An Approach for Formation of Vascularized Liver Tissue by Endothelial Cell–Covered Hepatocyte Spheroid Integration
Inamori, M., Mizumoto, H., & Kajiwara, T. (2009). An Approach for Formation of Vascularized Liver Tissue by Endothelial Cell–Covered Hepatocyte Spheroid Integration. Tissue Engineering, 15(00), 1-10. doi:10.1089=ten.tea.2008.0403
Summary:
A major hurdle in cell transplantation is in vitro vascularization of tissues. Vascularization is necessary for the successful culture and proper function of potential replacement tissues. Certain tissues, such as hepatocytes, require high cell densities for correct function post-transplantation. The only way to achieve such densities without starving cells is to vascularize the hepatocyte mass. Without vascularization, hepatocyte spheroids (which have a cell density close to native liver tissue) are limited to diameters of about 100 microns.
In this paper, rat hepatocyte were isolated from a 7-8 week old male Wistar rat. Viable cells were incubated on a rotary shaker, causing them to grow into spheroids. Spheroids of 100-150 microns were separated out and then coated with collagen using a Collagen Gel Culturing Kit. Human umbilical vein endothelial cells (HUVECs) that had been purchased and cultured separately were then cocultured with collagen-coated spheroids so that HUVECs could cover the spheroids. Lastly, the spheroids were inoculated into hollow fibers used for plasma separation. Filled fibers were incubated and their development monitored. Liver specific function of these fibers were also evaluated.
Without a collagen coating, HUVECs would normally invade the hepatocyte spheroids, inhibiting the vascularization of the tissue once packed into hollow fibers. An ECM, in this case collagen, plays a crucial role in the ability of packed spheroids to vascularize. The ECM allows HUVECs to remain on the surface of the spheroids and limits their migration into it during the initial days of growth. This HUVEC coating formed tubular structures where adjacent spheroids touched inside the packed hollow fiber. Over time, further vascularization occured as HUVECs eventually migrated into the spheroids, in a process similar to that of native liver tissue development.
Significance:
The use of HUVEC-coated spheroids is a promising strategy to create vascularized tissues. The critical component is to coat spheroids with an ECM component such as collagen to inhibit the migration of endothelial cells into the spheroid tissue unit.
5 comments:
The collagen ECM is used to prohibit the HUVECs from migrating into the spheriods which would inhibit vasculariztion. But then you mention vascualrization occured overtime as the HUVECs migrated into the spheroids. When, how, and why does the change occur? Is it only in the beginning of growth that the HUVECs need to be blocked from migrating into the spheroids? If so why?
How do you know that the it is the collagen ECM that prevents vascularization when in the end, HUVECs are still able to migrate into the spheroids that have collagen ECM? Also, just to clarify, does this mean that the collagen ECM isn't necessarily the agent that causes vascularization, but more the agent that temporarily keeps HUVECs from entering the spheroids so that they can then vascularize?
How does collagens prevent the HUVEC to grow into the spheroids and why does this occur only at the early stage? The cells will be able to survive without HUVEC penetration due to their small sizes. Can collagen prevent HUVEC from reaching in spheroids completely and how would it affect this experiment? Also, why are tubular structure required and how would the result be without the extra tubular structure?
Interesting stuff, especially because vascularization appears to be a major hurdle for the researchers in many of these posts. Did the paper mention if these or similar techniques might be applied to other types of tissue, or if this is a very hepatocyte-specific process?
Like Pete said, this is quite interesting. How come ECM or something is needed to prevent HUVECs to from migrating in around spheroids earlier? Is it trying to give them room for vascularization? Were there any important growth factors involved (e.g. VEGF)? Were there any particularly interesting future works that the authors are looking to do in the near future?
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