qPCR assay to evaluate nephron repair/failed repair using MACS-purification of tubular cells from cisplatin-injured organoids (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

Veronika Sander; Alan Davidson

Keywords

[‘Cell dissociation’, ‘cell sorting’, ‘culture’, ‘gene expression analysis’, ‘hiPSC’, ‘human’, ‘induced pluripotent stem cells (iPSC)’, ‘isolation’, ‘kidney’, ‘kidney injury’, ‘MACS cell separation’, ‘organoid’, ‘proximal tubule epithelial cells’, ‘RNA isolation’]

Subjects

[‘Cell biology’, ‘Cell culture’, ‘Gene expression analysis’, ‘Isolation, purification and separation’, ‘Toxicology’]

Release Date

2022-05-11

Abstract

This protocol describes Magnetic-activated cell sorting (MACS) of tubular epithelial cells from hPSC-derived kidney organoids. Magnetic beads conjugated with Epithelial Cell Adhesion Molecule (EPCAM) are used for the gentle yet highly efficient isolation of epithelial cells. The purified EPCAM-positive cell population contains cells of proximal and distal tubule identity, whereas the EPCAM-negative population (the ‘flow-through’) is composed of non-epithelial cell types, including endothelial cells, stromal cells and podocytes. qPCR analysis of the tubular cells can be performed to inform on changes of repair / failed repair marker expression after organoid injury. The EPCAM+ cells can also be cultured, e.g. in Renal Epithelial Growth Medium, as primary hPSC-derived renal epithelial cells.

Reagents

• DPBS 1x, no calcium, no magnesium, Thermo Fisher, Cat#14190250
• Ethylenediaminetetraacetic acid (EDTA), Sigma, Cat#E6758
• AlbumiNZ™ protease reduced, immunoassay (EIA) grade, ≥97% (BSA), MP Biomedicals, Cat#219989880
• TrypLE Express Enzyme (1X), Phenol Red, Thermo Fisher, Cat#12605010
• Easystrainer 40 µM, for 50 mL tubes, Greiner Bio-one, Cat#542040
• Easystrainer 100 µM, for 50 mL tubes, Greiner Bio-one, Cat#542000
• CD326 (EpCAM) MicroBeads, human, Miltenyi Biotec, Cat#130-061-101
• MS Separation columns, Miltenyi Biotec, Cat#130-042-201
• Lonza™ Renal Epithelial Growth Medium (REGM™) Bulletkit™, Lonza, Cat#CC-3190
• ROCK inhibitor Y-27632, STEMCELL Technologies, Cat#72304

Equipment

• MiniMACS Separator - Magnet, Miltenyi Biotec, Cat#130-042-102
• MACS® MultiStand, Miltenyi Biotec, Cat#130-042-303

Procedure

Before you start Prepare MACS buffer (50 ml): • 46.7 mL PBS • 200 μL 0.5 M EDTA • 3.3 mL of 7.5% BSA (in PBS) Store for up to 4 weeks at 4°C and keep on ice during the protocol.

MACS protocol

1. Place a minimum of 50 whole organoids in a 15 mL conical tube, wash once with 2 mL PBS
2. Remove PBS, then add 1-2 mL TrypLE Express for 10 min at 37˚C for dissociation into single cells. Pipet gently at 5 mins to help with the dissociation. Pipet/triturate again after the 10 min.
3. Inactivate the digest by adding 5 mL chilled MACS buffer
4. Sequentially pass the cell solution through 100 μm, 40 μm (optional 20 μm) cell strainers (sitting on 50 mL conical tubes) to remove debris and achieve single cells. Use 1 mL MACS buffer to wash through the strainers.
5. Pellet cells by centrifugation, 300 x g for 10 min at 4°C
6. Discard the supernatant, then resuspend cells in 250 μL of MACS buffer and add 50 μL of EPCAM+ (CD326) microbeads, mix gently but thoroughly by flicking the tube, refrigerate at 4°C for 30 min
7. Wash cells twice with by adding ~1.7 mL of MACS buffer and pellet by centrifugation, 300 x g for 7 min at 4°C, discard supernatant. Note: Perform this centrifugation in a 2 mL tube for better pelleting of the cells.
8. While the cells are centrifuging, place a MS Separation column in a MiniMACS Separator that is placed on a MultiStand
9. Pre-rinse the column with 500 μL MACS buffer
10. After the 2nd centrifugation, resuspend the pelleted cells in 500 μL MACS buffer
11. Load the cells onto the column
12. Optional: Collect flow-through for a negative sample
13. Wash the column 3x with 500 μL of MACS buffer
14. Remove the column from the separator and place into a 1.5 mL collection tube
15. Pipette 1 mL of MACS buffer onto the column then gently push the plunger into the column to elute the cells
16. Pellet cells at 300 x g for 10 min at 4°C
17. Remove the supernatant, then use the cells for RNA isolation (store in Trizol or Lysis buffer of an RNA isolation kit)
18. Alternatively, resuspend the pellet in 1 mL Renal Epithelial Growth Medium (REGM) plus ROCK inhibitor (1:2000 dilution), then plate in a T75 cell culture flask (pre-filled with 9 mL REGM plus ROCKi, and culture at 37˚C. Leave undisturbed over night to allow cells to attach. Change media after two days to remove dead cells.

