Glomeruli Isolation: A Technique to Obtain Intact Glomeruli from Murine Kidney

Overview

This video describes the method to isolate intact glomeruli from mouse kidneys. The encapsulated and decapsulated glomeruli thus obtained can further be used for culturing parietal epithelial cell outgrowths from the murine kidney.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

NOTE: Untreated, healthy wild-type (WT) mice (n = 4) and cd44-/- (n = 4) mice were sacrificed at the age of 12−16 weeks. Both male and female mice were used. All mice were on the C57Bl/6 background.

1. Mouse Kidney Dissection

1. Sacrifice healthy WT mice or genetically altered mice by cervical dislocation.
2. Dissect whole mouse kidneys directly after sacrificing the mice. For this, perform a median laparotomy using abdominal scissors, cutting the skin and then the abdominal muscles. Remove the intestine and place it next to the mouse.
3. Free the kidneys from connecting tissue and pull out the kidney using surgical forceps, cutting the renal artery, renal vein, and ureter with scissors.
4. Remove the renal capsules from the kidneys by holding the kidney with surgical forceps and pull off the capsule using another pair of forceps.
5. Place the kidneys in a 6-well cell culture plate (2 kidneys/well) prepared with 2 mL of Hanks' balanced salt solution (HBSS) per well and place on ice.

2. Isolation of Glomeruli from Mouse Kidney

1. Transfer the kidneys to a 100 mm Petri dish and mince the kidneys into small pieces of 1−2 mm using two scalpels. Keep the minced kidney pieces wet using 1−2 mL of HBSS.
2. Place the minced kidney pieces on top of a 300 µm metal sieve and press the kidney through the sieve using a plunger of a 20 mL syringe. Repeatedly rinse the sieve with HBSS in between and collect the flow-through in a clean Petri dish using a serological pipette. Collect also everything that remains/sticks to the bottom side of the sieve by scraping it off with the scalpel and transfer it to the collected flow-through (kidney homogenate).
3. Rinse the kidney homogenate through a 75 µm sieve with HBSS. Collect the flow-through and subsequently rinse this flow-through through a 53 µm sieve. Wash both sieves using HBSS to remove all smaller structures.
   NOTE: In this step, the flow-though is only rinsed but not pressed through the 75 µm and 53 µm sieve. Washing with HBSS is necessary to remove debris and smaller structures on the sieves. Therefore, normally 200−300 mL of HBSS are used in total.
4. Collect the kidney structures/material that remain on the 75 µm and 53 µm sieve by washing the upper surface of the sieves with Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum (FCS) and transfer the material into a 6-well ultra-low attachment microplate.
   NOTE: To wash the upper surface of the sieve, rinse the sieve in a tilted position (>45°) and collect the kidney material at the edge of the sieve. The collected material is enriched for encapsulated as well as decapsulated glomeruli, showing only a few tubular fragments. Encapsulated glomeruli are very sticky. To collect single glomeruli, it is therefore important to prevent glomeruli from adhering to the surface of a Petri dish or well plate. To avoid adherence, use medium with 20% FCS and use ultra-low attachment plates for this step.

3. Culturing of Glomerular Outgrowth

1. Bring the ultra-low attachment microplate to an inverted light microscope and collect single encapsulated and/or decapsulated glomeruli with a 20 µL pipette. Avoid pipetting other structures and debris. After collecting a single glomerulus in the pipette tip, add fresh DMEM medium without collected kidney material into the same pipette tip to a volume of 20 µL.
2. Transfer the single glomerulus with the 20 µL DMEM medium to the center of a well of a 24-well cell culture plate and incubate for 3 h at 37 °C and 5% (v/v) CO₂ to allow attachment of the glomerulus to the center of the well. Carefully move the plate to avoid floating of the glomerulus to the borders of the well.
3. After 3 h incubation, the glomerulus is attached to the center of the well. Carefully add 500 µL of endothelial basal medium (EBM) supplemented with a growth factor kit containing hydrocortisone, human endothelial growth factor, bovine brain extract, and gentamicin sulfate-amphotericin B (Table of Materials) and additional 5% (v/v) FBS and 1% (v/v) penicillin/streptomycin (pen/strep) to each well.
4. Culture the single glomeruli for 6 days at 37 °C, 5% (v/v) CO₂.
   NOTE: Within 6 days, the outgrowth consisting of parietal epithelial cells are formed. If one aims to test the effects of specific compounds or drugs on the parietal epithelial cells, it should be added to the medium within this 6-day period.

Materials

Name	Company	Catalog Number	Comments
24-well cell culture plate	Corning Costar
Digital inverted light microscope	Westburg, EVOS fl microscope
Dulbecco's Modified Eagle's medium	Lonza
Fetal Bovine Serum	Lonza
Hanks' Balanced Salt Solution	Gibco
Petri dish	Sarstedt		Size 100
Scalpel	Dahlhausen		Size 10
Sieves	Endecotts Ltd		Size: 300 µm, 75 µm, 53 µm, steel
Ultra-Low Attachment Microplates	Corning Costar		6-well plates
Syringe	BD Plastipak		Size: 20 mL