrine therapies.

Tricultured Cell Sheets Develop into Functional Pancreatic Islet Tissue with a Vascular Network.

Methods to induce islet ß-cells from induced pluripotent stem cells or embryonic stem cells have been established. However, islet ß-cells are susceptible to apoptosis under hypoxic conditions, so the technique used to transplant ß-cells must maintain the viability of cells in vivo. This study describes the development of a tricultured cell sheet, which was made by coculturing islet ß-cells, vascular endothelial cells, and mesenchymal stem cells for 1 day. The islet ß-cells in the tricultured cell sheet self-organized into islet-like structures surrounded by a dense vascular network in vitro. Triple-layered tricultured cell sheets engrafted well after transplantation in vivo and developed into insulin-secreting tissue with abundant blood vessels and a high density of islet ß-cells. We anticipate that the tricultured cell sheet could be used as an in vitro pseudo-islet model for pharmaceutical testing and may have potential for development into transplantable grafts for use in regenerative medicine. Impact statement This research assessed whether tricultured cell sheets containing islet ß-cells, vascular endothelial cells, and mesenchymal stem cells were able to form islet tissue. There were two main findings. First, the islet ß-cells in the tricultured cell sheet self-organized into islet structures surrounded by a dense vascular network in vitro. Second, triple-layered tricultured sheets engrafted well onto rat muscle and developed into insulin-secreting tissue with an abundance of blood vessels. The tricultured cell sheet could be used as a pseudo-islet model for pharmaceutical testing and may have potential for development into a transplantable graft for application in the clinical setting.

Networked lymphatic endothelial cells in a transplanted cell sheet contribute to form functional lymphatic vessels.

This study evaluated whether cell sheets containing a network of lymphatic endothelial cells (LECs) promoted lymphangiogenesis after transplantation in vivo. Cell sheets with a LEC network were constructed by co-culturing LECs and adipose-derived stem cells (ASCs) on temperature-responsive culture dishes. A cell ratio of 3:2 (vs. 1:4) generated networks with more branches and longer branch lengths. LEC-derived lymphatic vessels were observed 2 weeks after transplantation of a three-layered cell sheet construct onto rat gluteal muscle. Lymphatic vessel number, diameter and depth were greatest for a construct comprising two ASC sheets stacked on a LEC/ASC (3:2 ratio) sheet. Transplantation of this construct in a rat model of femoral lymphangiectomy led to the formation of functional lymphatic vessels containing both transplanted and host LECs. Further development of this technique may lead to a new method of promoting lymphangiogenesis.

External pressure dynamics promote kidney viability and perfusate filtration during ex vivo kidney perfusion.

Normothermic machine perfusion (NMP) has not yet been established as a technique for preserving organs for a day. A key contributing factor to the same is that the perfusing solutions cannot circulate continuously and evenly in the organs. Here, we conceived a method of applying intermittent air pressure from outside the organ to assist its circulatory distribution during perfusion. We used a perfusion culture system while applying external pressure to culture rat kidneys and compared the circulatory distribution in the kidneys, changes in tissue morphology due to injury, and perfusate filtration. The intermittent pressurization (IMP) (-) group showed markedly poorer circulation on the upper side compared with that in the lower side, alongside histological damage. On the other hand, the IMP (+) group showed improved circulation in the upper side and had lesser histological damage. Furthermore, the IMP (+) group maintained the ability to filter perfusate for 24 h. In transplantation medicine and regenerative medicine research, this method has the potential to contribute to more efficient organ preservation and more functional tissue regeneration in the future.

Development of appropriate fatty acid formulations to raise the contractility of constructed myocardial tissues.

