Injectable Liver:A Novel Approach Using Fibrin Gel as a Matrix for Culture and Intrahepatic Transplantation of Hepatocytes
Helge Bruns, Ulrich Kneser, Stephanie Holzhuter, Beate Roth, Jantjeline Kluth, Peter M.Kaufmann, Dietrich Kluth, and Henning C.Fiegel
TISSUE ENGINEERING Volume 11,Number 11/12,2005 ©Mary Ann Liebert,Inc.
http://www.liebertonline.com/doi/pdf/10.1089/ten.2005.11.1718
Summary:
This paper develops a new realm of liver therapy and transplantation. Currently established liver transplantation including liver, split-liver and living-related liver transplantation depend on donors. This dependence has an implicit limiting factor of organ donors. This paper looks at a cell-transplantation technique. Other liver cell-transplantation techniques include orthotropic cell transplantation and heterotropic cell transplantation. Orthotropic cell transplantation requires the implantation of liver cells to the spleen or portal vein and require a dependence on migration through these vessels to the liver. This corrects metabolic enzyme deficiencies but has high risk of portal vein thrombosis and pulmonary embolism, causing this to be unfavorable. Heterotropic cell transplantation requires an implantation underneath the kidney capsule or in other heterotropic locations. This allows for a transplant as large as the original organ and provides a successful 3-D formation of the cells, however the correct function is difficult to attain due to the lack of accurate liver signals during in-vitro growth. This paper discusses a new form of liver cell transplantation where the cells are directly injected into the liver with a 3-D matrix to allow them to be integrated into the liver chimerically.
Fibrin glue is used as the unique matrix for this experiment, as it has been shown to be successful in other medical needs including reducing blood loss after major surgeries and can be easily used as injection surgery with engineered tissue, however it had not been reviewed for liver cells. Hepatocytes were isolated using a two-step collagenase perfusion method with 80%-95% viability determined with trypan-blue exclusion. Using rats as donors and recipients, the authors injected 1mL of cell-matrix mixture with approximately 2.5e5 cells under the liver capsule of each animal and observed the effects over 0, 2, and 7 days.
The fibrin gel matrix was composed of 20mg/mL human fibrinogen. DNA was quantified using an extinction wavelength of 365nm and an emission wavelength of 460nm. RT-PCR was then performed to determine the RNA count in the samples to then determine the concentration of hepatocytes based on a standard curve of known amounts of RNA per hepatocytes under the same conditions. Two mice died in surgery and two more were explanted the same day as the surgery to determine the complications and effects on the liver. The fibrin matrix stopped the liver capsule from bleeding by clotting upon injection. Two days after the injection, the fibrin matrix was still visible and the implanted cells were still easily discernable from the native hepatocytes but some of the donor cells were detected as having been accepted into the liver with CK-18 staining. Seven days post-surgery, the fibrin matrix was degraded significantly and donor cells were detected as being integrated into the parenchyma of the liver, while some remained easily detectable outside the liver.
Fibrin gel was proven to be a successful matrix to grow hepatocytes in for transplantation based on in vitro testing done, though some limits were recognized as the cells began to degrade after three days. When the fibrin gel was injected it proved to be without serious side effects and formed a three dimensional scaffold for the transplanted cells to grow in. The authors suggest that this fibrin matrix presents a promising future for injectable liver cells to repair damage.
Relevance:
This area of study presents excellent resources where there is low donor availability. This involves cell tissue in a three dimensional matrix which allows for only a few donor cells to regenerate and form in the supporting matrix. This matrix also provides the scaffold for the injection which increases the ease of use. This study uses an already developed method for cartilage and other implants and reviews its use with another organ. Revealing the ability to review such methods for different uses readies new fields in a way others are already fully prepared. The authors connect the cell-culture and protein and RNA quantification we have been learning in this class to real-world problems.
