Reconstruction of Functional Tissues with Cell Sheet Engineering
Joseph Yang, Masayuki Yamato, Tatsuya Shimizu, Hidekazu Sekine, Kazuo Ohashi, Masato Kanzaki, Takeshi Ohki, Kohji Nishida and Teruo Okano
The approach of seeding cells into biodegradable scaffolds has become a hallmark of modern tissue engineering. Rather than using biomaterials as scaffolding materials for tissue reconstruction, the investigators have created an alternative approach using polymer-coated culture surfaces to facilitate the non-invasive harvest of cultured cells as intact tissue sheets. The culture surface is grafted with poly(N-isoproplyacrylamide) (PIPAAm), a temperature responsive polymer that allows controlled attachment and detachment of living cells via temperature changes.
With this new method, cell types that secrete significant amounts of extracellular matrix (ECM) proteins, are cultured for prolonged periods to create sheet-like structures for tissue reconstruction. By covalently immobilizing PIPAAm onto conventional culture surfaces, changes in the surface properties can be controlled by varying the incubation temperature. At temperatures above 32 °C (lower critical solution temperature), culture surfaces are slightly hydrophobic and enables various cell types to attach, spread and proliferate as if on normal tissue culture polystyrene. When the temperature is below 32 °C, the PIPAAm-grafted surfaces spontaneously become hydrophilic. A hydration layer forms between the culture surface and the attached cells, allowing the harvest of confluent cells as intact sheets. Since the grafted surfaces facilitate spontaneous cell detachment, scientists can avoid using proteolytic enzymes such as typsin and collagenase. With non-invasive cell harvest, cell-to-cell junction and ECM proteins can be maintained. Cell sheet engineering had been used to create functional tissue sheets to treat a wide range of diseases including corneal dysfunction, esophageal cancer, tracheal resection, and cardiac failure. The researchers have also developed methods to create thick vascular tissues as well as, organ-like systems for the heart and liver by generating 3D tissues of cultured cells and deposited ECM proteins with the cell sheets. The article goes in more detail about each of these applications.
Using cell sheet engineering, numerous cell types have shown the maintenance of differentiated functions after low-temperature cell sheet harvest due to the preservation of cell surface proteins, such as growth factor receptors, ion channels, and cell-to-cell junction proteins. In addition, cell sheets can be easily transferred and attached to other surfaces (culture dishes, host tissues). A disadvantage is that engineered tissues created using this cell sheet method contain relatively little ECM. Thus, the method may not be ideal for the creation of cell-sparse tissues like bone and cartilage. Regardless, cell sheet engineering seems to be able to provide new possibilities in regenerative medicine and tissue engineering.
I chose this article because cell sheet engineering seemed interesting and it could be useful for future projects. This method is like an extension of the basic cell culture techniques taught in class - some day, I would like to try it out and see the results. Cell sheet engineering is a method that could be implemented with regeneration of specific tissues without the risks of using traditional scaffold-based methods. In addition, cell sheet engineering is novel alternative for research projects that require the re-creation of functional tissue structures.
6 comments:
Are any growth factors added to increase the release of ECM material to form the sheets and would it help build stronger sheets if added?
This layer-by-layer approach to bioengineering organs sounds similar to the goal of bioprinting, which is designed to print out thin layers of cells to create 3D constructs. For organs with complex internal structures (such as the kidney), wouldn't it be difficult to pattern the cell types in individual sheets and align them with each other layer? This especially sounds like a big issue with sheet tissue engineering, where the researchers make little/no mention of using computers to lay out their designs.
Nice posting. I like the biodegradable idea. Also interesting the temperature-controled surfaces. However, I read that some cells attach better at temperatures below 32ºC. Do they culture the cells all on this conditions all the time? Would this matter when the tissue is transplanted into a body (e.g. animal)? Isn't body temperature ~37ºC.
It is a really interesting idea to be able to form layers of cells and detach them by altering temperature without the use of proteolytic enzymes. You mentioned that the researchers have developed methods to create thick vascular tissues. How did they include vascularization within the 3D constructs of tissue layers? Is it similar to the method used in Elise's paper in which vascularization was first induced in the tissue before the transplant of the 3D construct of tissue layers? Or is the vascularization within the 3D construct of tissue layers itself? The second method would probably be more useful since the first method would limit the thickness of the 3D construct.
to Albert: It does not seem like the paper discussed about the addition of growth factors, but it is something that they could have considered. I would think that more ECM material released by the growth factors would build stronger sheets - ECM material is what makes these sheets easily transferred according to the article, so that could imply that the sheets are strong due to the presence of ECM proteins.
to Brian: The article did not go in-depth about the entire process, it mainly focused on applications of cell-sheet engineering. The researchers could have possibly used computers, but it's not mentioned. However, you do bring up a good point about the alignment issue. This is an issue in cell-sheet engineering that could be investigated and resolved in the future.
to Vimalier: Not sure again whether or not they cultured cells under the specific temperature conditions all the time. Unfortunately, the article is pretty vague about that, but the researchers were indicating a general trend that they observed in experiments. As for whether would temperature be an issue when the tissue is transplanted in the body: so far, it seems like temperature has not had a profound effect, according to some of the applications that were mentioned in the article. But possibly, further investigation regarding temperature would be beneficial.
to Alice: It seems like the method the researchers used regarding vascularization is the one mentioned in Elise's article. I agree with you that the second method would be more useful, but cell sheet engineering is still a relatively novel idea. But the second method you proposed could be tested and possibly implemented once cell-sheet engineering gets even more refined.
I have a similar comment to vimalier's. Although the temperature-controlled substrate idea would be useful in vitro, what would the impact be in the body where the temperature is relatively stable? thank you for your reply that temperature so far has not has a significant effect, it would just be something to consider as another problem when tissue is transfer in vivo.
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