Introduction: Heart disease is a major cause of mortality worldwide, and the annual number of deaths due to heart disease has increased in recent years. Although heart failure is usually managed with medicines, the ultimate treatment for end-stage disease is heart transplantation or an artificial heart. However, the use of these surgical strategies is limited by issues such as thrombosis, rejection and donor shortages. Regenerative therapies, such as the transplantation of cultured cells and tissues constructed using tissue engineering techniques, are receiving great attention as possible alternative treatments for heart failure. Research is ongoing into the potential clinical use of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). However, the energy-producing capacity of cardiomyocytes maintained under previous culture conditions is lower than that of adult primary cardiomyocytes due to immaturity and a reliance on glucose metabolism. Therefore, the aims of this study were to compare the types of fatty acids metabolized between cardiomyocytes in culture and heart cells in vivo and investigate whether the addition of fatty acids to the culture medium affected energy production by cardiomyocytes. Methods: A fatty acid-containing medium was developed based on an analysis of fatty acid consumption by rat primary cardiomyocytes (rat-CMs), and the effects of this medium on adenosine triphosphate (ATP) production were investigated through bioluminescence imaging of luciferase-expressing rat-CMs. Next, the fatty acid content of the medium was further adjusted based on analyses of fatty acid utilization by porcine hearts and hiPSC-CMs. Oxygen consumption analyses were performed to explore whether the fatty acid-containing medium induced hiPSC-CMs to switch from anaerobic metabolism to aerobic metabolism. Furthermore, the effects of the medium on contractile force generated by hiPSC-CM-derived tissue were evaluated. Results: Rat serum, human serum and porcine plasma contained similar types of fatty acid (oleic acid, stearic acid, linoleic acid, palmitic acid and arachidonic acid). The types of fatty acid consumed were also similar between rat-CMs, hiPSC-CMs and porcine heart. The addition of fatty acids to the culture medium increased the bioluminescence of luciferase-expressing rat-CMs (an indirect measure of ATP level), oxygen consumption by hiPSC-CMs, and contractile force generated by cardiac tissues constructed from hiPSC-CMs. Conclusions: hiPSC-CMs metabolize similar types of fatty acid to those consumed by rat-CMs and porcine hearts. Furthermore, the addition of these fatty acids to the culture medium increased energy production by rat-CMs and hiPSC-CMs and enhanced the contractility of myocardial tissue generated from hiPSC-CMs. These findings suggest that the addition of fatty acids to the culture medium stimulates aerobic energy production by cardiomyocytes through ß-oxidation. Since cardiomyocytes cultured in standard media rely primarily on anaerobic glucose metabolism and remain in an immature state, further research is merited to establish whether the addition of fatty acids to the culture medium would improve the energy-producing capacity and maturity of hiPSC-CMs and cardiac tissue constructed from these cells. It is possible that optimizing the metabolism of cultured cardiomyocytes, which require high energy production to sustain their contractile function, will improve the properties of hiPSC-CM-derived tissue, allowing it to be better utilized for disease modeling, drug screening and regenerative therapies for heart failure.

Organ Bioluminescence Imaging Under Machine Perfusion Setting for Assessing Quality of Harvested Organ Preservation.

Determination of organ viability over a period of time is a key technology in the process of organ preservation. However, a robust methodology to address this issue has not been established. Luciferase-expressing organs enable the assessment of the variances in organ viability over time as well as the visualization of a damaged tissue region. Herein, we introduce the assessment method for organ viability in detail using luciferase-expressing organs harvested from transgenic Lewis rats (Luc-LEW Tg rats). We exemplify the femoral muscle pedicle flap for the methods of tissue preparation, of setting up the machine perfusion system, and of measuring emitted light to assess organ viability. This evaluation method would be applicable to other organ-preservation studies as an innovative tool for developing a profound understanding of organ preservation.

Tubular Cardiac Tissue Bioengineered from Multi-Layered Cell Sheets for Use in the Treatment of Heart Failure.

This chapter describes a method for creating tubular cardiac tissue in vitro. Thick cardiac tissue in a tubular configuration is prepared by stacking cell sheets stepwise on the inner wall of a segment of small intestine, which functions as a blood vessel bed. The capillaries of the small intestinal segment are fed by an artery and drained by a vein. Therefore, perfusion culture of the cardiac tissue is achieved by continuously infusing culture medium into the arterial vessel that supplies the segment of small intestine. The aim of this technique is to fabricate tubular cardiac tissue that can function as a pump by sequentially implanting and culturing cardiac cell sheets on the inner wall of a perfused segment of small intestine.

A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapy.

