Kidney tubuloid cultures can be established from healthy kidney tissue and from urine allowing for the generation of representative models to study kidney disease and physiology. This protocol allows for the generation of healthy kidney tubuloids from tissue and urine, the latter being a noninvasive and patient-friendly way of obtaining material for generation of cultures. Tubuloid cultures can be used to study kidney physiology and for disease modeling, such as infectious, malignant, and hereditary kidney diseases.
To generate human kidney tubuloids from tissue, place the kidney tissue sample into one milliliter of advanced DMEM plus plus plus medium in a 10 centimeter Petri dish and use scalpels to mince the tissue into pieces of an approximately one cubic millimeter size. Use forceps and scalpels to transfer the tissue pieces to a 15 milliliter tube and wash the dish with five milliliters of fresh medium. Use a sterile 10 milliliter pipette to transfer the medium to the tube.
Sediment the tissue pieces by centrifugation and resuspend the pellet in three to four milliliters of collagenase solution. Incubate the tissues on a horizontal shaker at 37 degrees Celsius and 250 revolutions per minute for 45 to 60 minutes with vigorous shaking every 15 minutes. When the suspension is homogenous and most of the tissue pieces have digested, fill the tube with medium and mix five to 10 times by inversion.
Centrifuge the suspension. If the pellet is red, add one milliliter of red blood cell lysis buffer to the tube for a five-minute incubation at room temperature. At the end of the incubation, wash the tube with fresh medium.
If pieces of tissue are still visible, resuspend the pellet with five milliliters of fresh medium and filter the suspension through a 100 micron cell strainer into a 50 milliliter tube. Transfer the filtered suspension to a new 15 milliliter tube for centrifugation and use a P1000 pipette set to a known volume to measure the volume of the cell pellet. Carefully resuspend the pellet without creating air bubbles and incubate the cells for one minute on ice.
At the end of the incubation, resuspend the pellet in a 70 to 75%volume of basement membrane extract and plate 15 microliter droplets of the resulting suspension in a 37 degree warmed multi-well cell culture plate. When all of the droplets have been plated, incubate the plate upside down at 37 degrees Celsius for 15 to 20 minutes, then add the appropriate volume of 37 degrees Celsius warmed culture medium, and inspect the cultures under a brightfield microscope. To generate human kidney tubuloids from urine, divide the urine sample equally between 50 milliliter tubes and wash the samples two times with 10 to 20 milliliters of fresh washing medium per wash.
After the second wash, resuspend the pellets in their residual supernatants and pool the samples in a single 15 milliliter tube for a third centrifugation. Measure the cell pellet volume as demonstrated and carefully resuspend the pellet without creating air bubbles. After one minute incubation in ice, resuspend the pellet in a 70 to 75%volume of basement membrane extract and plate 15 microliter droplets of the resulting suspension in 37 degrees Celsius warmed multi-well cell culture plates as demonstrated, then incubate the droplets upside down for 15 to 20 minutes before adding fresh medium and viewing the cultures by light microscopy.
After one to two weeks of either type of culture, use a P1000 pipette to disrupt the tubuloid droplets in each well using the tip to scrape the bottoms of the wells to collect the attached cells. Pull the well contents in a single 15 milliliter tube containing 10 milliliters of advanced DMEM plus plus plus medium and collect the tubuloids by centrifugation. Based on the size of the pellet, add trypsin replacement agent supplemented with 10 micromolar Y27632 for a five-minute incubation at 37 degrees Celsius.
At the end of the incubation, use a 1, 000 microliter pipette tip with a 10 microliter pipette tip placed over the tip to pipette the tubuloid suspension 20 to 30 times. Check under the microscope to see if any intact tubuloids are still present within the tube. It fewer than 10%of intact tubuloids are present, fill the tube with fresh advanced DMEM plus plus plus, and collect the tubuloids by centrifugation to allow the pellet volume to be measured as demonstrated.
Resuspend the pellet in a 70 to 75%volume of basement membrane extract and plate 15 microliter droplets in a 37 degree Celsius warmed multi-well cell culture plate. After a 15 to 20-minute upside down incubation at 37 degrees Celsius, add pre-warmed culture medium to the droplets and inspect the culture by light microscopy. Tubuloids generated from kidney tissue samples typically appear within seven days of culture establishment and need to be passaged within one to two weeks of plating.
In urine cultures, compact tubuloid structures and adherent cells appear approximately 14 to 21 days after plating and first passaging generally occurs between three and four weeks. The presence of cystic epithelial tubuloids increases with each passage. The successful generation of human kidney tubuloid cultures can be assessed by immunohistochemical staining for markers expressed in the tubular kidney epithelium, such as paired box gene 8 protein.
A correct timing of enzymatic digestion of tissue material and fast processing of urine samples are crucial steps for the successful outcome of these protocols. The high efficiency of establishment of kidney tubuloid cultures can pave the way for patient-specific testing of drug-induced nephrotoxicity, a common side effect of many chemotherapeutics.
