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Organoids from asynchronously mixed directed differentiation cells (Version 1.0) | ATLAS-D2K Center

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

Organoids from asynchronously mixed directed differentiation 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; Ashwani Kumar Gupta

Keywords

[‘organoid’, ‘nephron progenitor’, ‘nephron’]

Subjects

[‘Cell biology’, ‘Developmental biology’, ‘Tissue culture’]

Release Date

2021-09-21

Abstract

The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including in vitro tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.

Introduction

Here, we provide an efficient method of asynchronous mixing of the kidney progenitors at the air-liquid interface that potentiates nephrogenesis to produce tightly packed nephron epithelia with more tubular clusters in kidney organoids. These kidney organoids were filled with glomerular podocytes, proximal tubules, distal tubules, stromal cells, connecting tubule or collecting ducts. The protocol yields a complex and extensive network of endothelial cells. In addition, proximal tubules in kidney organoids were mature and functional, showing endocytic function confirmed by alexa flour 488 (AF488) labeled dextran uptake. In this protocol, a step by step methodology of asynchronous mixing of kidney progenitors to generate kidney organoids is presented which we recently published elsewhere (Kumar Gupta et al. 2020).

Reagents

    • Geltrex (Thermo fisher Scientific, Cat. # A1413301)
  • DMEM/F12 (Thermo fisher Scientific, Cat. # 11330-032)
  • StemFit (amsbio, Cat. # SFB-500)
  • FGF2 (R&D systems, Cat. # 234-FSE-025)
  • Rock inhibitor Y-27632 (EMD Millipore, Cat. # 688002-1mg)
  • H9 cell line (WiCell, Cat. # WA09)
  • Accutase (STEMCELL Technologies, Cat. # 7920)
  • Advanced RPMI 1640 (Thermo fisher Scientific, Cat. # 12633-012)
  • Glutamax (Thermo fisher Scientific, Cat. # 35050061)
  • CHIR99021 IN SOLUTION (Reprocell, Cat. # 04-0004-10)
  • Activin A (R&D systems, Cat # 338-AC-010)
  • FGF9 (R&D systems, Cat. # 234-F9-025)
  • Heparin, (Sigma-Aldrich, Cat. # H3393-25KU)
  • TrypLe express ((Thermo fisher Scientific, Cat. # 12563029)
  • FBS (Atlanta Biologicals, Cat # S11550)
  • APEL2 (STEMCELL Technologies, Cat. # 5270)
  • PFHM II (Thermo fisher Scientific, Cat. # 12040077)
  • BMP7 (R&D systems, Cat. # 354-BP-010)
  • Reconstitution Buffer 4, BSA/HCl (R&D systems, Cat. # RB04)
  • Dextran, AF 488, 10000 MW (Thermo fisher Scientific, Cat. # D22910)
  • Paraformaldehyde (Sigma-Aldrich, Cat. # P6148-500G)
  • DPBS (Thermo fisher Scientific, Cat. # 14190144)

Equipment

  • 6 well plate (VWR, Cat. # 29442-042)
  • 15 ml falcon tube (VWR, Cat. # 62406-200)
  • 50 ml falcon tube (VWR, Cat. # 21008-940)
  • Strainer 40 micron (VWR, Cat. # 21008-949)
  • Isopore membrane (EMD Millipore, Cat. # VCTP01300)
  • 24 well plate (VWR, Cat. # 29443-952)
  • 96 well Clear Round Bottom Ultra-Low Attachment Microplate (Corning, Cat. # 7007)
  • Refrigerated Benchtop Centrifuge (Beckman Coulter, Type: X-15R)
  • Inverted Phase Contrast microscope (Nikon, Type: Eclipse TS100)
  • CO2 incubator (Thermo fisher Scientific, Type: HERAcell 150i)
  • Sunflower Mini-Shaker (Grant-bio, Type: PS-3D)