8 comments:
Its a scientific breakthrough to see scientists developing liver implantation methods. This discovery has a great potential to alleviate many patients suffering from liver diseases. It also will expedite the availability of liver transplant as supposed to waiting for a donor. This will definitely help many patients who are currently and in the future are in need of a liver transplant.
As Tizita stated, this new implantable liver therapy seems very promising and will significantly broaden the therapeutic window for treating patients with liver failure or liver damage. However, since two mice in this study died during the implantation surgery, is it possible to conclude that this new liver transplantation technique poses greater risk than existing transplantation therapies or were the deaths of these mice due to external confounding factors? Additionally, since the fibrin gel began to degrade after three days in vitro, might this treatment be beneficial for only mild cases of liver damage?
Tizita: This is a very promising breakthrough, however there will be much more research to verify the ability of medical professionals to use this treatment in humans. With this treatment, the donor problem and waiting list would be resolved as you mentioned, and this paper suggests the treatment would require a much shorter recovery time and fewer follow-up procedures and check-ups once the injectable liver treatment is fully developed
Rina: The authors suggest that the deaths of the two mice were not related to the injectable liver directly. One mouse did not recover from anesthesia, and was autopsied and the death was found to have no connection to the liver. The second mouse developed severe bleeding and was killed and removed from the study, however the authors do not suggest the bleeding was in response to the liver directly, but rather the surgical procedure, any surgery is risky.
The cells did begin to degrade after three days, so this may suggest that this procedure can only be used for mild liver problems, however this is one of the first times this procedure was performed, thus there are many things that will be changed throughout the course of the development. One action the researchers may take is determining a way to make the fibrin gel more porous to allow liver signals and nutrients to reach the cells more easily, or perhaps including a non-toxic nutrient in the gel for the cells to survive on before being accepted into the liver.
The fibrin gel is designed to degrade to allow the liver to use the new cells to grow, so the loss of the gel is caused by the growth of the cells and the ability of the body to absorb and remove the fibrin without an immune response, this allows the patient to grow a new, functional liver and thus not need a transplant.
Is it an innate property of fibrin to become a gel when injected into the liver? Is it the temperature change (room temp to body temp) that causes gelation?
Further research into this technique is needed, especially how to reduce the risk in the surgery. I think the "life" of the fibrin matrix can be lengthened by adding other biodegradable or resorbable materials into original cell mixture. Did the authors discuss possibly adding growth factors or signaling molecules to allow better differentiation or integration into the liver?
Hey Sydney,
This new implantable liver therapy seems like it could help a lot of people. In regards to commments made about the fibrin gel matrix, I thought it was interesting that in their future work, the scientists considered making the gel more porous to increase surface area and thus increase the flux of nutrients and signals to the cells. I was wondering, given that the gel degrades after 3 days, do you think that a porous gel would be able to perform the same way as the fibrin gel already used considering that increased porosity might change the material's mechanical properties?
-Robert
Lavanya: The gel is injected in liquid form while it is solidifying, thus allowing the 3D shape to form a scaffold for the cell growth. The authors discuss the difficulty in creating conditions close enough to in vivo liver to fully prepare the cells outside of the body, but do not further discuss the topic of growth factors or other signaling mechanisms to aid in the differentiation of the cells.
RobbedSmiler and Lavanya: The degradation of the fibrin gel is a desired trait, although a combination of your posts can create a more suitable gel, if different biodegradable materials are introduced into a more porous system that is then used to inject the liver. This would allow the cells to grow in the 3D structure while acquiring sufficient nutrients and signals from the liver for differentiation. This may change the mechanical properties, but the 3D biodegradable scaffold was as much detail as the authors supplied so this may be a good solution to the problems of cell degradation due to lack of nutrients and gel degradation over time.
Hey Sydney, I had a question about the fibrin glue used in the experiment. Where did the researchers obtain the glue? They were working with mice... does it matter what species the fibrin glue came from? What exactly is fibrin "glue" and how is it different than just a culture of fibrin?
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