Introduction: Lung transplantation is the only effective treatment option for many patients with irreversible pulmonary injury, and the demand for lung transplantation is increasing worldwide and expected to continue to outstrip the number of available donors. Regenerative therapy with alveolar epithelial cells (AECs) holds promise as an alternative option to organ transplantation. AECs are usually co-cultured with mouse-derived 3T3 feeder cells, but the use of xenogeneic tissues for regenerative therapy raises safety concerns. Fabrication of AEC sheets under feeder-free conditions would avoid these safety issues. We describe a novel feeder-free method of fabricating AEC sheets that may be suitable for pulmonary regenerative therapy. Methods: Lung tissues excised from male outbred rats or transgenic rats expressing green fluorescent protein (GFP) were finely minced and dissociated with elastase. The isolated AECs were cultured under four different feeder-free conditions according to whether a rho kinase (ROCK) inhibitor was included in the low-calcium medium (LCM) and whether the tissue culture dish was coated with recombinant laminin-511 E8 fragment (rLN511E8). The expanded cells were cultured on temperature-responsive dishes and subsequently harvested as AEC sheets. Engraftment of GFP-AEC sheets after their transplantation onto a partially resected region of the left lung was assessed in athymic rats. Results: AECs proliferated and reached confluence when cultured in LCM containing a ROCK inhibitor on tissue culture dishes coated with rLN511E8. When both the ROCK inhibitor and rLN511E8-coated culture dish were used, the number of AECs obtained after 7 days of culture was significantly higher than that in the other three groups. Immunohistochemical analyses revealed that aquaporin-5, surfactant protein (SP)-A, SP-C, SP-D and Axin-2 were expressed by the cultured AECs. AEC sheets were harvested successfully from temperature-responsive culture dishes (by lowering the temperature) when the expanded AECs were cultured for 7 days in LCM + ROCK inhibitor and then for 3 days in LCM + ROCK inhibitor supplemented with 200 mg/L calcium chloride. The AEC sheets were firmly engrafted 7 days after transplantation onto the lung defect and expressed AEC marker proteins. Conclusions: AEC sheets fabricated under feeder-free conditions retained the features of AECs after transplantation onto the lung in vivo. Further improvement of this technique may allow the bioengineering of alveolar-like tissue for use in pulmonary regenerative therapy.

Bioartificial pulsatile cuffs fabricated from human induced pluripotent stem cell-derived cardiomyocytes using a pre-vascularization technique.

There is great interest in the development of techniques to bioengineer pulsatile myocardial tissue as a next-generation regenerative therapy for severe heart failure. However, creation of thick myocardial grafts for regenerative medicine requires the incorporation of blood vessels. In this study, we describe a new method of constructing a vascular network in vivo that allows the construction of thick human myocardial tissue from multi-layered cell sheets. A gelatin sheet pre-loaded with growth factors was transplanted onto the superficial femoral artery and vein of the rat. These structures were encapsulated together within an ethylene vinyl alcohol membrane and incubated in vivo for 3 weeks (with distal superficial femoral artery ligation after 2 weeks to promote blood flow to the vascular bed). Subsequently, six cardiomyocyte sheets were transplanted onto the vascular bed in two stages (three sheets, two times). Incubation of this construct for a further week generated vascularized human myocardial tissue with an independent circulation supplied by an artery and vein suitable for anastomosis to host vessels. Notably, laminating six cell sheets on the vascular bed in two stages rather than one allowed the creation of thicker myocardial tissue while suppressing tissue remodeling and fibrosis. Finally, the pulsatile myocardial tissue was shown to generate auxiliary pressure when wrapped around the common iliac artery of a rat. Further development of this technique might facilitate the generation of circulatory assist devices for patients with heart failure.

Perfusable vascular tree like construction in 3D cell-dense tissues using artificial vascular bed.

