Development of Human Renal Tubular Epithelial Cell Primary Cultures in Monolayers and Three-Dimensional Conditions

Human renal tubular epithelial cells established in both primary and three dimensional cultures, were developed as a model to investigate the cytotoxic effects of Shiga toxin, which induces pathophysiological alterations, culminating in hemolytic uremic syndrome. No specific treatment exists for hemolytic uremic syndrome. Therefore, the current challenge lies in evaluating drugs in HRTEC primary cultures to inhibit the effects of Shiga toxin on the human kidney.

Shiga toxin has been shown to inhibit cell viability and proliferation, induce apoptosis in HRTEC primary cultures and suppress the development of tubular structures in three dimensional HRTEC cultures. The protocols offer the advantage of enabling the study of epithelial cell functional mechanisms in cell cultures that retain the original characteristics of the human renal proximal tubule. To begin, dissect the renal capsule using forceps.

Separate the renal cortex from the medulla and cut the cortex into small fragments of approximately one millimeter. Place the kidney fragments in sterile hanks solution without calcium and magnesium supplemented with 0.1%collagenase type one to perform the digestion. Incubate the renal fragments for 30 minutes at 37 degrees Celsius with shaking.

To stop digestion, add one volume of PBS, containing 10%fetal bovine serum or FBS. Wash the sample three times by centrifuging at 160G for 10 minutes each in a centrifuge refrigerated at four degrees Celsius, discarding supernatant and resuspending the pellet in the same solution each time. Pass the digested product through a 70 micrometer poor mesh to filter the tubules and discard the glomeruli that remained retained in the filter.

Centrifuge the filtered tubules at 160G for 10 minutes at four degrees Celsius. After discarding the supernatant, resuspend the pellet in sterile hanks solution without calcium and magnesium supplemented with 0.2%collagenase type one. Incubate the sample for 30 minutes at 37 degrees Celsius with shaking.

To stop digestion, add one volume of PBS containing 10%FBS before washing the sample by centrifugation as shown previously. After the last centrifugation, resuspend the cell pellet in five milliliters of complete culture medium in a 25 square centimeter culture flask with a vented lid. Close the lid of the culture flask and incubate the cells at 37 degrees Celsius in a humid atmosphere with 5%carbon dioxide.

After adding the culture medium and incubating the cells again at 37 degrees Celsius, observe the cells under an inverted microscope. Take an aliquot of liquid basement membrane matrix at a volume of 150 microliters per square centimeter and dispense it into each well of a multi-well plate to cover the bottom. Incubate the plate in a cell culture incubator at 37 degrees Celsius for one hour to allow the matrix to solidify into a gel.

To detach confluent primary HRTEC cultures after discarding the culture medium from the flask, add one milliliter of 0.25%trypsin with 0.02%EDTA solution. Spread the solution evenly across the surface of the flask before incubating it for five minutes at 37 degrees Celsius. Observe the cells under an inverted microscope to confirm complete cell detachment.

After resuspending the detached cells in 10 milliliters of PBS or culture medium, centrifuge at 160G for 10 minutes at four degrees Celsius. Once the supernatant is discarded and the pellet is resuspended in complete culture medium with the same supplements used for primary HRTEC cultures, count the resuspended cells using a Neubauer chamber. Seed 40, 000 cells in 150 microliters of medium per well onto the solidified basement membrane matrix and incubate the cell cultures for several days at 37 degrees Celsius in a humid atmosphere with 5%carbon dioxide.

Monitor the cultures regularly using an inverted optical microscope to observe cell aggregation and formation of tubular structures. HRTEC seeded on the basement membrane matrix initiated tubular genesis by migrating and organizing into multicellular cords within the first four to 10 hours of culture. By one day after seeding, cellular aggregates emerged from the cords and began forming early tubular structures.

The 3D tubular structures continued to elongate and mature progressively by three days and eight days of culture. Hematoxylin and eosin staining of the 3D HRTEC structures revealed a central lumen surrounded by epithelial cells. Immunofluorescence confirmed the expression of aquaporin one protein in both apical and basolateral membranes of the 3D HRTEC epithelial cells mirroring the expression pattern observed in proximal tubules of the human kidney.