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How to Generate hPSC-Derived 3D Kidney Organoids Using STEMdiff™

3 Parts 15 Materials Print

The STEMdiff™ Kidney Organoid Kit provides a simple workflow for the differentiation of kidney organoids from a 2D monolayer of human pluripotent stem cells using 96-well plates or other standard cell culture plate formats. While this approach is compatible with high-throughput imaging and assays, it limits the ability to culture organoids in suspension, e.g. to facilitate cyst formation in polycystic kidney disease (PKD) studies or to embed them in matrices for vascularization studies.

This protocol addresses these limitations by modifying the original STEMdiff™ Kidney Organoid Kit workflow to generate 3D organoids. The procedure begins by differentiating hPSCs for seven days in 2D to reach the nephron progenitor stage. The cells are then dissociated, re-aggregated, and further differentiated for 11 days into kidney organoids of uniform size and number using AggreWell™ Microculture Plates.

3D kidney organoids can be generated in bulk using formats such as the AggreWell™ 800, yielding 5,900 organoids per six-well plate, or as smaller replicates using the AggreWell™ HT, producing 32 organoids per 96-well plate.

A flow chart of the cell culture timeline for the differentiation stages of the hPSC-derived kidney organoids and representative brightfield microscopy images of the kidney organoids.

Figure 1. Workflow Schematic for Generation of 3D Human Kidney Organoid Cultures Using the STEMdiff™ Kidney Organoid Kit

hPSCs previously maintained in mTeSR™1 are plated into Corning® Matrigel®-coated plates on Day -3. After 24 hours (Day -2), adherent cells are overlaid with an additional layer of Matrigel®, which results in the formation of cavitated PSC spheroids within the next 48 hours. On the following day (Day 0), differentiation of cavitated PSC spheroids is initiated by changing the medium from mTeSR™1 to STEMdiff™ Kidney Organoid Kit. During the next 18 days, the cells are directed through to the late primitive streak, followed by reaggregation using AggreWell™ microwell plates to give rise to 3D kidney organoids.

Navigation: Materials Part I: Seeding hPSCs As Single Cells and Formation of Cavitated Spheroids Part II: Kidney Organoid Differentiation Stage 1 Part III: Kidney Organoid Differentiation Stage 2

Materials


Part I: Seeding hPSCs As Single Cells and Formation of Cavitated Spheroids

The protocol is initiated from single-cell seeding of high-quality hPSCs from a 6-well plate to a 96-well plate (Day -3), followed by the formation of cavitated PSC spheroids (Day -2 to Day 0), which become visible on Day -1.

Day -3

  1. Starting with high-quality hPSCs in a 6-well maintenance plate, use a microscope (4X magnification) to visually identify regions of differentiation in the hPSC culture. Mark these with a felt tip or lens marker on the bottom of the plate.
  2. Remove regions of differentiation by scraping with a pipette tip or aspiration. Wash with 2 mL/well of D-PBS (Without Ca++ and Mg++).
    Note: It is critical that the cells are of high quality (less than 5% differentiation). Removal of differentiated cells will result in better organoid formation efficiency.
  3. Aspirate the D-PBS and add 0.5 mL ACCUTASE™ per well. Incubate at 37°C for 10 minutes.
  4. During incubation, add 2 mL of room temperature DMEM/F-12 per well harvested to a 50 mL conical tube.
  5. After incubation, use a 1 mL pipettor to transfer cells into the tube containing DMEM/F-12.
  6. Break up the colonies into single cells by pipetting up and down 3 - 4 times.
  7. Centrifuge the cells at 300 x g for 5 minutes.
  8. Aspirate the supernatant and resuspend the cells in 1 mL mTeSR™1 + 1:10 diluted CloneR™2 per well harvested.
  9. Filter the cell suspension through a 37 μm Reversible Strainer and retain the flow-through.
  10. Count viable single cells in the flow-through using a Nucleocounter® or hemocytometer.
  11. Add 2 mL/well of mTeSR™1 + 1:10 diluted CloneR™2, then directly seed 30,000 cells into each well of a Matrigel®-coated 6-well plate (for coating instructions, please refer to the ). Ensure cells are evenly distributed by sliding the plate back and forth several times on the biosafety cabinet surface in a cross shape.
    Note: An initial experiment is recommended to determine the optimal single cell seeding density for the cell line being used.
  12. Transfer the plate and incubate at 37°C and 5% CO2 with 95% humidity overnight.

    13. Day -2 (hPSCs to Cavitated Spheroids)

  13. Perform a full medium change to overlay cells with Matrigel® as follows:
    1. Thaw one aliquot of Matrigel® on ice.
    2. Add Matrigel® to cold (2 - 8 °C) mTeSR™1 (without CloneR™2) to give a protein concentration of 0.25 mg/mL.
    3. Immediately remove medium from wells, then add 2 mL cold mTeSR™1 + Matrigel® per well (25 µg protein/well).
    4. Incubate at 37°C for 24 hours.

    14. Figure 2. At 24 Hours Post-Seeding, Cells Begin to Form Small Separate Colonies

    Morphology of (A, B) human embryonic stem cells (hESCs; H9 line) and (C,D) human induced pluripotent stem cells (hiPSCs; WLS-1C line) on Day -2 at the start of the kidney organoid differentiation protocol. Both cell lines were seeded into a 6-well plate at 3 x 104 cells/well using mTeSR™1. Scale bars = 200 µm.

    Day -1

  14. Perform a full medium change with 2 mL/well of mTeSR™1 (room temperature). Incubate at 37°C for 1 day.
    Note: At Day 0, hPSC colonies should begin to form round, cavitated hPSC spheroids of 50 - 100 µm in size. See Figure 3 for representative images.

