人类肾脏血管周围间质细胞新型临床分级分离方法

The overall goal of this clinical grade isolation method is to isolate human kidney perivascular stromal cells, or kPSC, for the cell therapeutic treatment of various kidney diseases. This method can help answer key questions in the field of regenerative medicine, such as a human kPSCs suitable for center nerve therapy for kidney diseases and transplantation. The main advantages of this technique are that it's clinical, and that it can be used to generate a sufficient number of kPSCs for cell therapy purposes.

Ellen Lievers, a technician in our laboratory will demonstrate the functional testing of kPSCs. Before beginning the kidney cell isolation procedure, connect a sterilized perfusion tray to a water bath in the vertical position. When the perfusion tray has been warmed by the water, change the tray to the horizontal position, but still connected to the water bath, then place an L/S 25 tube into the pump.

Place cultures forceps on one end of the tube and allow the tube to rest on the bottom of the tray. Place sterile gauze on top of the tube to prevent obstruction, and attach a luer connector to the other end of the tube, then add approximately 100 milliliters of medium to the tray and begin flushing the L/S 25 tube at a pump speed of 100 milliliters per minute. To isolate the kidney cells, transfer the kidney from the double sterile bags onto the sterile perfusion tray, and use scissors and gentle tearing to remove the perirenal adipose tissue and kidney capsule.

Upon location of the renal artery on the aortic patch, use the accessory spike to cannulate the renal artery, and secure the spike with a sterilized tie wrap. With the pump on, attach the pump tube end to the luer connector. And flush the kidney with the medium in the tray at a 100 milliliter per minute flow rate.

Next add 20 milliliters of collagenase and 2.5 milliliters of DNAse to the perfusion tray, and allow the enzymes to digest the kidney, gently turning the organ from time to time by hand until it becomes soft, and the perfusion liquid begins turning opaque. The duration of collagenase digestion is critical, too short will lead to cell clumps, while too long will lead to cell death. At the end of the enzymatic perfusion, gently massage the kidney until a cell suspension is obtained, and remove the non-digested material and the spike.

Add 50 milliliters of normal human serum to the cell suspension, then with the pump at a low flow rate, place the tube connected to the kidney above several 50 milliliter collection tubes to allow the cell suspension to drain from the perfusion tray into the collection tubes. When all of the cells have been collected, centrifuge the cell suspensions, and wash the pellets two times with washing medium, supplemented with 10%normal human serum. After the second wash, resuspend the pellets in an equal volume of cell culture medium to that of the cell pellets.

To culture the crude kidney cells, add approximately one milliliter of cell suspension to 24 milliliters of alpha-MEM 5%platelet lysate cell culture medium per T 175 cell culture flask, and incubate the flasks at 37 degrees Celsius and 5%carbon dioxide. After two days remove the supernatant and cell debris, and feed the cells with 25 milliliters of fresh cell culture medium. When the cells are confluent, isolate the NG2 positive fraction by magnetic cell separation, according to standard protocols.

Then seed four times 10 to the third NG2 positive cells per centimeter squared in an appropriately sized cell culture flask, and place the flask in the cell culture incubator. When the cells reach 90 to 100%confluency, harvest the supernatant and wash the adherent cells two times in PBS. Detach the cells with the appropriate volume of trypsin according to the flask size, stopping the reaction with the reserved supernatant after five minutes.

Gently resuspend the cells, then transfer the cell suspension to an appropriately sized conical tube for centrifugation, and replate the cells at a four times 10 to the third cells per centimeter squared density. Sometimes after NG2 enrichment the cells stop proliferating, or start to grow in 3D structures. This can be resolved by trypsinizing the cells and replating them.

After four to five passages, seed the kPSCs in a six-well plate at two times 10 to the fifth cells per two milliliters of medium per well concentration, and seed five times 10 to the fifth proximal tubular epithelial cells in two milliliters of proximal tubular epithelial cell medium per well in a separate six-well plate. Place both plates in the cell culture incubator for 48 hours, harvesting the kPSC cell supernatant at the end of the incubation, then remove the proximal tubular epithelial cell supernatant and use a yellow pipette tip to make a scratch in the cell monolayer at the bottom of each well. Wash the scratched cells with PBS, and add the condition to medium from the kidney vascular stromal cell cultures.

Then image the scratch at four, eight, 12 and 24 hours at the same marked position on an inverted brightfield microscope to assess the percentage of closure over time. Kidney perivascular stromal cells are plastic adherent, spindle-shaped cells that are positive for stromal and perivascular cell markers, and negative for the markers CD31, CD34, CD45, and HLA-DR. Typically a homogeneous population of kidney perivascular stromal cells can be reached at passage four, with the cells reaching senescence around passage nine to 10.

In this representative wound scratch assay, to assess the functional capacity of the kPSCs, when conditioned medium from a kidney perivascular stromal cell culture was added, the wound closed significantly faster than in wells cultured with the control medium. Once mastered the primary isolation technique will take about two hours, it will take you one to two months to generate a homogenous kPSC population. While attempting this procedure, it's important to stop the collagenase treatment as soon as the kidney becomes soft, and the fluids become less transparent.

In addition to the procedures demonstrated here, these techniques could also be used for the isolation of perivascular stromal cells from other solid organs. The kPSCs isolated with this technique can be used to study the regenerative potential of these cells in several different models of kidney disease and transplantation. After watching this video you should be able to isolate and culture kPSCs in a clinical grade manner.

Don't forget that working with human material carries the risk of virus transmission, always take precautions like testing for HIV, hepatitis B, hepatitis C when performing this procedure.