Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets

Kazyo Ohashi, Takashi Yokoyama, Masayuki Yamato, Hiroyouki Kuge, Hiromichi Kanehiro, Masahiro Tsutsumi, Toshihiro Amanuma, Hiroo Iwata, Joseph Yang, Teruo Okano, and Yoshiyuki Nakajima

link: http://www.nature.com/nm/journal/v13/n7/pdf/nm1576.pdf

There are currently two major methods for implanting hepatic tissue in patients who do not require whole liver organ transplants. In one treatment, hepatocytes, suspended in extracellular matrix components, are injected into the body via the portal vein. In the second method, hepatocytes are implanted with a biodegradable scaffold that serves as a structural foundation for cell attachment. Unfortunately, both treatments have many risks and disadvantages. For example, the first treatment may result in embolization – the injected cells obstruct portal circulation. The second method may lead to fibrosis, inflammatory reactions, or immunogenic reactions, as a consequence of introducing a foreign object into the body, the. Furthermore, hepatic cell viability is short (on the scale of days) in both treatments because the body does not well vascularize the engineered tissue. As a result, multiple transplantations are required.

To address these issues, the investigators developed protocols to produce long living and functional 2D and 3D hepatic tissues, to optimize vascularization of the engineered tissues, and to implant them without biodegradable scaffolds. To create the tissues, the investigators isolated primary hepatocytes, expressing human alpha 1-antitrypsin (hA1AT), from transgenic mice. They grew the cells on culture dishes coated with temperature-sensitive polymer poly(N-isopropylacrylamide) or (PIPAAm). At 37°C, the cells grew and remained attached to the slightly hydrophobic polymer. After the cells reached confluency, the investigators lowered the temperature. Below 32°C, the polymer became hydrated and caused the cells to detach uniformly as a 2D sheet. To make the 3D tissues, 2D sheets were layered on top of one other. In the control, the scientists used the proteolytic enzyme collagenase to separate the tissue from the polymer. The scientists found that the tissue prepared by varying temperature had better structural and functional integrity than the control. The engineered tissues produced more hA1AT, had better drug metabolism, and retained intercellular connectivity or extracellular matrix components. In the control, the enzymes destroyed fibronectin which was important for cell adhesion. However, in the cells detached by lowering temperature, fibronectin was still present.

Vascularization of transplanted tissue is essential for prolonged tissue viability. In the study, the investigators inserted the engineered hepatocyte tissue into the subcutaneous area of mice. In order to vascularize the region, the scientists implanted a device that secreted fibroblast growth factor. After a vascular network had formed, the scientists removed the device and transplanted the tissue. In the mouse, these cells had longer viability than those implanted using conventional methods. The engineered cells also maintained hepatocyte-specific functions. For example, using BrdU and immunofluorescence staining, the scientists found that the cells were able to profilerate in the mouse. They were also able to detect plasma exchange between the engineered tissue and the body.

The advantages of the new protocol are that it is simple, minimally invasive, and free of materials that can elicit a detrimental immune response. There are, however, some disadvantages to the technique. These tissues were uniform and did not have structural polarity. The formation of sinusoidal and biliary surfaces is necessary to create tissue that can replace whole organs. Thus, this technique can not yet be used to replace whole organ transplantation. Nevertheless, I chose this paper because the technology has potential to benefit patients with liver diseases that do not require an organ transplants, including those with hemophilia and congenital metabolic liver diseases, such glycogen storage disease and congenital deficiency of coagulation factor VII. Since the cells must be incubated on the plates for a few days to reach confluency, scientists can genetically modify the cells to optimize treatment for particular liver diseases. In the future, this technology can be extended to develop larger sections of liver that would lower the demand for organ transplantation.