Perfusable vascular structures in cell-dense tissues are essential for fabricating functional three-dimensional (3D) tissues in vitro. However, it is challenging to introduce functional vascular networks observable as vascular tree, finely spaced at intervals of tens of micrometers as in living tissues, into a 3D cell-dense tissue. Herein, we propose a method for introducing numerous vascular networks that can be perfused with blood into 3D tissues constructed by cell sheet engineering. We devise an artificial vascular bed using a hydrogel that is barely deformed by cells, enabling perfusion of the culture medium directly beneath the cell sheets. Triple-layered cell sheets with an endothelial cell network prepared by fibroblast co-culture are transplanted onto the vascular bed and subjected to perfusion culture. We demonstrate that numerous vascular networks are formed with luminal structures in the cell sheets and can be perfused with India ink or blood after a five-day perfusion culture. Histological analysis also demonstrates that perfusable vascular structures are constructed at least 100 µm intervals uniformly and densely within the tissues. The results suggest that our perfusion culture method enhances vascularization within the 3D cell-dense tissues and enables the introduction of functional vasculature macroscopically observable as vascular tree in vitro. In conclusion, this technology can be used to fabricate functional tissues and organs for regenerative therapies and in vitro experimental models.

Continuous measurement of surface electrical potentials from transplanted cardiomyocyte tissue derived from human-induced pluripotent stem cells under physiological conditions in vivo.

Recording the electrical potentials of bioengineered cardiac tissue after transplantation would help to monitor the maturation of the tissue and detect adverse events such as arrhythmia. However, a few studies have reported the measurement of myocardial tissue potentials in vivo under physiological conditions. In this study, human-induced pluripotent stem cell-derived cardiomyocyte (hiPSCM) sheets were stacked and ectopically transplanted into the subcutaneous tissue of rats for culture in vivo. Three months after transplantation, a flexible nanomesh sensor was implanted onto the hiPSCM tissue to record its surface electrical potentials under physiological conditions, i.e., without the need for anesthetic agents that might adversely affect cardiomyocyte function. The nanomesh sensor was able to record electrical potentials in non-sedated, ambulating animals for up to 48 h. When compared with recordings made with conventional needle electrodes in anesthetized animals, the waveforms obtained with the nanomesh sensor showed less dispersion of waveform interval and waveform duration. However, waveform amplitude tended to show greater dispersion for the nanomesh sensor than for the needle electrodes, possibly due to motion artifacts produced by movements of the animal or local tissue changes in response to surgical implantation of the sensor. The implantable nanomesh sensor utilized in this study potentially could be used for long-term monitoring of bioengineered myocardial tissue in vivo under physiological conditions.

Capillary Networks for Bio-Artificial Three-Dimensional Tissues Fabricated Using Cell Sheet Based Tissue Engineering.

One of the most important challenges facing researchers in the field of regenerative medicine is to develop methods to introduce vascular networks into bioengineered tissues. Although cell scaffolds that slowly release angiogenic factors can promote post-transplantation angiogenesis, they cannot be used to construct thick tissues because of the time required for sufficient vascular network formation. Recently, the co-culture of graft tissue with vascular cells before transplantation has attracted attention as a way of promoting capillary angiogenesis. Although the co-cultured vascular cells can directly contribute to blood vessel formation within the tissue, a key objective that needs to be met is the construction of a continuous circulatory structure. Previously described strategies to reconstruct blood vessels include the culture of endothelial cells in a scaffold that contains microchannels or within the original vascular framework after decellularization of an entire organ. The technique, as developed by authors, involves the progressive stacking of three-layered cell sheets onto a vascular bed to induce the formation of a capillary network within the cell sheets. This approach enables the construction of thick, functional tissue of high cell density that can be transplanted by anastomosing its artery and vein (provided by the vascular bed) with host blood vessels.

A novel method to align cells in a cardiac tissue-like construct fabricated by cell sheet-based tissue engineering.

Fabrication of cardiac tissue from human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has received great interest, but a major challenge facing researchers is the alignment of cardiomyocytes in the same direction to optimize force generation. We have developed a novel method of fabricating a cardiac tissue-like construct with aligned cells based on the unidirectional stretching of an hiPS-CM sheet. A square cell sheet was harvested from a temperature-responsive culture dish and placed on a silicone surface, and an extending force was imposed on the silicone to stretch the cell sheet along one direction. To enable evaluation of cardiomyocyte morphology in vitro, a cell sheet was constructed by coculture of hiPS-CMs and human adipose-derived stem cells. In separate experiments, a stretched double-layered cell sheet constructed from hiPS-CMs alone was transplanted onto the muscle of an athymic rat, and its features were compared with those of a nonstretched (control) cell sheet. Immediately after stretching, the stretched cell sheet was significantly longer than the control cell sheet. Immunohistological analysis revealed that the cardiomyocytes showed unidirectional alignment in the stretched cell sheet but random directionality in the control cell sheet. Two weeks after transplantation, immunohistology demonstrated that the stretched cell sheet had retained the unidirectionality of its myocardial fibers and had an orientation intensity that was higher than that of the control cell sheet after transplantation or the stretched cell sheet before transplantation. Our technique provides a simple method of aligning an hiPS-CM-derived cardiac tissue-like construct without the use of a scaffold.

