Immortalized glomerular endothelial cells with stable fluorescent mitochondria are a great tool to examine the effect of different stimuli on mitochondrial structure in vitro. The described method in the current article yields a high number of glomerular endothelial cells. By testing small molecules on a GEC, we could screen for monotherapy for diabetic kidney disease where the GECs are affected.
The described method is easy to perform and doesn't require specific equipment. However, it is important to follow the described steps and sterilize the equipment to avoid contamination. To begin, place the perfusion and isolation materials required to isolate kidney glomerular cells on a workspace.
Filter the freshly prepared Hanks'Balanced Salt Solution, or HBSS, with a 0.2-micron filter, and keep it under the hood to avoid contamination. Then, mix 200 microliters of magnetic beads with 20 milliliters of HBSS. Prewarm the RPMI with 10%FCS and endothelial cell culture medium in a water bath at 37 degrees Celsius.
Then, pre-coat two wells of a six-well plate with two milliliters of 10 micrograms per milliliter collagen IV for 45 minutes at room temperature. Wash the plate twice with PBS to remove the acetic acid used to dilute the collagen. Use the coated plates immediately, or store them at two to eight degrees Celsius for up to four weeks.
Clean the surgical instruments and workspace with 70%ethanol. Subsequently, autoclave the surgical instruments for 30 minutes. After opening the abdomen of an anesthetized mouse, insert a butterfly needle into the left ventricle.
To inject 100 microliters of the bead solution to expand the blood circulation, carefully break the inferior vena cava below the kidneys with a 19-gauge needle and continue the perfusion of the bead solution. Next, gently remove the fat tissue with tweezers, excise both kidneys with thin surgical scissors C, and put them on a plate with one milliliter of HBSS on ice. Mince the kidney with a sterile scalpel into one-millimeter small pieces.
Incubate the minced kidney with one millimeter of freshly prepared collagenase type II for 35 minutes at 37 degrees Celsius with gentle tilting on a horizontal shaker. After digestion, add four milliliters of RPMI with 10%FBS to neutralize the collagenase. Filter the digested tissue through a sterile 100-micron cell strainer into a 50-milliliter tube.
Use the bottom of a sterile 1.5-milliliter tube to stir the digested tissue against the strainer, allowing the glomeruli to go through. Rinse the cell strainer with 15 milliliters of HBSS. Then, centrifuge the flow-through at 200 XG for five minutes at room temperature.
At this stage, the tube contains three layers. The upper layer contains smaller structures such as tubules, the middle layer is beads with glomeruli, and the bottom layer contains debris. Carefully aspirate the supernatant and the top white layer.
Then, resuspend the pellet containing beads bearing the glomeruli and debris in 1.5 milliliters of HBSS and transfer it into a two-milliliter tube. Place the tube in a magnetic concentrator and aspirate the supernatant before washing the beads twice with one milliliter of HBSS. Place 20 microliters of cell suspension onto a slide.
Then, observe the slide under a microscope for glomeruli's presence. Next, resuspend the pellet in an endothelial cell growth medium and transfer the suspension to a two-milliliter tube. Add one microliter of interferon gamma for each three milliliters of medium, and place the cells in a six-well plate for incubation at 33 degrees Celsius.
After three days of culture, carefully replace the one milliliter medium with a fresh medium. At this stage, the cells are heterogeneous with a mixture of glomerular cells. After seven days, replace the medium with two milliliters of fresh endothelial cell growth medium, supplemented with interferon gamma to start cells'proliferation.
After 10 days, when the cells reach 80%confluency, passage them using trypsin and transfer them into a T-25 flask coated with collagen IV.After 21 days of culture, transfer cells to a T-75 flask. Add three milliliters of 0.25%trypsin to a T-75 flask and incubate the cells at 37 degrees Celsius for five minutes. Then, neutralize the trypsin with three milliliters of endothelial growth medium and transfer the suspension to a 15-milliliter tube for centrifugation at 200 XG for five minutes.
Aspirate the supernatant and wash the cells in one milliliter of PBS containing 1%BSA, two-millimolar EDTA, 1%penicillin/streptomycin. Again, centrifuge and resuspend the pellet in 200 microliters of CD31 antibody coated beads and 800 microliters of PBS. Incubate the cells for 45 minutes with continuous shaking at 37 degrees Celsius and 5%carbon dioxide.
Next, place the tube in the magnetic concentrator and wash the cells four times with PBS containing BSA, EDTA, and penicillin/streptomycin. After the last wash, resuspend the cells in three milliliters of endothelial growth medium, supplemented with interferon gamma. Plate 1.5 milliliters of cells per well in collagen IV coated wells on a six-well plate and culture under permissive conditions at 33 degrees Celsius.
After four days, replace 750 microliters of the medium with a fresh endothelial growth medium, supplemented with interferon gamma. After 10 to 14 days of culture, using an epifluorescence microscope with a 488-nanometer filter, observe for growing CD31 positive glomerular endothelial cell, or GECs, colonies that express fluorescent mitochondria. After 21 days, or when the cells have reached 80 to 90%confluency, transfer them into a T-25 flask.
Maintain the culture with an RPMI growth medium containing 10%FCS. Alternatively, cells can be cryopreserved. Passage cells using trypsin, as demonstrated earlier.
Then, centrifuge the cells and resuspend the pellet in three milliliters of freezing medium. Aliquot the cells in cryo tubes and store them in liquid nitrogen, vapor temperature. This protocol described a method for the isolation of glomerular endothelial cells.
From the isolated glomeruli, cells started growing slowly after three days of culture. After seven days, cells appeared heterogeneous and showed other glomerular cell types, such as podocytes, parietal, epithelial, and mesangial cells. After reaching 70 to 80%confluency, other glomerular cells were removed using a positive selection of endothelial cells.
The MitoDendra2 GECs expressed CD31 and were negative for podocyte marker, as shown by synaptopodin negative staining. The effect of glucose concentration on the mitochondria structure was examined. Compared to the elongated mitochondria visible in cells under normal glucose, high glucose induced fragmentation or fission of mitochondria, as observed by prominent spheroid shaped mitochondria.
Furthermore, a 405-nanometer laser was used to photoconvert a selected subpopulation of mitochondria in a single live MitoDendra2 GEC. A successful photoswitching of mitochondria from green to red in the selected area for normal and high glucose concentration treated cells is shown. This switching allowed witnessing the fusion events in MitoDendra2 GECs.
Under normal glucose, yellow merged green and red fluorescence were observed due to mitochondria matrix fusion. In contrast, in the high glucose treated GECs, the mitochondria were mainly fragmented. Placing the cells in one well of six-wells plate with a small volume of growth medium is essential to let the cells attach to the dish.
The isolated endothelial cells could be used to perform mitochondrial function and structural studies. The use of immortalized endothelial cells with fluorescent mitochondria features will pave the way toward drug discovery. The small molecules could be tested on the cells, and live imaging is performed to examine their effect on mitochondria function.
