The overall goal of this method to make customized hydrogels is to study cells and culture conditions that replicate the complexity of the lung extracellular matrix. This method can help answer key questions in the pulmenary field such as how do lung cells interact with ECM under healthy and pathalogical conditions. The main advantage of this technique is that the lung ECM hydrogels are tailorable to your application in 2D or 3D culture.

Generally, individuals new to this method will struggle because of the complexity of the decellularization techniques, sometimes requiring multiple people and attention to detail to optimize the amount of viable tissue. For this protocol, have a porcine lung ready for decellularization. Details on its preparation can be found in the text protocol.

Once the lung is prepared, use a hand pump that is cannulated to fit the pulmonary artery. First, perfuse around one liter of water into the vasculature, and then 1.5 liters into the trachea. Do this three times, increasing the volume of water perfused as cellular debris is removed.

Next, perfuse the tissue with Triton X-100, also using the hand pump. Then, submerge the tissue in a bath of Triton X-100 solution at four degrees Celsius for 24 hours. After 24 hours, rinse the vasculature and trachea three times with distilled water.

After the rinses, use the hand pump to perfuse the tissue with deoxycholate. And then submerge tissue into deoxycholate for 24 hours at four degrees Celsius. The next day, rinse the tissue three times with distilled water, as before.

After the rinses, use the hand pump to perfuse the tissue with filtered sodium chloride. Then submerge the tissue in filtered sodium chloride for an hour at four degrees Celsius. After the bath, perfuse the vasculature and trachea three times with distilled water to rinse.

After the rinses, perfuse the tissue with DNase solution, and then submerge it in DNase solution for one hour at four degrees Celsius. After an hour, perfuse the vasculature and trachea five times using PBS, not water. Now, only using opaque white tissue, dissect away all the tubules that are two millimeters in diameter or larger.

They are primarily found around the hilum and medial portions of the lungs. Leave the respiratory zones intact, which are mostly in the periphery. After removing the tubules, dissect the tissue into one inch sections or smaller.

The orientation of these blocks is not important. After draining the tissue blocks, transfer them to a 50 milliliter conical tube and freeze them at 80 degrees Celsius. A few blocks can be set aside for histology to confirm the removal of the cells and debris.

To process the lung tissue into decellularized powder, replace the cover on the frozen tissue tube with a filter paper, and secure this with an elastic band. Then, lyophilize the tissue until all the excess liquid is gone using a freeze dryer according to manufacture's directions. Then, store the tissue at 80 degrees Celsius until it can be milled.

To ready the mill, first load it with liquid nitrogen, leaving room for the mill tube. Next, from the mill tube, remove the magnetic mill bar and cover the bottom of the tube with tissue. Then, replace the mill bar and loosely pack in more tissue to fill the tube.

Then, close the mill tube, place it in the freezer/mill, and fill the mill with liquid nitrogen up to its maximum capacity. Now, run the mill for approximately five minutes with an impaction rate of about 600 per minute. Then, store the decellularized powder at 80 degrees Celsius, until needed.

To make the pre-gel material, add decellularized tissue powder and pepsin to a conical tube. Then add 0.01 molar hydrochloric acid to the tube, for a final concentration of 1%decellularized tissue powder, and 0.01%pepsin. Keep this solution under constant agitation at room temperature for 48 hours to digest the tissues.

After 48 hours, put the solution on ice for five minutes. Now adjust the solution's pH to 7.4 using cold 0.1 molar sodium hydroxide, and then bring the solution's PBS concentration to 1X by adding 10X PBS. We have some experience with altering times and the pH of the pre-gel.

There is some flexibility in the range that can be used, but the properties can change dramatically if the procedure is altered in minor ways. The solution is not considered pre-gel and it can be used to coat non-treated plates to form hydrogels to form cells on, to form hydrogels to form cells in, or it can be further modified before forming hydrogels to increase the hydrogels'rigidity. To culture cells on two-dimensional micro-porous gel coating, add 20 microliters of pre-gel solution to wells of a 96-well non-treated tissue culture plate.

Then, refrigerate the plate overnight at four degrees Celsius so proteins are absorbed onto the plate. The next day, aspirate the pre-gel solution and rinse the wells with PBS. Then, add 3, 200 cells to each well and top up the media volume in each well to 100 microliters.

The plates can then be incubated. For a three-dimensional cell culture with cells inside the micro-porous gel, resuspend the cells of interest at one million per milliliter in pre-gel solution. Then, quickly dispense 16 microliters of the cell suspension into each well of a 96-well plate to form a 3D cell culture that is about 500 microns thick.

The gel will form in 30 minutes once incubated at 37 degrees Celsius. After the gels form, add media to the wells up to the 100 microliter marks. For a three-dimensional cell culture with cells on top of the micro-porous gel, add the 100 microliters of the pre-gel solution to the wells of an untreated 96-well plate.

Then, incubate the plate at 37 degrees Celsius for 30 minutes for gelation. Once the gels form, add 3, 200 cells in 100 microliter media on top of the hydrogels and proceed to incubate the plate. Hydrogels were produced from normal pig, rat, and mouse lungs using the described method.

Rheological testing confirmed that over 90%of the gelling occurs in the first minutes after reaching 37 degrees Celsius. The formed hydrogels were stable for long periods in PBS, but cell attachment and proliferation may change with time. The pre-gel solution was tested as a coding to improve cell attachment.

Human umbilical vein endothelial cell attachment and proliferation was measured using an MTTSA. Improvements were observed by coating the wells with ECM. In making 3D gels, adding Genipin increased the hydrogel crosslinking.

Using frequency sweeps on a rheometer, hydrogel stiffness was directly correlated to the Genipin concentration, with the median and most relevant concentration being 0.01%Testing the more rigid gels, cell proliferation was measured to be higher by an MTTSA on the gels with increased crosslinking. Once mastered, decellularization can be performed in eight hours over four days. The pre-gel solution can be made in one hour over two days, and the gel formation can be accomplished in two hours over two days.

Following this procedure, other methods like AFM, western blots, staining, and immunohistochemistry can be performed in order to answer additional questions, like elastic modulus, protein composition, and cellular response to the material.