Partial Decellularized Scaffold Combined with Autologous Nasal Epithelial Cell Sheet for Tracheal Tissue Engineering

Decellularization has emerged as a potential solution for tracheal replacement. As a fully decellularized graft failed to achieve its purposes, the de-epithelialization partial decellularization protocol appeared to be a promising approach for fabricating scaffolds with preserved mechanical properties and few immune rejection responses after transplantation. Nevertheless, a lack of appropriate concurrent epithelialization treatment can lead to luminal stenosis of the transplant and impede its eventual success. To improve re-epithelialization, autologous nasal epithelial cell sheets generated by our cell sheet engineering platform were utilized in this study under an in vivo rabbit model. The newly created cell sheets have an intact and transplantable appearance, with their specific characteristics of airway epithelial origin being highly expressed upon histological and immunohistochemical analysis. Subsequently, those cell sheets were incorporated with a partially decellularized tracheal graft for autograft transplantation under tracheal partial resection models. The preliminary results two months post operation demonstrated that the transplanted patches appeared to be wholly integrated into the host trachea with adequate healing of the luminal surface, which was confirmed via endoscopic and histologic evaluations. The satisfactory result of this hybrid scaffold protocol could serve as a potential solution for tracheal reconstructions in the future.

Chemically Crosslinked Methylcellulose Substrates for Cell Sheet Engineering

Methylcellulose (MC) hydrogels have been successfully proposed in the field of cell sheet engineering (CSE), allowing cell detachment from their surface by lowering the temperature below their transition temperature (Tt). Among the main limitations of pristine MC hydrogels, low physical stability and mechanical performances limit the breadth of their potential applications. In this study, a crosslinking strategy based on citric acid (CA) was used to prepare thermoresponsive MC hydrogels, with different degrees of crosslinking, to exploit their possible use as substrates in CSE. The investigated amounts of CA did not cause any cytotoxic effect while improving the mechanical performance of the hydrogels (+11-fold increase in E, compared to control MC). The possibility to obtain cell sheets (CSs) was then demonstrated using murine fibroblast cell line (L929 cells). Cells adhered on crosslinked MC hydrogels’ surface in standard culture conditions and then were harvested at selected time points as single CSs. CS detachment was achieved simply by lowering the external temperature below the Tt of MC. The detached CSs displayed adhesive and proliferative activity when transferred to new plastic culture surfaces, indicating a high potential for regenerative purposes.

Multi-layered in vitro 3D-bone model via combination of osteogenic cell sheets with electrospun membrane interlayer

In this study, it was aimed to present an approach for the development of multi-layered tissue engineering constructs by using cell sheet engineering. Briefly, MC3T3-E1 mouse pre-osteoblast cells were cultured in temperature-responsive plates (Nunc Upcell®) in the presence of osteogenic medium and the resulting cell sheets were laminated with electrospun poly(L-lactic acid) (PLLA) membranes to obtain viable three-dimensional, thick constructs. The constructs prepared without PLLA membranes were used as control. The cell viability and death in the resulting structures were investigated by microscopic and colorimetric methods. The in vitro performance of the structures was discussed comparatively. Alkaline phosphatase (ALP) activity, collagen and sulfated glycosaminoglycan (sGAG) content values were calculated. The presented approach shows potential for engineering applications of complex tissues with at least two or more microenvironments such as osteochondral, corneal or vascular tissues.

Self-assembly of tessellated tissue sheets by growth and collision

Tissues do not exist in isolation; they interact with other tissues within and across organs. While cell-cell interactions have been intensely investigated, less is known about tissue-tissue interactions. Here, we studied collisions between monolayer tissues with different geometries, cell densities, and cell types. First, we determine rules for tissue shape changes during binary collisions and describe complex cell migration at tri-tissue boundaries. Next, we demonstrate that genetically identical tissues displace each other based solely on cell density gradients, and present a physical model of tissue interactions that allows us to estimate the bulk modulus of the tissues from collision dynamics. Finally, we introduce TissEllate, a design tool for self-assembling complex tessellations from arrays of many tissues, and we use cell sheet engineering techniques to transfer these composite tissues like cellular films. Overall, our work provides insight into the mechanics of tissue collisions, harnessing them to engineer tissue composites as designable living materials.

Design of Temperature-Responsive Cell Culture Surfaces for Cell Sheet Engineering

Temperature-responsive cell culture surfaces, which modulate cell attachment/detachment characteristics with temperature, have been used to fabricate cell sheets. Extensive study on fabrication of cell sheet with the temperature-responsive cell culture surface, manipulation, and transplantation of the cell sheet has established the interdisciplinary field of cell sheet engineering, in which engineering, biological, and medical fields closely collaborate. Such collaboration has pioneered cell sheet engineering, making it a promising and attractive technology in tissue engineering and regenerative medicine. This review introduces concepts of cell sheet engineering, followed by designs for the fabrication of various types of temperature-responsive cell culture surfaces and technologies for cell sheet manipulation. The development of various methods for the fabrication of temperature-responsive cell culture surfaces was also summarized. The availability of cell sheet engineering for the treatment and regeneration of damaged human tissue has also been described, providing examples of the clinical application of cell sheet transplantation in humans.

3D Bioprinting Strategies for the Regeneration of Functional Tubular Tissues and Organs

It is difficult to fabricate tubular-shaped tissues and organs (e.g., trachea, blood vessel, and esophagus tissue) with traditional biofabrication techniques (e.g., electrospinning, cell-sheet engineering, and mold-casting) because these have complicated multiple processes. In addition, the tubular-shaped tissues and organs have their own design with target-specific mechanical and biological properties. Therefore, the customized geometrical and physiological environment is required as one of the most critical factors for functional tissue regeneration. 3D bioprinting technology has been receiving attention for the fabrication of patient-tailored and complex-shaped free-form architecture with high reproducibility and versatility. Printable biocomposite inks that can facilitate to build tissue constructs with polymeric frameworks and biochemical microenvironmental cues are also being actively developed for the reconstruction of functional tissue. In this review, we delineated the state-of-the-art of 3D bioprinting techniques specifically for tubular tissue and organ regeneration. In addition, this review described biocomposite inks, such as natural and synthetic polymers. Several described engineering approaches using 3D bioprinting techniques and biocomposite inks may offer beneficial characteristics for the physiological mimicry of human tubular tissues and organs.

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

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

Epidermal Cell Sheet Transplantation on an Anastomotic Site of the Small Intestine in an Experimental Animal Model

Objective: The present study was performed to examine the effects of anastomotic site tissue reconstruction by transplantation of epidermal cell sheets onto the small intestine in an animal model. Cell sheet engineering using cell sheets are used to construct monolayers and bilayers, which are then transplanted into organs. Clinical trials of the application of cell sheets to the cornea, esophagus, lung, and heart muscle are currently underway. Methods: The small intestine in female pig (20 kg) was cut 1.5 cm vertically at 6 points at 10-cm intervals, and Gambee sutures were applied at 5-mm intervals. The suture line was covered by epidermal cell sheets. Resection was performed 1 week after the operation. Results: Cell sheets applied to sutures in the small intestine survived and differentiated 1 week after transplantation. The small intestine showed marked thickening in the region of cell sheet transplantation, and the amount of connective tissue in the transplanted specimens was 2.54 times that in controls. Conclusions: Further studies are necessary to identify the strength of anastomosis and substances that may enhance collagen synthesis and healing at sites of anastomosis.