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Isolation and culture of CITED1+ nephron progenitor cells (Version 1.0) | ATLAS-D2K Center

PLEASE NOTE: ATLAS-D2K closed July 31, 2025 and this website is for reference purposes only.

Isolation and culture of CITED1+ nephron progenitor cells (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

Leif Oxburgh

Keywords

[‘nephron progenitor’, ‘mouse’]

Subjects

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

Release Date

2017-02-08

Abstract

Abbreviations

  • FBS – Fetal Bovine Serum
  • NPC – Nephron Progenitor Cell
  • NPEM – Nephron Progenitor Expansion Medium NZC – Nephrogenic Zone Cell
  • RT – Room Temperature

Reagents

  • Collagenase A (Roche, 11 088 793 001)
  • Pancreatin (porcine) (Sigma, P1625)
  • Matrigel (Corning, 354277)
  • HBSS without calcium or magnesium (Life Technologies, 14175-095)
  • PBS without calcium or magnesium (Lonza, 17-513F)
  • FBS (Biowest, S1620)
  • AutoMACS running buffer (Miltenyi, 130-091-221)
  • Pre-Separation Filters (30 μm) (Miltenyi, 130-041-407)
  • anti-CD105-PE (Miltenyi, 130-102-548)
  • anti-CD140-PE (Miltenyi, 130-102-502)
  • anti-Ter119-PE (Miltenyi, 130-102-893)
  • anti-CD326-PE (Miltenyi, 130-102-265)
  • anti-PE microbeads (Miltenyi, 130-048-801)
  • APEL Medium (Stemcell Technologies, 05210)
  • Human recombinant FGF9 (R&D, 273-F9-025)
  • Human recombinant BMP7 (R&D, 354-BP-010)
  • Human recombinant BMP4 (R&D, 314-BP-010)
  • Human recombinant IGF1 (R&D, 791-MG-050)
  • Mouse recombinant IGF2 (R&D, 792-MG-050)
  • CHIR99021 (Stemgent, 04-0004)
  • LDN-193189 (Stemgent, 04-0074-02)
  • Y27632 (Millipore, 688001)
  • Heparin (Sigma, H3393)
  • TrypLETM dissociation solution (Life Technologies, 12563029)

Equipment

  • Nutator placed at Room Temperature (RT)
  • Nutator placed at 37°C
  • AutoMACS Pro Separator (Miltenyi, 130-092-545)
  • 96, 24 or 6 well culture plates (Corning, 3595, 3524, 3516)
  • Transfer Pipette (Corning, 357575)

Procedure

Preparation of reagents prior to isolation

  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. 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 and 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 laminar flow cabinet for cell culture for at least 1 hour.
  3. Prepare round bottomed 5 ml tubes to receive dissected kidneys by filling them with 2 ml HBSS without magnesium or calcium.
  4. Transfer 50 ml of autoMACS running buffer to a 50 ml conical tube and warm to RT.
  5. Start autoMACS instrument: check solutions, install autoMACs running buffer bottle, run clean program.
  6. Make appropriate volume of NPEM in a 50 ml conical tube.

Nephron progenitor expansion medium (NPEM)

tablefactorconcentrations.png{width=800 height=auto}

Figure1: Factor concentrations

Make NPEM in a laminar flow cabinet for cell culture and thoroughly mix by inverting several times.

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 HBSS-containing tube using a transfer pipette with an orifice cut to at least twice the diameter of the kidneys to be transferred. 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. Remove the HBSS and add another 2 ml of HBSS to wash. Be careful not to damage the kidneys with pipette tip.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  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. 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.
  11. 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.
  12. Transfer 10 μl of the cell suspension into a microfuge tube containing 90 μl autoMACS running buffer and determine cell count using a hemocytometer. 13a. For a mixed population of NZCs: Spin cells at 300g for 5 minutes to pellet and resuspend in medium for plating. 13b. For NPC purification proceed to Step 1 in Section 1.6.

NPC purification from a mixed population of NZCs

This section describes a magnetic depletion protocol based on antibody labeling and cell capture using a Miltenyi Biotec autoMACS Pro Separator. For separations using manual columns it is recommended to consult Miltenyi Biotec for protocol modifications.