Part II: Kidney Organoid Differentiation Stage 1

Note: After initiation of differentiation (Day 0, Steps 1 - 2 below), an incubation period of 36 hours is optimal (up to 40 hours can be acceptable). It is recommended to perform the following steps at the end of Day 0, based on when you would like to reach the 36- to 40-hour timepoint.

Day 0 (Initiate Kidney Organoid Differentiation)

  1. Use sterile technique to prepare Stage 1 Medium (STEMdiff™ Kidney Basal Medium + STEMdiff™ Kidney Supplement SG). For detailed instructions, please refer to the .
  2. Carefully remove medium from each well of the 6-well plate. Add 200 µL of Stage 1 Medium (room temperature) to each well. Incubate at 37°C for 36 - 40 hours.

Figure 3. Day 0 Colonies in Matrigel Formed Small Cavitated Spheroids Prior to Stage 1 of Kidney Organoid Differentiation

Morphology of (A, B) hESCs (H9 line) and (C, D) hiPSCs (WLS-1C line), cultured in mTeSR™1, on Day 0 following successful formation of cavitated spheroids. Scale bars = 200 µm.


Part III: Kidney Organoid Differentiation Stage 2

Day 1.5 (36 Hours After Initiation of Differentiation)

  1. Use sterile technique to prepare Stage 2 Medium (STEMdiff™ Kidney Basal Medium + STEMdiff™ Kidney Supplement DM). For detailed instructions, please refer to the .
  2. Perform a full medium change with 200 µL/well of Stage 2 Medium (room temperature). Incubate at 37°C for 2 days.

    3. Figure 4. Morphology of hPSCs at Day 1.5 Following the Induction of the Late Primitive Streak Phase During Kidney Organoid Differentiation

    Morphology of (A, B) hESCs (H9 line) and (C, D) hiPSCs (WLS-1C line) on Day 1.5 during Stage 1 differentiation where the late primitive streak is induced. Cells will undergo a significant amount of cell death, which results in partial cell detachment and reduced colony sizes. Scale bars = 200 µm.

    Day 4

  3. Carefully perform a full medium change with 100 µL/well of Stage 2 Medium (room temperature). Incubate at 37°C for 3 days.

    4. Day 7 (Reaggregation)

  4. Prepare AggreWell™HT plate by rinsing with 200 µL/well Anti-Adherence Rinse Solution (AARS, ϳԹ, Catalog #07010). Perform 2 washes with D-PBS (Without Ca++ and Mg++) to remove excess AARS.
    Note: If a larger number of organoids per well is desired, differentiations can also be performed in AggreWell™800 plates.
  5. Wash each well gently with 2 mL/well room temperature D-PBS (Without Ca++ and Mg++).
  6. Aspirate D-PBS and add 0.5 mL room temperature ACCUTASE™ per well. Incubate at 37°C for 10 minutes.
  7. Add 1 mL room temperature Stage 2 Medium per harvested well to a 15 mL conical tube.
  8. After incubation, use a 1 mL pipettor to rinse cells off the plate and transfer them into the tube containing DMEM/F-12.
  9. Break up clumps into single cells by pipetting up and down 3 - 4 times.
  10. Centrifuge cells at 300 x g for 5 minutes.
  11. Aspirate supernatant and resuspend in fresh 1 mL Stage 2 Medium per well harvested.
  12. Filter cell suspension through a 37 μm Reversible Strainer and retain the flow-through.
  13. Count viable single cells in the flow-through using a Nucleocounter® or hemocytometer.
  14. Seed 5000 viable cells per microwell, e.g. 1.6 x 105 live cells per well of an AggreWell™HT plate or 1.5 x 106 cells per well of an AggreWell™800 24-well plate (5000 cells per microwell), then top up each well with Stage 2 medium to 200 µL or 2 mL, respectively.
  15. Triturate the cell suspension in the wells 2 times without creating bubbles to ensure even cell distribution. Let the cells settle by gravity at room temperature for 10 - 15 minutes without moving the plate, then transfer the plate into the 37°C incubator.

    16. Figure 5. Morphology of hPSC-Derived Monolayers at Day 7 Prior to Harvesting for Reaggregation into AggreWell™

    Morphology of (A, B) hESCs (H9 line) and (C, D) hiPSCs (WLS-1C line) on Day 7 prior to harvesting for reaggregation. Emergence of early organoid structures may be observed. Scale bars = 200 µm.

    Day 7 - 18

  16. Every 2 - 3 days, until Day 18, perform a 50% medium change, e.g. 100 µL/well of (room temperature) Stage 2 Medium for the AggreWell™HT plate or 1 mL/well for the AggreWell™800 24-well plate. Incubate at 37°C.
    Note: Medium changes are best performed with a liquid handling robot or an automatic pipettor.
    Note: Dark tubular structures should become visible between Day 8 and 10.

    17. Figure 6. Morphology of hPSC-Derived 3D Kidney Organoids at Day 18 Demonstrate a Spherical Shape with a Convoluted Tubular Interior

    Brightfield images of (A, B) hESC- (H9 line) and (C, D) hiPSC- (WLS-1C line) derived 3D kidney organoids at Day 18. The organoids were harvested from an AggreWell™HT microwell plate into a flat-bottom 96-well plate for imaging. Scale bars = 200 µm.

    Figure 7. 3D Kidney Organoids at Day 18 Demonstrate Presence of Podocytes, Proximal Tubules, and Distal Tubules

    Immunofluorescent images of (A) hESC- (H9 line) and (B) hiPSC- (WLS-1C line) derived 3D kidney organoids at Day 18. The organoids demonstrate markers for podocytes (red), proximal tubules (green), distal tubules (white), and nuclei (blue). Scale bars = 200 µm.

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  • Document #PR00098
  • Version 1.0.0
  • April 2025
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