The overall goal of this procedure is to generate a primary culture of nephrogenic zone cells from the developing kidney. This is accomplished by first dissecting out kidneys from day 17.5 embryos. The next step of the procedure is to remove the kidney capsules.
A limited enzymatic digestion of the organs is then performed to dislodge cap mesenchyme and corticals stroma cells from the nephrogenic zone. The final step of the procedure is to plate the cells in fibronectin and culture in serum free medium. Ultimately, results can be obtained that demonstrate a purified nephrogenic zone cell population by immuno staining of specific markers.
Though this method can be used to study cell signaling, it can also be applied to understanding substrate requirements for cellular expansion. For example, for engraftment techniques, demonstrating the procedure will be Derek Adams, who is a technician in my lab and Aaron Brown, who is a postdoc in my lab. After preparing the necessary solutions and treating the tissue culture dishes according to the written protocol, begin the mouse embryonic kidney dissection by sacrificing the mother at E 17.5.
According to your institutional guidelines, remove the uterus and place it into a Petri dish containing several milliliters of docos sate, buffered saline or DPVS with calcium and magnesium. Under a dissecting microscope, use watchmaker's forceps to remove each embryo and another pair of forceps to decapitate. Once the entire litter has been removed from the uterus and decapitated, transfer the embryos to a clean Petri dish containing enough DPBS with calcium and magnesium to cover the embryos.
Leave behind as much blood and tissue debris as possible for each embryo, make a circumferential incision in the abdominal wall at the level of the umbilicals with forceps. Then place the embryo on its back and pin down the shoulders with one set of forceps. With a second set of forceps, remove the internal organs from the lungs to the bladder.
The kidneys will be attached to the back of the mass of internal organs. Next, gently tease away the kidneys from the rest of the organs. Taken care to keep the kidneys connected to each other.
Then hold the tissue between the kidneys with one set of forceps and grab the adrenal gland attached to the capsule on one of the kidneys with the other forceps. Gently peel the capsule from the kidney. Be careful not to damage the kidney.
Be sure to remove any remaining capsule from the kidney as well as the utero if still attached. Repeat with remaining kidney. Finally, cut a transfer pipette to an appropriate diameter and gently transfer the kidneys into a five milliliter polystyrene tube of HBSS.
Repeat the kidney isolation for each embryo and pool the kidneys in the tube up to eight kidneys per tube in a sterile environment. Begin the enzymatic digestion process by first removing the HBSS solution from the five milliliter tube containing the kidneys with a pipette. Be careful to avoid contact with the kidneys and immediately add 1.5 milliliters of collagenase, a Pancreatin digest solution.
Repeat for each tube of kidneys and cap the tubes. Then place on a mutator in a 37 degree Celsius incubator for 15 minutes. Just prior to the completion of the incubation, add three microliters of one unit per microliter DNAs to a fresh five milliliter polystyrene tube for each digest being performed.
After the incubation, quickly remove the digest from the incubator and add 75 microliters FBS invert three times to mix. And let's stand at room temperature for two minutes. Then transfer 1.4 milliliters of the cell suspension to a five milliliter polystyrene tube containing DNAs Mix and place on a mutator in a 37 degree Celsius incubator for 10 minutes.
Following the incubation, transfer each cell suspension to a 1.5 milliliter einor tube and centrifuge at 325 times G for five minutes. Next, remove the supernatant centrifuge, the tubes at 325 times G for five minutes. Resus, suspend the pellet in one milliliter of 5%F-B-S-H-B-S-S by pipetting up and down five to six times.
Cap each tube before moving to the next sample Following centrifugation, remove the SNAT and add 500 microliters of KSFM with additives to each tube after medium has been added to all tubes. Gently resus suspend each cell pellet by pipetting up and down five to six times with a one mil liter micro pipette. Combine all cell suspensions in a fresh five milliliter polystyrene tube.
Now filter the cell suspension through a pre wetted 40 micron cell strainer cap and collect the flow through in a new polystyrene tube. Avoid touching the strainer with the pipette tip once all the flow through has been collected. Refilter the flow through a second time through a new pre wetted cell strainer.
Remove the strainer and cap the tube with a fresh sterile from a new tube. Count the cells and plate at a density of 100, 000 to 200, 000 cells per square centimeter in KSM with additives in a fiber encoded culture plate. Allow the nephrogenic zone cells to recover and attach in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide for two to four hours prior to any stimulation for experimentation.
Shown here is a phase contrast. 20 x image of nephrogenic zone cells plated at 200, 000 cells per square centimeter and cultured for 24 hours. In this image, nephrogenic zone cells were stained with anti PAX two antibody, which is specific for nephron progenitor cells and shown in red while nuclei in blue were counterstain with dapi.
A phase contrast view of a cluster of nephron progenitors is shown at 40 x magnification. To further demonstrate specificity, nephrogenic zone cells were isolated from embryos that have laxy under the control of the Fox D one promoter, which is expressed in the stromal cell population. Within the nephrogenic zone of the developing kidney cells were then stained with DAPI Titan fi nuclei in blue foin for F actin in green and anti beta lactase in red.
Once mastered a monolayer primary culture of nephrogenic zone cells can be generated in approximately two hours. These cultures facilitate rapid and high throughput studies of cell signaling pathways in nephron progenitor development as a highly conducive to virus transduction treatment with recombinant proteins and small molecule treatment. Further cell sorting procedures can also be used to isolate specific cell types from these cultures.
