A Commercially Available Kit to Generate Human Kidney 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
Benjamin S. Freedman
Keywords
[‘organoid’, ‘stem cell differentiation’, ‘hiPSC’]
Subjects
[‘Cell culture’]
Release Date
2021-09-20
Abstract
The STEMdiff™ Kidney Organoid Kit is a complete, serum-free cell culture medium system that supports highly efficient and reproducible generation of human pluripotent stem cell (hPSC)-derived kidney organoids in a simple, two-stage differentiation protocol, based on Freedman et al., 2015. Kidney organoids are composed of podocytes, proximal tubules, distal tubules, endothelium, mesenchyme, and off-target cells. Kidney organoids generated with STEMdiff™ Kidney Organoid Kit grow in three-dimensional aggregates on adherent culture plates (24, 96, and 384-well) that are convenient for immunofluorescence analysis. They are tested for compatibility with phenotypic high-throughput assays such as nephrotoxic compound screening. Compared to the original protocol, the commercially available kit comprises only three reagents, which reduces complexity. Each batch undergoes quality control validation for organoid differentiation, to reduce variability and the need for troubleshooting. For certain applications, for instance to test custom media formulations, it may be desirable to use the original protocol rather than the commercially available kit. In addition to the manual for the commercially available kit, a book chapter describing the detailed method, as well as the original paper describing both method and applications, are appended here.
Introduction
There is substantial need for reproducibility, simplicity, and user friendliness in generating kidney organoids. The well-established Freedman et al. protocol for nephron organoid differentiation has been recently optimized and adapted for commercial production in the form of an organoid differentiation kit. The manufacturer, STEMCELL Technologies, is well-known in the field for high quality reagents. The kit provides a ready entry point for investigators interested in growing kidney organoids.
Reagents
The kit consists of the following reagents: COMPONENT # COMPONENT NAME SIZE 05161 STEMdiff™ Kidney Basal Medium 100 mL 05162 STEMdiff™ Kidney Supplement SG (100X) 200 µL 05163 STEMdiff™ Kidney Supplement DM (50X) 1.6 mL
Sold separately are the following:
- Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free (Corning 356231)
- Standard reagents for feeder-free human pluripotent stem cell culture (mTeSR1 system) and passaging as single cells (Accutase).
Equipment
No specialized equipment is needed. The organoids can be stained in whole mount form and visualized on standard immunofluorescence microscopes without the need for clearing or sectioning. The use of a multi-channel pipettor (20 - 200 µL) (e.g. Catalog #38064) and reagent reservoirs (e.g. Catalog #38080) may facilitate workflows.
Recommended standard equipment include:
• Biosafety cabinet certified for Level II handling of biological materials • Incubator with humidity and gas control to maintain 37°C and 95% humidity in an atmosphere of 5% CO2 in air • Low-speed centrifuge with a swinging bucket rotor • Pipette-aid with appropriate serological pipettes (e.g. Catalog #38002) • Hemocytometer or Nucleocounter® • Pipettor with appropriate tips (e.g. Catalog #38058) • Inverted microscope • -20°C freezer • Refrigerator (2 - 8°C)
Procedure
The product manual, attached, outlines clearly the specific steps involved in generating kidney organoids using this method, and should be referred to for the detailed protocol. Essential steps include:
- Pre-coating plates with Matrigel (Day-4 or earlier)
- Seeding dissociated human pluripotent stem cells onto these plates at specific cell densities in the mTeSR1 + ROCK inhibitor (Day -3)
- Sandwiching these cells with a thin layer of Matrigel to form epiblast spheroids (Day -2 to Day -1)
- Inducing differentiation with Stage 1 Medium (STEMdiff™ Kidney Basal Medium + STEMdiff™ Kidney Supplement SG) for 36 hours (Day 0 to Day 1.5)
- Changing to Stage 2 Medium (STEMdiff™ Kidney Basal Medium + STEMdiff™ Kidney Supplement DM) and changing the media thereafter every 2-3 days (Day 1.5 to Day 18)
Stages of organoid differentiation (mesendoderm –> renal vesicle –> nephron) should be monitored daily and are helpful in predicting whether the protocol is working. An immunofluorescence protocol is included in the manual. We recommend the fixation process described in the methods chapter.
