The overall goal of this procedure is to use cell sheets to construct a tissue engineered patch. This is accomplished by first treating the cell culture surface with the thermo sensitive polymer poly propyl acrylamide, also referred to as p nipam. The second step is to culture the cells of interest in a high density on the polymer coated surface to create cell sheets.
Next, the sheets are lifted by lowering the plate temperature, which allows the cells to detach from the plate as a sheet of cells. The final step is to move the intact cell sheets without tearing them onto a construct for implantation. Ultimately, simple light microscopy can be used to show that the cell sheet has been created and maintained during its manipulation while creating an implantable tissue engineered material.
The main advantage of this technique over existing methods, which will embed cells into matrix like hydrogels, is that in this technique, the cells lateral borders can be generated and maintained as as a functional tissue. These methods can also enable further studies in the field of tissue engineering. For example, how well do the cells survive after transplantation when they're in this organized state?
Demonstrating this procedure will be Dr.William Turner, a postdoctoral fellow in my laboratory and Ms.Snapshot sand do. To begin this procedure dissolve 2.6 grams of P nipam in two milliliters of a 60%toluene, 40%hexane solution. Next, heat the mixture to 60 degrees Celsius for 10 minutes and stir until the P nipam is dissolved.
Then cut the filter paper into a 60 millimeter diameter circle and place it in a buchner funnel. Filter the solution through the Buchner funnel into a pre weighed glass beaker. Keep the beaker and contents in a bell vacuum overnight.
Afterward, weigh the beaker with p nipam. Subsequently add isopropyl alcohol to P nipam to create a 50 50 weight weight solution. Next place two milliliters of the solution on the surface of the tissue culture plate and coat it for five minutes under UV light.
Then wash the plate with two milliliters of warm PBS twice before using it for cell culture. In this procedure, first, isolate the rat aortic smooth muscle cells by washing the cells with two milliliters of warm PBS. Next, add three milliliters of trypsin or another cleaving dissociating solution to the cells and incubate for five minutes.
Then add three milliliters of the culture media or PBS containing 10%FBS to the cells. After that, collect the cells in a conical tube and count them. Subsequently, spin the cells at 1000 RPM for five minutes.
Aspirate the supernatant and resuspend the cells in the growth media. Next place the media containing the cells on a 35 millimeter thermo sensitive plate at a concentration that will achieve 100%Confluence, then incubate at 37 degrees Celsius overnight to maintain the cell adhesion to the plate. Now collect the endothelial cells and disperse them into a single cell solution.
Using one x trypsin, acuate or cell dissociation buffer could also be used for single cell dispersion. Next, deactivate the trypsin enzyme with an equal amount of soybean trypsin inhibitor or 10%FBS in PBS. Then collect the cells in a 15 milliliter conical tube.
Count the cells with a hemo cytometer and calculate the volume needed for the patch dimensions for a four millimeter patch, 2 million endothelial cells are used after that, extract 2 million cells and place them in a new 15 milliliter conical tube. Then centrifuge the cells for five minutes, aspirate the supernatant, leaving the cells as a pellet in the conical tube. Now prepare the high aurin hydrogel in accordance with the company protocol.
Mix high aurin and liquid gelatin in a one-to-one ratio. Subsequently transfer 80%of the total final volume into the conical tube containing the pellet, resuspend the endothelial cells in the high aurin gelatin mixture. Next, transfer the suspended cells into the fibrous matrix.
Add 20%of the total final volume of the crosslinker and incubated at 37 degrees Celsius for one hour. In this step, remove the 35 millimeter P nypa treated plate containing the cells from the incubator and place it in a cell culture hood. At room temperature, quickly aspirate the media from the cells.
Then add two milliliters of 37 degrees Celsius, 6%normal gelatin to the plate while the gelatin is still warm, place the metal lattice into the gelatin, submerging it below the surface of the normal gelatin. Afterward, place the entire plate on ice for five to seven minutes, allowing the gelatin to harden. After seven minutes, use a spatula to carefully separate the gelatin edges from the side of the plate.
Next, lift the metal lattice from the plate with forceps. Transfer the cell sheet to the dish and place it on top of the fibrous matrix hydrogel combination. Add two milliliters of 37 degrees Celsius media to the tissue culture plate and incubate overnight to allow the sheet of cells to adhere to the hydrogel surface.
In the meantime, remove the metal lattice after the solution has warmed up. Here, cells were cultured on a Pippa coated surface and then moved as a sheet to the surface of the fibrous matrix. Early trials that were not transferred with the gelatin metal lattice resulted in small tattered patches.
This image represents an early patch design composite picture combining the mito tracker, red dyed rat aortic smooth muscle cells, calcium am green fluorescent human umbilical vein endothelial cells, and the transmitted light image. And this is the composite of cell sheet combined with the stronger base fibrous matrix hydrogel combination. This composite image was taken from the bottom of the patch through a stronger base fibrous matrix, and this composite picture of the cell sheets was taken over the base fibrous matrix.
Hydrogel combination cells in the sheet cover the base fibrous matrix hydrogel combination, which show increased fluorescence at the edges due to bunching of the cells. This is a transmission image of the construct and the autofluorescent natural fibrous base matrix in DPI is shown here. Following this procedure, other tissue engineering experiments can answer other questions such as how to cell alignment and communication affect transplantation success.
This technique allows researchers in tissue engineering to explore complex ways of transplanting multiple cell types. Don't forget that working with solvents can be extremely hazardous. Precautions, such as wearing proper masks and chemical hoods should always be taken while performing this procedure.