This protocol was modified from Forbes et al. Patient-iPSC-Derived Kidney Organoids Show Functional Validation of a Ciliopathic Renal Phenotype and Reveal Underlying Pathogenetic Mechanisms. Am J Hum Genetics 102: 816–831, 2018. doi: 10.1016/j.ajhg.2018.03.014. and Phipson et al. Evaluation of variability in human kidney organoids. Nat Methods 16: 79–87, 2019. doi: 10.1038/s41592-018-0253-2.

Timing

2-3 weeks for organoid generation using the Davidson protocol (Refs 1,2)

~10 min for MACS buffer preparation

~2 h for MACS

Critical_Steps

Critical consideration throughout the protocol include:

• gentle handling of the cells, i.e. gentle pipetting, no vortexing
• filtration of the organoid digests to achieve single cells will increase the purity of the isolated cell populations

Trouble_Shooting

• ‘Younger’ organoids, i.e. day 12 - 15 of our protocol, will be easier to dissociate, whereas organoids of day 20+ will contain more matrix that won’t be fully disintegrated after the 10 min TrypLE digest. That is normal and the undigested debris will be filtered out with the 100 and 40 micrometer straining step (step 4).
• Due to the gentle centrifugations at 300 x g, the cell pellets may be fragile and not well formed. If that is the case and it’s difficult to remove the supernatant completely, leave some supernatant on the pellet to avoid cell loss, and continue with a larger volume.
• If more organoids or larger organoids generated with a different protocol are used per sample, it may be necessary to scale up to a larger size MACS column.

Anticipated_Results

To determine the purity of the of the isolated EPCAM- and + cell fractions, specific proximal and distal tubule markers, such as LRP2, CUBN, SLC12A1 etc, as well as non-tubular markers, e.g. endothelial cell marker PECAM1, can be measured by qPCR. Tubular markers should be non detectable or very low in the EPCAM- fraction, and highly expressed in the EPCAM+ fraction, and vice versa for non-tubular markers.

References

1. Przepiorski A, Sander V, Tran T, Hollywood JA, Sorrenson B, Shih J-H, Wolvetang EJ, McMahon AP, Holm TM, Davidson AJ. A Simple Bioreactor-Based Method to Generate Kidney Organoids from Pluripotent Stem Cells. Stem Cell Reports , 2018. doi: 10.1016/j.stemcr.2018.06.018
2. Sander V, Przepiorski A, Crunk AE, Hukriede NA, Holm TM, Davidson AJ. Protocol for Large-Scale Production of Kidney Organoids from Human Pluripotent Stem Cells, STAR protocols 2020
3. Forbes et al. Patient-iPSC-Derived Kidney Organoids Show Functional Validation of a Ciliopathic Renal Phenotype and Reveal Underlying Pathogenetic Mechanisms. Am J Hum Genetics 102: 816–831, 2018. doi: 10.1016/j.ajhg.2018.03.014.
4. Phipson et al. Evaluation of variability in human kidney organoids. Nat Methods 16: 79–87, 2019. doi: 10.1038/s41592-018-0253-2.

Consortium

(Re)Building a Kidney (RBK) Consortium