One of the greatest challenges facing tissue engineers is the production of a 3D tissue culture for physiological testing. Two dimensional cell cultures such as those traditionally grown on culture dishes simply do not demonstrate similar physiological function compared to the 3D tissue they are meant to represent, which limits the ability of researchers to test new drugs or therapies. Also, the removal of such cells from a tissue dish can only be accomplished through cellular trypsinization, which separates the cells not only from the growth surface but also from each other. This compromises the integrity of cell-cell structure, highlighting the need for a 3D tissue culture.
Methods already in place to form said cultures generally rely on a re-creation of the extracellular matrix found in the body using natural or synthetic polymer materials such as collagen gel or polyvinyl alcohol (PVA). Said scaffolds work by giving cells a matrix to grow upon, which with the addition of proliferative growth factors can act as a cellular skeleton for 3D tissue fabrication. The main limitation of this approach is that cells which have attached and multiplied on these scaffolds cannot be removed and the scaffold becomes part of the tissue culture, potentially detracting from implantation or experimental viability.
In 2003, researchers at Tokyo Women’s Medical University began developing a new cellular layering technique, allowing entire sheets of cells to be grown, removed from the growth surface and layered together, creating an effective 3D tissue. The secret: a temperature sensitive polymer called poly(N-isopropylacrylamide) (PIPAAm). PIPAAm changes its physical properties in response to temperature alteration, the most important change being its attraction to water. At higher temperatures such as 37 degrees Celsius (the temperature of the human body, and an effective temperature for cellular growth), PIPAAm is hydrophobic, meaning it repels water, allowing cells to attach to the surface via intracellular proteins. As the temperature is lowered PIPAAm becomes more hydrophilic, meaning it attracts water. This causes the cells to be pushed away from the surface by the attachment of water, while maintaining intercellular bonds. The result is a complete sheet of cells held together by natural cell-produced extra cellular matrix. When these sheets are layered together they adhere to form a functional 3D tissue.
The implications of this advancement are broad, and have already been put to test in clinical trials.
- A 3D Corneal Limbal Stem Cell culture was grown, implanted into a rat cornea, and shown to regenerate limbal tissue (the clear, fibrous tissue of the outer eye). This was made possible by the absence of sutures or carriers.
- A 3D Cardiac Myocyte (muscle) Cell culture was fabricated into a cardiac patch, which when placed on a damaged rat heart, showed regenerative properties and enhanced heart function.
- Aortic Cells were cultured, layered, and wrapped around a polymer tube to form an aortic graft, which showed functionality in animal studies.
- This technology has also shown potential in bladder reconstruction through the culture of Urothelial Cells.
Although this method shows great promise for the field of tissue engineering it is not without limitation, the greatest of which is vascularization (the production or presence of blood vessels). Cells need the ability to exchange gases, proteins, and other cytoplasmic elements to maintain normal function and survival. In cell sheet layered engineering, in the absence of vascularization the maximum number of layers without cell death is three. This is relatively thin and may not possess the mechanical strength required of its purpose, such as a large animal cardiac patch. In the future, it may be possible to induce vascularization before implantation, thereby allowing the number of layers to be greatly increased. The current solution to this limitation is a procedure called polysurgery, in which three layer patches are implanted periodically, giving time for blood vessels to form before placing the next layer.
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