The overall goal for this procedure is to prepare 3D fibrin scaffolds for culturing and differentiating pluripotent stem cells, including embryonic and induced pluripotent stem cells. This is accomplished by first making fibrinogen solutions and dialyzing them overnight to remove molecules that inhibit polymerization. The second step of the procedure is to polymerize a bottom base layer of fibrin in each well of a 24 well tissue culture plate.
The third step of the procedure is to seed. Single embryo body is derived from pluripotent stem cells onto the base layer of scaffold, followed by addition of second layer of fibrin to complete encapsulation. The final step of the procedure is to add the appropriate cell culture media to the scaffolds.
Depending on the application, ultimately results can be obtained that show embryo bodies derived from pluripotent stem cells can be successfully cultured inside of 3D fibrin, biomaterial based scaffolds. The main advantage of this technique over traditional 2D cell culture methods is that you can observe and manipulate stem cell behavior in three dimensions. This method can help answer key questions about stem cell biology by screening factors such as chemical compounds and growth factors that influence stem cell behavior In a 3D microenvironment, Fibrinogen is a blood derived protein and thus appropriate safety training must be completed before handling.
To begin, remove lyophilized fibrinogen from the refrigerator and allow it to sit for 20 minutes to come to room temperature, add three milliliters of tris buffered saline to each 35 millimeter Petri dish. Weigh out approximately 100 to 130 milligrams of fibrinogen and sprinkle it onto the surface of the TBS in each dish. Wait five minutes for the fibrinogen to begin to go into solution.
Then cover the Petri dish with a lid. Incubate the Petri dishes at 37 degrees Celsius for two hours to allow the fibrinogen to fully go into solution. Next wet dialysis tubing with TBS fold the bottom end of the tubing and clamp the end shut with dialysis clamps, pipette the fibrinogen solution from the dishes into the dialysis tubing.
Then fold over the top end and clamp shut with a dialysis clamp. Place the dialysis tubing containing fibrinogen solution into a four liter container of TBS. Mix the solution.
Using a stir plate set to low speed dialyze the solution overnight on the plate for at least 12 hours. The T Bs solution does not need to be changed over this time under a sterile tissue culture hood. Remove the fibrinogen solution from the dialysis tubing and place it into a conical tube.
Filter the fibrinogen solution with a five micron syringe filter to remove large impurities in the solution. Next, use a point 22 micron filter to filter, sterilize the fibrinogen solution into a 50 milliliter conical tube. After determining the total volume measure the absorbance of the fibrinogen solution at 280 nanometers, a dilution factor of 50 is often necessary to obtain an absorbance under one.
Calculate the concentration of protein present in the fibrinogen solution using the following equation where the concentration of fibrinogen in milligrams per milliliter equals a two 80 times dilution factor divided by 1.55, where 1.55 is the extinction coefficient at a two 80 for human fibrinogen. Next, calculate the amount of TBS needed to dilute the concentration of the solution to 11.1 milligrams per milliliter of fibrinogen based on the initial volume. The final concentration of protein in the fibrin scaffolds will be 10 milligrams per milliliter.
After polymerization, add 15 microliters of a 40 units per milliliter thrombin solution to the right side of each well in the first row of the 24 well plate, then add 15 microliters of a 50 millimolar calcium chloride solution to the left side of each. Well finally add 270 microliters of the fibrinogen solution to the plate. Shake the plate from side to side and back to front to mix the solutions.
Let the row containing the mixtures polymerize for five minutes At room temperature, the scaffolds become more opaque as they polymerize continue to make rows of polymerized fibrinogen one at a time until the desired number of fibrin scaffolds is obtained. After the last five minute incubation, incubate the plates at 37 degrees Celsius for one hour to completely polymerize the scaffolds. When the one hour incubation is complete.
Seed the scaffold by using a 20 microliter pipette man to select an individual embryo body and place it in the center of the fibrin scaffold. Check with a microscope that each well contains a single embryo body. Add five microliters of thrombin solution and five microliters of 50 millimolar calcium chloride solution on top of each embryo body, followed by 90 microliters of fibrinogen solution to each well, the solution should form a bubble encapsulating the embryo body.
Incubate the plate for an additional hour at 37 degrees Celsius to ensure polymerization after incubation, add one milliliter of the appropriate cell culture medium to each well and place the plate back into the 37 Celsius incubator. Here, representative images of mouse induced pluripotent stem cells cultured on mouse embryonic fibroblast feeder layers are shown. Using an eight day retinoic acid treatment protocol, the cells are induced to form embryo bodies, aggregates of cells containing neural progenitors as shown here.
This figure shows the appearance of an IPS derived embryo body after three days of culture inside of 3D fibrin scaffolds. Similar results have been obtained previously using mouse embryonic stem cells. After watching this video, you should have a good understanding of how to seed embryo bodies into 3D fibrin scaffolds using this two day process.
Don't forget that working with fibrinogen can be extremely hazardous and proper precautions, including wearing personal protective gear and proper waste disposal should be taken while performing this procedure.