  1. For each 10 million NZCs, gently resuspend cell pellet to a total volume of 76 ul (taking into consideration the volume of pellet) with autoMACS running buffer in a 1.5 ml microfuge tube and add the following antibodies for a total volume of 110 μl: a. 9 μl of anti-CD105-PE b. 9 μl of anti-CD140-PE c. 8 μl of anti-Ter119-PE d. 8 μl of anti-CD326-PE
  2. Triturate cell/antibody suspension gently with a 200 μl pipettor and incubate at 4°C for 13 minutes without agitation.
  3. Add 1 ml autoMACS running buffer and triturate gently 3 times. Centrifuge sample at 300g for 5 minutes.
  4. Discard supernatant and gently resuspend pellet in 1 ml autoMACS running buffer. Centrifuge sample at 300 g for 5 minutes.
  5. Discard supernatant and gently resuspend the pellet in a total volume of 80 μl autoMACS running buffer (taking into consideration the volume of pellet).
  6. Add 20 μl of anti-PE microbeads to cell suspension. Gently mix by triturating several times and incubate at 4°C for 18 minutes without agitation.
  7. Add 1 ml autoMACS running buffer and triturate gently 3 times. Centrifuge sample at 300 g for 5 minutes.
  8. Discard supernatant and resuspend cell pellet in 1 ml autoMACS running buffer. Centrifuge again at 300 g for 5 minutes.
  9. During centrifugation, label five 15 ml conical tubes: Input, Pos1, Neg1, Neg2, and Neg3. Three sequential autoMACs Pro Separator runs must be performed in order to obtain the purified CITED1+ cell population (see Figure 2).

conicaltubes.png

Figure2: Conical tubes

AutoMACs run 1:

  1. Discard supernatant and resuspend the pellet in 500 μl autoMACS buffer and transfer to the 15 ml conical tube labeled “input”.
  2. Place “input” tube into position 1 of the autoMACS Pro Separator, and Neg1 and Pos1 in positions 2 and 3.
  3. Start separation using the Deplete(s) program and run a Qrinse (quick rinse) at the end of each separation. The NPCs will be collected as the negative fractions. AutoMACs run 2:
  4. After the first separation place the Pos1 tube with positive fraction in the inlet position with Neg2 in position 2 and an empty tube in position 3 and rerun the Deplete(s) program. In this step the positive fraction from the first separation will be run again to collect many non-labeled NPCs that are still present in the positive fraction. This step can increase the yield by up to 50%. Any remaining positive fractions can be discarded.

AutoMACs run 3:

  1. Combine the Neg1 and Neg2 fractions that contain the NPCs into the Neg1 labeled conical tube and place this tube as the input (position 1). Place the Neg3 tube in position 2 and an empty tube in position 3. Rerun the Deplete(s) program to further enrich the NPC fraction to near 100 % CITED1-expressing cells, which will be deposited in the Neg3 tube.
  2. Spin the Neg3 fraction at 300 g for 5 minutes to pellet the purified NPCs.
  3. Remove the supernatant and resuspend the NPCs in 1 ml of NPEM. Use 10 μl of the suspension for a cell count. The average yield of NPCs recovered from 5 million NZCs is about 1.2 million in our hands.

Cell plating and culture (when plating freshly harvested cells use 25,000/cm2)

  1. Suggested cell seeding density is between 5,000 and 25,000 cells per cm2. Increase the volume of NPC suspension with NPEM such that the desired cell density per ml is achieved for plating.

Note: To prevent clustering of cells near the edge of the well, which will result in earlier cellular overgrowth and the need for more frequent passaging, it is highly recommended to initially plate the cells in a high volume of NPEM. This will reduce the meniscus effect, which results in a higher cell density at the edge of the well due to the greater vertical column height of cells in suspension.

meniscuseffect.jpg

Figure3: The meniscus effect at different culture medium volumes in a 24 well plate. The ratio of the meniscus height (yellow) to total vertical height (red) at well edge decreases at higher volumes.

The following volumes can be used as a guide: Medium volume for various well sizes: 96 well plate – 200 μl 24 well plate – 2 ml 6 well plate – 4 ml

  1. Immediately prior to plating, remove Matrigel from the culture plate wells using a vacuum aspirator connected to a sterile pipette tip and immediately add the desired volume of cell suspension in NPEM to each well. Agitate the plate to spread cells evenly.
  2. Several hours after plating when cells have adhered to the culture plate, excess NPEM can be removed and stored at 4 °C for several days to be used for the next passage. Prior to NPEM storage, pellet any dead cells by centrifugation at 300 g and transfer the supernatant to a new tube while leaving pelleted cells or cellular debris behind. For example, cells are plated a 2 ml volume in a 24 well culture plate and 1.5 ml of NPEM is removed after 2 hours.
  3. Change 100% of the medium every 48 hours.

