This protocol is the autologous system for human fibroblast culture with the patient's own platelet-rich plasma and a standardized preparation for five minutes. PRP promotes a massive cell expansion of fibroblast in culture. I would recommend you to use this method using PRP as a cell supplement when you need massive creek and safe cell expansion for autologous cell therapies.
When you attempt this protocol, I would recommend you to check the PRP integrity. Your plasma has to be very clean with absence of red blood cells. When you transfer it, you have to do it very carefully.
And concerning the digestion of your tissue, it has to be very clean before proceeding. To begin, carefully turn the blood-filled platelet-rich plasma, or PRP, tubes upside down three times to mix the blood with anticoagulant. Centrifuge the blood samples at 1500g for five minutes in a fixed 45-degree-angled rotor and balance the tubes.
After centrifugation, the fractionated blood has red and white blood cells trapped under the separating gel and platelets on the surface of the gel. Gently invert the PRP tube 20 times to resuspend the platelet deposit in the plasma supernatant. Ensure that the platelets are fully detached from the gel and the plasma should change from clear and transparent to turbid.
Collect the plasma solution from the tube using a transfer device connected to a 10-milliliter syringe. Place the skin samples in a 50-milliliter polypropylene tube with PBS and shake the tube gently. If the skin sample is relatively large, place it on the lid of a 150-square-centimeter tissue culture dish with the epidermal side down.
Remove the subcutaneous fat tissue using a pair of forceps and scissors. To prevent the tissue from drying out, rinse the tissue every few minutes in PBS. When fat removal is complete, use a scalpel to cut the skin samples into strips, approximately 0.5 by 1.5 centimeters.
Next, add five milliliters of Collagenase/Dispase mix to a sterile 15-milliliter tube. Transfer the cut tissue to the tube. Ensure that the tissue pieces are submerged in the solution and cap the tube securely.
Place the tubes in an incubator at 37 degrees Celsius with orbital shaking for 150 minutes. After incubation, move the tube containing the tissue to the biosafety cabinet. Place the lid of a sterile 100-millimeter culture dish upside down in the biosafety cabinet.
Transfer the tissue suspension to the bottom of the 100-millimeter culture dish without splashing. If any pieces of tissue remain in the tube, use a sterile one-milliliter pipette or sterile forceps to transfer the pieces to the bottom of the culture dish. With the epidermis facing up, hold the dermis with a pair of forceps.
Grasp the edge of the epidermis with another pair of forceps and peel the dermis and epidermis apart. Keeping the separated pieces on the same lid, place the dermal pieces in a new 100-millimeter culture dish containing PBS. Next, using laboratory forceps, transfer the dermal pieces to a 15-milliliter polypropylene tube containing three milliliters of 0.3%Trypsin PBS.
Incubate the tube in a 37-degree-Celsius water bath and mix the contents every two to three minutes by inverting the tube several times. After 10 to 20 minutes, stop the reaction by adding three to five milliliters of ice-cold complete growth medium. Vortex the tube vigorously several times.
Filter the suspension, which contains the fibroblasts, through an 85-micron nylon mesh into a 50-milliliter tube. Centrifuge the fibroblast solution at 150g for 10 minutes. Aspirate the supernatant and resuspend the cell pellet in 100 to 200 microliters of complete growth medium for cell count.
Plate the cells in a 25-square-centimeter tissue culture flask. Then, incubate the flask at 37 degrees Celsius. Change the medium which contains non-adherent cells on the following day, and again, every three to four days.
When the fibroblasts reach 70 to 80%confluency, wash them with PBS and add Trypsin-EDTA solution. After incubating the flask for three minutes at 37 degrees Celsius, stop the reaction by adding three to five milliliters of warm complete growth medium. Then, centrifuge the cell suspension at 200g for five minutes.
Remove the supernatant by aspiration. Culture the fibroblasts in PRP medium and incubate at 37 degrees Celsius. The minimum cell density recommended is 4, 000 viable cells per centimeter squared.
A dedicated medical device was used to produce PRP by processing whole blood from 10 different patients. The device removed the majority of the red blood cells and white blood cells. The mean platelet concentration in the PRP was approximately 50%higher than the platelet concentration in whole blood.
Autologous fibroblasts were cultivated in medium with 10%FBS or in media with PRP concentrations ranging from one to 50%After seven days of culture, without changing the medium, the cultures primed with 20%PRP exhibited a higher number of viable fibroblasts. PRP had potent effects as a proliferation booster up to 7.7 fold compared to FBS. Phalloidin staining showed that the morphological change observed upon PRP activation was related to F-actin reorganization, which is a signature of fibroblast activation.
A major concern of rapid cell expansion is the risk of genomic modification. In order to assess this, you can perform comparative genomic hybridization assay to assess the genomic stability of your cells. The main advantage of this technique is to replace FBS by a PRP, which is a more potent autologous biological supplement.
With this, researcher can grow more cell faster without any addition of a xenogeneic substances. We may use this technique for other cell types that need ex vivo expansion before clinical application, for example, keratinocyte or mesenchymal stem cell.