Procedure

1. Plating hESCs for directed differentiation 1.1. Dilute the matrix in DMEM/F12 (1:100) and add 1 ml/well into 3 wells each of two 6 well plates. 1.2. Incubate the coated plate, undisturbed at 37°C for 1–2 h for effective coating. 1.3. Prepare 18 ml of prewarmed basic culture medium supplemented with 100 ng/ml FGF2 and 10 µM Rock inhibitor Y-27632. 1.4. Remove the medium from 1 well of the 6 well hESCs culture plate and then wash the cells once with DPBS. After washing, add 1 ml prewarmed cell detachment solution (e.g., Accutase) to detach the cells from the plate. The 2nd well can be used to freeze cells or to propagate cell culture. 1.5. Incubate in a 37°C incubator for 10 min. 1.6. Triturate 3–4 times gently and then transfer the cells into a 15 ml conical tube. 1.7. Adjust the volume to 3 ml with medium. Take 10 µl cell suspension to count cells. 1.8. Spin the cell suspension at 1000 g for 3 min to pellet the cells. 1.9. Discard the supernatant and resuspend the cell pellet in 1 ml of medium and triturate 2 times. 1.10. For the 1st batch of asynchronous mixing, plate 1.7 × 105 hESCs /well in 2 ml medium (Plate 1) and culture at 37°C, 5% CO2. 1.11. For the 2nd batch of asynchronous mixing, plate 0.45 × 105 hESCs/well in 2 ml medium in a 6 well plate (Plate 2) and culture at 37°C, 5% CO2. 1.12. Change the medium of both plates after 48 h with fresh basic culture medium supplemented with 50 ng/ml FGF2 without Rock inhibitor Y-27632. 1.13. After 72 h, ensure that cells in Plate 1 are ~50% confluent. This is the correct time to start the first directed differentiation. 1.14. Ninety-six hours after seeding, change the medium in Plate 2 with fresh basic culture medium supplemented with 25 ng/ml FGF2. 1.15. One hundred and twenty hours after seeding, ensure that cells of the Plate 2 are ~50% confluent. This is the correct time to start the second directed differentiation. 2. Directed differentiation of hESCs into kidney progenitors (perform this procedure on both batches of cells staggered 2 d apart) 2.1. The hESCs should be ~50% confluent at the start of differentiation. Remove the medium and wash the cells once with DPBS. 2.2. Add 2 ml of advanced RPMI 1640 containing 1× L-glutamine supplement (e.g., GlutaMax) and 8 µM CHIR into each well of the 6 well plate and culture at 37°C, 5% CO2. This will be day 0 of differentiation for Plate 1. 2.3. On day 2, of CHIR 99021 treatment, hESCs colonies may break apart and disperse into single cells throughout the well of the 6 well plate. Change the medium with fresh advanced RPMI 1640 containing 1× L-glutamine and 8 µM CHIR very carefully to avoid dislodging the cells that still adherent to the plate. 2.4. On day 4, change the medium with fresh advanced RPMI 1640 containing 1× L-glutamine and 10 ng/ml Activin A. 2.5. On day 6, change the medium with fresh advanced RPMI 1640 containing 1× L-glutamine and 10 ng/ml Activin A. 2.6. On day 7, change the medium with fresh advanced RPMI 1640 containing 1× L-glutamine and 10 ng/ml FGF9. 2.7. After 9 d of this treatment protocol (day 9), ensure that the cells are differentiated to kidney progenitors and adopt renal vesicle-like morphology. Now this 2D culture is ready for transition into 3D culture at the air-liquid interface to generate kidney organoids. For Plate 1—proceed to section 5. For Plate 2—proceed to section 6. 3. Making kidney progenitor cell aggregates at the air-liquid interface 3.1. Prepare a 24 well plate for culture of cells at the air-liquid interface. 3.1.1. Prepare the medium containing APEL2, 1.5% PFHM II, 100 ng/ml BMP7, 100 ng/ml FGF9 and 1 µg/ml Heparin and add 1 ml/well into the 24 well plate. 3.1.2. Float polycarbonate membrane on the medium in each well of the 24 well plate, using sterile forceps. Make sure not to flood the filters—wells with partially or entirely submerged filters should not be used. Keep plate aside in the hood for later use. 3.2. Wash the wells of the Plate 1 two times with DPBS and add 1 ml of cell dissociation enzyme (e.g., TrypLe express) into each well of the 6 well plate. 3.3. Incubate the plate for 5 min at 37°C. Triturate 3–4 times to disperse cell clusters into single cell suspension. 3.4. Neutralize the cell dissociation enzyme by adding 8 ml/well of advanced RPMI 1640 with 1 ml of FBS (total 10% FBS) in a 50 ml conical tube. 3.5. Strain the cells through a 40 µm strainer and use 10 µl of the strained cell suspension to count the cells. 3.6. Centrifuge the cells at 300 g for 5 min. 3.7. Resuspend the cells at 2.5 × 10e5 cells/µl in APEL2 medium containing 1.5% PHFM II. 3.8. Spot 2 µl of the cell suspension onto the polycarbonate membrane floating on the medium in the 24 well plate. Spot 6–8 cell aggregates/membrane. 3.9. Culture for 2 d at 37°C, 5% CO2 in the incubator. 4. Asynchronous mixing of kidney progenitors to generate kidney organoids 4.1. Prepare a 24 well plate to spot cells following step 5.1. 4.2. Harvest and count the kidney progenitors in the Plate 2. 4.3. Remove membranes one at a time from the 24 well plate seeded in step 5.8 and break the cell aggregates into small fragments using a 200 µl micropipette by triturating 7–10 times. 4.4. Mix these small fragments with fresh kidney progenitors from Plate 2 at a 1:1 ratio. (e.g., mix fragments from 1 cell aggregate of 5 × 10e5 cells with 5 × 10e5 cells of newly differentiated kidney progenitors from Plate 2, resuspend in 4 µl and make two cell aggregates on the membranes). 4.5. Spot the mixed cells onto the membranes floating at the surface of the medium in a 24 well plate. 6–8 cell aggregates/the membrane can be spotted. 4.6. Change the medium on day 13 with fresh prewarmed APEL2 containing 1.5% PHFM II without any growth factors. 4.7. Change the medium every 48 h with APEL2 containing 1.5% PHFM II. 4.8. On day 18, image the organoids under a stereo microscope and proceed to section 8 for marker expression analysis. In the absence of densely packed tubular clusters differentiation can be continued for the next 3–4 d.

