RPE related disorders remain a major part of blending diseases, yet without effective remnants in virtual culture of primary RPE cells to serve as a good model for physiological and pathological studies of RPE related disorders. In this protocol, we provide an easy to follow protocol to culture primary IPCs, which could quickly restore and maintain later RP characteristics, we to We believe that by using this method people could quickly generate RP cells from macistate studies and the protective drug screenings which could facilitate the development of new treatment for RPE disorders. A step by step value demonstration will make this method much easier to replicate in different laboratories that want to study RP cells.
To begin thaw the tissue digestion enzyme malaquad and sterilize 1X PBS. Supplemented with 2%penicillin and 2%streptomycin by filtering the solution through a 0.22 micrometer syringe filter unit. Wash the wells of the culture plate and transwell inserts with 1X PBS.
Remove the PBS from the wells and transwells with a pipette and add one milliliter of fresh 1X PBS to the lower chamber and 600 microliters of 10 micrograms per milliliter Laminin solution to the upper chamber. Incubate the plates and transwell inserts in this Heliciculture Incubator overnight at 37 degrees Celsius and 5%carbon dioxide. After that, remove the laminate solution from the upper chamber and wash with one milliliter of cold 1X PBS twice before seating the cell Clean the laminate flow hood with 75%ethanol and UV light.
Prepare three 50 milliliters sterile centrifuge tubes containing around 25 milliliters of 75%Ethanol, three 50 milliliters sterile centrifuge tubes containing around 25 milliliters of 1X PBS, and three 10 centimeter sterile cell culture dishes containing around 15 milliliters of 1X PBS. Soak four porcine eyeballs into about 15 milliliters of 75%Ethanol in a 10 centimeter Petri dish and use a pair of scissors and forceps to cut off all the residual connective tissues and muscles. To decontaminate the eyeballs, soak, and wash four eyeballs in three 50 milliliters sterile centrifuge tubes filled with 75%ethanol sequentially.
Then wash the eyeballs in three 50 milliliters sterile centrifuge tubes filled with 1X PBS sequentially. Invert the tubes every minute to wash the eyeballs thoroughly. Move the four eyeballs into a 10 centimeter sterile cell culture dish containing 1X PBS.
Trim the outer surface of each eyeball again, to remove the optic nerve and small debris. Use scissors to make a small cut at the intersection of the limbus and sclera. And then remove the cornea, iris, lens, vitreous body, and neural retina.
Transfer the RPE choroid sclera complex into a new 10 centimeter cell culture dish and make four cuts to flat the eye cup into the shape of a four-leaf clover. Perform trips in EDTA solution digestion by placing the four RPE choroid sclera complexes into a new 10 centimeter dish and pouring 20 milliliters of fresh trypsin EDTA solution to merge the RPE choroid sclera complexes. Place the dish into the cell culture incubator at 37 degrees Celsius for almost 30 minutes.
After 30 minutes, take the dish out of the incubator and, add 20 milliliters of pre-warm cell culture media with 10%FBS to the dish. To neutralize the trips in EDTA solution use a five milliliter transfer pipette to dissociate the RPE cells by gently pipetting several times. Collect the cell suspensions into 15 milliliters centrifuge tubes, and then gently pipette another 10 milliliters of fresh culture media with FBS to wash the RPE Choroids sclera complexes on the dish.
Collect the RPE cells by centrifuge the tubes at 300 G for five minutes at room temperature. Aspirate the SuperAgent using a pipette and add another five milliliters of culture media with FBS to re-suspend the cells. Centrifuge at 300 G for another five minutes.
Decant the SuperAgent and re-suspend the cells with 12 milliliters of culture media. Next seed 100, 000 to 200, 000 cells per well into 12 Well culture plates or transwell inserts. Change the culture media every two days and reduce the serum concentration to 1%when the cells fully cover the surface of the wells per insert.
Change the culture media every two days until the cells are harvested. The representative images of primary porcine RPE cells on day two, day six and day 10 are shown here. The QRTPCR analysis of mRNA levels of signature genes in primary porcine RPE cells, primary human RPE cells, ARPE 19 cells and porcine RPE choroid tissue is detected by this graphical image.
Here GAP DH was used as the housekeeping gene for RTPCR. Four biological replicates were used for primary porcine RPE cells and a RPE 19 cells where three biological replicates were used for primary human RPE cells and porcine RPE choroid tissue. The gene expression levels in ARPE 19 cells were set as controls.
The Western blood analysis of RPE 65 proteins in ARPE 19 and porcine RPE cells and in primary human RPE cells and porcine RPE choroid tissues are shown in this figure. Vinculin was used here as a loading control. This figure shows a two week culture of porcine RPE cells in DMEM Basic media with 1%FBS.
Cells cultured with or without Lamin were stained with RPE 65, sodium potassium APTAs, Z01, and Hex. TR measurements in cell sheets, cultured with or without laminin are depicted in this figure. The western blood analysis of vascular endothelial growth factor or VEGF and pigment epithelium derived factor or PEDF in primary porcine RPE and ARPE 19 cells are shown in this figure.
The sectorial functions of cultured primary porcine RPE and ARPE 19 cells are presented in these images. The great part of this protocol is to dissolve RP cells from RPE chide glial complexes. We recommend using fresh trips and solution each time and the properly controlling the digestion time, to dissolve eight RP cells.
