Isolation, propogation and differentiation of mouse Nephrogenic Zone Cells (NZCs) (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Thomas Carroll; Alicia Fessler

Keywords

[‘3D culture’, ‘Cell dissociation’, ‘culture’, ‘isolation’, ‘kidney’, ‘kidney digestion’, ‘nephron progenitor’, ‘nephrogenesis’, ‘organoid’, ‘stem cell’, ‘tissue dissociation’, ‘mouse’]

Subjects

[‘Cell biology’, ‘Cell culture’, ‘Developmental biology’, ‘Tissue culture’, ‘Isolation, purification and separation’]

Release Date

2021-08-02

Abstract

Isolation, propogation and differentiation of mouse Nephrogenic Zone Cells (NZCs)

Reagents

• Collagenase (Roche, 10 103 578 001)
• Pancreatin, porcine (Sigma, P1625)
• HBSS without calcium or magnisium (Gibco, 14175-079)
• PBS without calcium or magnisium
• FBS
• AUTOMACS running buffer (Miltenyi, 130-091-221)
• APEL2 (Stemcell Technologies, 05270)
• ROCK inhibitor Y-27632 (Millipore, 688001)
• Human recombinant FGF9 (R&D, 273-F9-025)
• BD Matrigel™ hESC qualified (corning, 354277)
• CHIR (StemGent 04-0004)
• Heparin (Sigma, H3393)
• TrypLETM dissociation solution (Life Technologies, 12563029)
• DMEM/F12 (ThermoFisher, 11320033)
• PenStrep

Equipment

• pre-separation filter 30um (MiltenyiBiotec, 130-041-407)
• 96, 24 or 6 well plates (Conrning 353072, 35047, 353224)
• Nutator placed at 37°C
• hemocytometer
• Round bottomed 5ml falcon tubes (Fisher Scientific, 14-959-1A)
• 50mL conical tubes (Fisher Scientific, 14-432-22)
• 15mL conical tubes (Fisher Scientific, 14-959-53A)
• 1.5mL eppendorf tubes (Fisher Scientific, 14-282-300)
• Nuclepore Track-Etch Membrane (Whatman, 110409)
• Bench top centrifuge

Procedure

Isolate nephrogenic zone cells according to Brown et al. 2015. This can also be found at [[N-H9AM]].

Preparation of reagents prior to isolations:

1. Prepare collagenase A/pancreatin enzyme digest solution 2 hours before kidney dissection by adding 25 mg collagenase A to 10 ml PBS without calcium or magnesium. Place on a nutator at RT for 15 minutes or until collagenase A is dissolved. Add 100 mg pancreatin and place on nutator until kidney harvest is complete. Filter sterilize (0.2 μm filter) before use to remove any undissolved particles. Alternatively, large batches of the enzyme digest solution can be made, filter sterilized, and frozen at -20°C for up to 6 months.
2. If plating cells in monlayer for culture, dilute Matrigel 1:25 in cold DMEM/F12 and coat plates. - Volumes of Matrigel for coating are: 50 μl/well for 96 well 300 μl/well for 24 well 1 ml/well for 6 well. -Make sure Matrigel is distributed over the entire surface of the well and allow to sit undisturbed in a for at least 1 hour.
3. Prepare round bottomed 5 ml falcon tubes to receive dissected kidneys
4. Transfer 50 ml of autoMACS running buffer to a 50 ml conical tube and warm to RT.
5. Make appropriate volume of culture media. If uncertain of the number of cells, there is time in the procedure to do this later.