Cell passaging

  1. When the cells have reached 70-80% confluence at the areas of the well with the highest cell density they must be passaged for further expansion. The highest cell densities usually occur at the edge of the well, but sometimes can occur in the middle of the well. If cells are allowed to become too dense, they will substantially lose the potential for multiple passages. Figure 4 can be used as a guide to show the optimal density that cells should be allowed to grow to before passage.

optimalpassagedensity.jpg

Figure4: Optimal passage density

  1. To passage, warm autoMACS running buffer to RT and TrypLE dissociation solution to 37 °C.
  2. Aspirate NPEM and add pre-warmed TrypLE solution in the following amounts based upon well size. 96 well – 50 μl 24 well – 250 μl 6 well – 500 μl
  3. Place culture plate in a 37 °C incubator for 2 minutes and then check that the cells have started to dislodge from the plate surface under a light microscope. If not, extend the treatment in 1 minute increments until cell detachment becomes noticeable.
  4. Add the following volumes of autoMACS running buffer to each well and wash cells from plate by triturating gently. 96 well – 200 μl 24 well – 1000 μl 6 well – 2000 μl
  5. Transfer the cells to an appropriate tube and spin at 300 g for 5 minutes to pellet the cells.
  6. Remove the supernatant and resuspend the CITED1+ progenitors in 1 ml of NPEM. Perform a cell count on a 1:10 dilution of cells.
  7. Increase the volume of NPEM and plate cells at the desired density on Matrigel coated culture plates (see “Cell plating and culture” above).

Cryopreserving CITED1+ progenitors

  1. NPCs can be frozen in cryopreservation medium consisting of 10% DMSO, 40% FBS and 50% DMEM either immediately after isolation (uncultured) or following propagation and passaging in NPEM. 100,000 to 1 million cells per vial is recommended.
  2. Pellet uncultured cells or TrypLE dissociated cells that have been diluted in autoMACS running buffer by centrifugation at 300g and remove supernatant. If cryopreserving cultured cells, wash one additional time in autoMACS running buffer prior to adding freeze medium to ensure that all TrypLE has been removed. Cells have been successfully cryopreserved from 80% confluent cultures in 24 well and 6 well plates up to passage 5, with subsequent expansion in NPEM.
  3. Resuspend cell pellet in an appropriate volume of chilled cryopreservation medium depending on the number of cells per vial needed and transfer cell suspension to screw cap cryovials.
  4. Place cryovial containing cells in a Styrofoam tube holder with cover at -70 °C overnight to slowly freeze the cells. The next day transfer frozen cells to liquid nitrogen for long term storage.

Thawing CITED1 progenitors

  1. Thaw cells rapidly in a 37 °C water bath by swirling until just a small sliver of ice is left in the cryovial. This takes approximately 90-120 seconds.
  2. Triturate once with 1 ml micropipette and transfer to a 15 ml conical tube. Slowly drip 2 ml RT autoMACS running buffer into the thawed cell suspension. After 2 ml has been added by dripping, add additional autoMACS running buffer to 10 ml.
  3. Spin cell suspension at 300 g for 5 minutes to pellet cells.
  4. Remove the majority of autoMACS running buffer (leave 50 μl behind to avoid disrupting the pellet) and wash pellet with 1 ml of NPEM by triturating gently several times.
  5. Transfer cells to a 1.5 ml microfuge tube and spin in a microfuge for 5 minutes at 300 g.
  6. Discard NPEM and resuspend cell pellet in the appropriate volume of fresh NPEM for plating (see “Cell plating and culture” above). It is important that cells do not clump as they will then cluster, causing focal overgrowth.
  7. Plate cells on Matrigel-coated culture plates as described in “Cell plating and culture” above.
  8. We typically observe greater than 90 % recovery of viable cells after thawing.

References

  1. Brown et al., 2015, Developmental Cell 34, 229–24

Associated_Publications

Long-Term Culture of Nephron Progenitor Cells Ex Vivo. Brown, Aaron C.; Gupta, Ashwani K.; Oxburgh, Leif. Methods Mol Biol. vol. 1926, 63–75. 2019.

Consortium

(Re)Building a Kidney (RBK) Consortium