Critical_Steps

Follow step 2.1 only when cells reach ~50% confluence. Follow step 3.1.2 very carefully to float membranes at the surface of the medium. If there is an air bubble below the floating membrane, remove the bubble with 1 ml pipette or replace membrane with a new one CAUTION: Change medium with growth factors carefully and gently during differentiation. Harsh pipetting may dislodge cells from the culture plate surface and once cells start to float in the medium, they subsequently die.

Trouble_Shooting

Abundant cell death and detachment from the surface of the plate during first 4 days of CHIR treatment. Start CHIR treatment once cells reach ~50% confluence. Change medium gently. No “loosely dense clusters” on day 4. Wait 2–4 h to get “loosely dense clusters” OR start new differentiation with fresh cell culture. Renal vesicle-like morphology absent. Wait 12–24 h to get “renal vesicle-like morphology” OR start new differentiation with fresh cell culture. Membrane submerged in medium. **Remove any visible air bubble below the membrane and transfer to fresh membrane if partially or entirely submerged **Air bubble or few tubular clusters in the organoids. Cell suspension should not have any visible air bubble. Wait 3–4 d more to get densely packed tubular clusters

References

Kumar Gupta A, Sarkar P, Wertheim JA, Pan X, Carroll TJ, Oxburgh L. Asynchronous mixing of kidney progenitor cells potentiates nephrogenesis in organoids. Commun Biol. 2020 May 11;3(1):231. doi: 10.1038/s42003-020-0948-7. PMID: 32393756; PMCID: PMC7214420. Morizane R, Lam AQ, Freedman BS, Kishi S, Valerius MT, Bonventre JV. Nephron organoids derived from human pluripotent stem cells model kidney development and injury. Nat Biotechnol. 2015 Nov;33(11):1193-200. doi: 10.1038/nbt.3392. PMID: 26458176; PMCID: PMC4747858.

Associated_Publications

Gupta AK, Ivancic DZ, Naved BA, Wertheim JA, Oxburgh L. An efficient method to generate kidney organoids at the air-liquid interface. J Biol Methods. 2021 Jun 30;8(2):e150. doi: 10.14440/jbm.2021.357. PMID: 34258308; PMCID: PMC8270790.

Consortium

(Re)Building a Kidney (RBK) Consortium