Engineering of functional cardiac tubes by stepwise transplantation of cardiac cell sheets onto intestinal mesentery.

Implantable organ-like grafts made using tissue engineering techniques could potentially be used as circulatory assist devices in people with heart failure. The aims of this study were to engineer implantable, thick cardiac tubes by the stepwise transplantation of cardiac cell sheets onto intestinal mesentery and confirm that these cardiac tubes exhibited pulsatile activity and generated an internal pressure. Cell sheets were created by culturing neonatal rat cardiac cells on temperature-responsive dishes. After harvesting, three cell sheets were stacked, and the triple-layered cell sheet was rolled around a section of endotracheal tube. The resulting construct was cultured to generate a cardiac tube. In the single-step group (n = 6), a cardiac tube was implanted onto the intestinal mesentery of a rat. In the double-step group (n = 6), a cardiac tube was implanted onto the intestinal mesentery of a rat, and another new cardiac tube was inserted into the original cardiac tube one day later. The pulsations and internal pressures of the implanted cardiac tubes were evaluated 1, 2 and 4 weeks after transplantation. Histology and immunohistochemistry were used to confirm whether vasculature was present in the cardiac tubes at 4 weeks after transplantation. We found that the cardiac tubes developed spontaneous pulsations from 1 week after transplantation. The average internal pressures of the cardiac tubes at 4 weeks after transplantation were 1.8 ± 1.0 mmHg in the single-step group and 2.5 ± 0.3 mmHg in the double-step group. The cardiac tubes in the double-step group contracted in response to electrical stimulation at 4 weeks after transplantation. Histological and immunohistochemical analyses revealed engraftment of the transplanted cardiac cell sheets and neovascularization of the cardiac tubes in both groups. Our findings demonstrate that it is feasible to generate functional cardiac tubes using the mesentery as a vascular bed. Further development of this technique will include the creation of a thicker tube, transplantation of the tube into major vessels and evaluation of the function of the tube under physiological conditions.

Intermittent application of external positive pressure helps to preserve organ viability during ex vivo perfusion and culture.

The perfusion of medium through blood vessels allows the preservation of donor organs and culture of bioengineered organs. However, tissue damage due to inadequate perfusion remains a problem. We evaluated whether intermittent external pressurization would improve the perfusion and viability of organs in culture. A bioreactor system was used to perfuse and culture rat small intestine and femoral muscle preparations. Intermittent positive external pressure (10 mmHg) was applied for 20 s at intervals of 20 s. Intermittent pressurization resulted in uniform perfusion of small intestine preparations and minimal tissue damage after 20 h of perfusion, whereas non-pressurized (control) preparations exhibited significantly worse perfusion of the upper surface than the lower surface and histologic evidence of tissue damage. Longer term studies were undertaken in luciferase-expressing rat femoral muscle preparations. Compared with non-pressurized controls, intermittent pressurization led to better perfusion throughout the 14-day experimental period, improved organ viability as indicated by a higher bioluminescence intensity after perfusion with luciferin, and reduced levels of tissue necrosis with better preservation of vascular structures and skeletal muscle nuclei (histologic analyses). Therefore, intermittent application of external positive pressure improved the perfusion of small intestine and skeletal muscle preparations and enhanced tissue viability when compared with controls. We anticipate that this innovative perfusion technique could be used to improve the preservation of donor organs and culture of bioengineered organs.

Correction to: Intermittent application of external positive pressure helps to preserve organ viability during ex vivo perfusion and culture.

article Intermittent application.

Microfluidic vascular-bed devices for vascularized 3D tissue engineering: tissue engineering on a chip.

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.