Timing
In total the procedure takes approximately 21 days.
Critical_Steps
The most critical steps in the protocol are:
- initial plating of the stem cells on Day -3. It is recommended to try a few different cell densities e.g. 1000, 3000, and 5000 single cells per well.
- exchange of media on Day 1.5. The cells are delicate at this stage. Aspiration and feeding should be done gently and rapidly.
Trouble_Shooting
It is recommended to confer with the manufacturer for troubleshooting. A troubleshooting guide is also included in the manual. Most issues can be dealt with by adjusting cell seeding practices. As every cell line is different, it is recommended to use as a positive control iPS or ES cell lines that are known to be capable of differentiating into organoids, such as:
- WA09 (WiCell)
- GM25256 (Coriell)
Anticipated_Results
Translucent, tubular structures should first become apparent by phase contrast microscopy by approximately Day 12.
Immunofluorescence can be used to verify organoid differentiation based on the appearance of podocytes, proximal tubules, and distal tubules in a proximal-to-distal arrangement. A recommended list of immunofluorescence markers can be found in the attached protocols and papers.
References
Freedman BS, Brooks CR, Lam AQ, Fu H, Morizane R, Agrawal V, et al (2015). Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids. Nat. Commun., doi: 10.1038/ncomms9715.
Cruz NM and Freedman BS. Differentiation of Human Kidney Organoids from Pluripotent Stem Cells (2019). Thomas Weimbs (Ed.), Methods in Cell Biology 153:133-150.
Generation of Human Kidney Organoids Using STEMdiff™ Kidney Organoid Kit (Technical Manual). STEMCELL Technologies, 2020.
Associated_Publications
Representative publications include:
Cruz NM, Song X, Czerniecki SM, Gulieva RE, Churchill AJ, Kim YK, Winston K, Diaz M, Fu H, Finn LS, Pei Y, Himmelfarb J, Freedman BS (2017). Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease. Nature Materials 16:1112–1119.
Kim YK, Refaeli I, Brooks CR, Jing P, Gulieva RE, Hughes MR, Cruz NM, Liu Y, Churchill AJ, Wang Y, Fu H, Pippin JW, Lin LY, Shankland SJ, Vogl AW, McNagny KM, Freedman BS† (2017). Gene-edited human kidney organoids reveal mechanisms of disease in podocyte development. Stem Cells 35:12, 2366-2378.
Czerniecki SM, Cruz NM, Harder JL, Menon R, Annis J, Otto EA, Gulieva RE, Islas LV, Kim YK, Tran LM, Martins TJ, Pippin JW, Fu H, Kretzler M, Shankland SJ, Himmelfarb J, Moon RT, Paragas N, Freedman BS† (2018). High-throughput screening enhances kidney organoid differentiation from human pluripotent stem cells and enables automated multidimensional phenotyping. Cell Stem Cell 22:929-940.
Harder JL, Menon R, Otto EA, Zhou J, Eddy S, Wys NL, O’Connor C, Luo J, Nair V, Cebrian C, Spence JR, Bitzer M, Troyanskaya OG, Hodgin JB, Wiggins RC, Freedman BS, Kretzler M, European Renal cDNA Bank (ERCB), Nephrotic Syndrome Study Network (NEPTUNE). Organoid single-cell profiling identifies a transcriptional signature of glomerular disease (2019). JCI Insight 4(1):e122697.
Nam SA, Kim JW, Kim HW, Kim HL, Kim K, Kim TM, Ju JH, Gomez IG, Uchimura K, Humphreys BD, Yang CW, Lee JY, Kim J, Cho DW,° Freedman BS,° and Kim YK.° Graft immaturity and safety concerns in transplanted human kidney organoids (2019). Exp. Mol. Medicine, 51:145.
Liu E, Radmanesh B, Chung BH, Donnan MD, Yi D, Dadi A, Smith KD, Himmelfarb J, Li M, Freedman BS, ° Lin J °. Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics (2020). Kidney360 1:203-215.
Acknowledgement
We thank UC2DK126006 for support in preparing this protocol. Additional support is listed in the attached publications.
Consortium
(Re)Building a Kidney (RBK) Consortium