NZC Culture Media

NZC propagation Media

• 1x APEL2
• 200ng/mL FGF9
• 10uM Y-27632
• 1ug/mL Heparin
• 1x PenStrep

NZC Differentiation Media

CHIR99021 Media - 1 hour

• 1x APEL2
• 8uM CHIR99021

FGF9 Media - 5 days

• 1x APEL2
• 200ng/mL FGF9

No Growth Factor Media

• 1x APEL

Isolation of nephrogenic zone cells (NZCs) from developing kidneys

1. Dissect the kidneys from E13 to P1 mice in a 10 cm dish containing a sufficient volume of PBS without calcium or magnesium to cover the embryos. Completely remove the ureter and kidney capsule to expose the nephrogenic zone and transfer kidneys to the 5ml falcon tube containing PBS using a transfer pipette. Discard ruptured or broken kidneys as they will reduce the purity of the final NPC preparation. Up to 24 kidneys can be placed in one tube.
2. Incubate the tube with kidneys in HBSS on a nutator for 2 minutes to dislodge debris that may still be attached to the kidneys (e.g. red blood cells and remaining capsule fragments).
3. Carefully remove as much of the HBSS as possible and add 2 ml of RT collagenase A/pancreatin enzyme digest solution. Place tube on the nutator at 37 °C for 12-15 minutes. Digestion times can vary greatly depending on the enzyme activities of different lots of collagenase A and pancreatin. Adjust digestion times to obtain 3-7 million NZCs from 20 E17 kidneys.
4. After digestion, remove tube containing kidneys and immediately add 125 μl of FBS to stop enzyme reaction. Invert to mix. Perform steps from here on in a laminar flow cabinet for cell culture or on a sterilized bench with a flame on.
5. Allow kidneys to sink to the bottom of tube and remove any floating particles with a 1 ml micropipette while removing as little of the cell suspension as possible. Floating particles at this stage in the purification are usually capsule fragments that were not removed prior to the digestion.
6. Remove remaining cell suspension with a 1 ml micropipette. There is no need to take all of the solution and leaving 200 μl behind is recommended to avoid unwanted particles at the bottom of the tube. (Do not touch the undigested kidenys).
7. Transfer the cell suspension evenly to two 1.5 ml microfuge tubes and spin in a microfuge at 2,000 rpm (300 g) for 5minutes.
8. Place a 30 μm pre-separation filter on a 15 ml conical tube and wash with 4 ml of autoMACS running buffer. Discard the flow through.
9. Discard supernatant, gently resuspend each cell pellet in 500 μl autoMACS running buffer, then combine cell suspensions into one 1.5 ml microfuge tube.
10. Add cell suspensions to the washed 30 μm pre-separation filter and wash with 500 μl of autoMACS running buffer. Transfer the 1.5 ml of filtered cell suspension to a 1.5 ml microfuge tube.
11. Transfer 10 μl of the cell suspension into a microfuge tube and determine cell count using a hemocytometer.

For Culturing a Mixed population of NZCs in Monolayer

1. Spin cells at 2000rpm/300g for 5 minutes to pellet
2. Resuspend in medium for plating

-Seed the cells between 5,000 and 25,00 cells per square centimeter -Medium volume for various well sizes

• 96 well plate - 200uL
• 24 well plate - 1mL
• 6 well plate - 2mL

1. Immediately prior to plating, remove Matrigell from the culture plate wells and immediately add the desired volument of cell suspension in NZC propagation media to each well. Agitate the plate to spread cells evenly.
2. Change the media every 48 hours.
3. Follow “Cell passaging” from [[N-H9AM]] to passage NZCs

For Culturing a Mixed Population of NZCs in an aggregate

1. Spin cells at 2000rpm/300g for 5 minutes to pellet
2. Prepare 1mL of CHIR pulse differentiation media for each 24 well. Using sterile technique, float a Nucleopore membrane in each 24 well.

*Alternatively, a Corning Transwell 0.4um pore polycarbonate membrane cell culture insert (Sigma, CLS3413) can be used.

1. Resuspend cells in 250,000 cells/uL autoMACS running buffer
2. Carefully add 2uL of the cell suspension to the top of each filter.
3. Change media to FGF9 differentiation media after 1 hour
4. Change media every 48 hours.
5. Change media to no growth factor media after 5 days.
6. Change media every 48 hours until ready to assay aggregate.

*Aggregates begin to die in the center around day 15.

References

Brown, A. C., Muthukrishnan, S. D., & Oxburgh, L. (2015). A synthetic niche for nephron progenitor cells. Developmental cell, 34(2), 229–241. https://doi.org/10.1016/j.devcel.2015.06.021

Takasato, M., Er, P. X., Chiu, H. S., Maier, B., Baillie, G. J., Ferguson, C., Parton, R. G., Wolvetang, E. J., Roost, M. S., Chuva de Sousa Lopes, S. M., & Little, M. H. (2015). Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature, 526(7574), 564–568. https://doi.org/10.1038/nature15695

Consortium

(Re)Building a Kidney (RBK) Consortium