Parsing Glomerular and Tubular Structure Variability in High-Throughput Kidney Organoid Culture
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethics
2.2. Cell Culture
2.3. Generation and Characterization of Healthy Control iPSC Line HEL61 and GRACILE Syndrome iPSC Line HEL124
2.4. Reverse Transcription and Quantitative PCR (qPCR)
2.5. Embryoid Bodies
2.6. Immunocytochemistry of iPSCs and Embryoid Bodies
2.7. Differentiation of iPSCs Towards Hepatocyte Lineage Cells
2.8. Western Blotting
2.9. Differentiation of Pluripotent Stem Cells into Kidney Organoids
2.10. Immunofluorescence Staining of Kidney Organoids
2.11. Microscopy and Morphological Analyses of Kidney Organoids
2.12. Opera Phenix High-Content Screening and Image Analysis
2.13. Statistical Analysis
3. Results
3.1. Generation and Characterization of iPSC Lines
3.2. The GRACILE Syndrome Molecular Phenotype Is Replicated in Patient-Derived iPSC-Lines Differentiated into Hepatocyte Cells and Kidney Organoids
3.3. The APEL/Air-Medium Interface Protocol Shows Variability in Kidney Organoid Structure Development Across Cell Lines
3.4. Optimization of the APEL/Air-Medium Interface Protocol Towards Higher Throughput Shows Association Between Culture Conditions and Kidney Organoid Structure Development
4. Discussion
Supplementary Materials
Author Contributions
Funding
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AMI | Air-medium interface |
APEL | Albumin polyvinyl alcohol essential lipids |
BSCC | Biomedicum Stem Cell Center |
D | Day |
DMEM | Dulbecco’s modified Eagle’s medium |
E8 | Essential-8 medium |
ECAD | Epithelial cadherin |
EDTA | Ethylenediaminetetraacetic acid |
EGTA | Ethylene glycol tetra acetic acid |
FBS | Fetal bovine serum |
FGF | Fibroblasts growth factor |
FMSC | Finnish Microarray and Sequencing Centre |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
hESCs | Human embryonic stem cells |
hiPSC | Human induced pluripotent stem cell |
iPSC | Induced pluripotent stem cell |
KOSR | KnockOut-Serum replacement |
LTL | Lotus tetragonolobus lectin |
ML | Monolayer |
PBS | phosphate-buffered saline |
PFA | paraformaldehyde |
PFHM | Protein-free hybridoma medium |
RISP | Rieske iron-sulfur protein |
ROCK | Rho-associated protein kinase |
RT-qPCR | Quantitative reverse transcription PCR |
S | Spheroid |
SeV | Sendai virus |
ULA | Ultra-low attachment |
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Cell Lines | All | <50% Cell Plating Density at D0 | >50% Cell Plating Density at D0 | |||||
---|---|---|---|---|---|---|---|---|
Success Rate (%) 1 | Number of Samples 2 | Success Rate (%) 1 | Number of Samples 2 | Number of Experiments | Success Rate (%) 1 | Number of Samples 2 | Number of Experiments | |
hiPSC | ||||||||
HEL24.3 | 69 | 35 | 95 | 21 | 6 | 29 | 14 | 4 |
HEL47.2 | 0 | 3 | ND | ND | NA | 0 | 3 | 2 |
HEL61.2 | 100 | 47 | 100 | 29 | 7 | 100 | 18 | 5 |
HEL124.1 | 0 | 5 | 0 | 1 | 1 | 0 | 4 | 2 |
HEL124.2 | 96 | 45 | 97 | 30 | 9 | 93 | 15 | 5 |
HEL11.4 | 100 | 3 | 100 | 3 | 1 | ND | ND | NA |
hESC | ||||||||
H9 | 92 | 12 | 100 | 6 | 2 | 83 | 6 | 2 |
S+AMI (1) and S (4) (Organoids n = 149) | ML+S+AMI (2) and ML+S (3) (Organoids n = 143) | |
---|---|---|
Variables | n | n |
Approach | ||
S+AMI (1) | 26 | |
ML+S+AMI (2) | 29 | |
ML+S (3) | 114 | |
S (4) | 123 | |
Cell Line | ||
HEL24.3 | 62 | 78 |
HEL61.2 | 87 | 65 |
Experiment | ||
A | 86 | 54 |
B | 63 | 89 |
Cell no. | ||
2 k | 7 | |
5 k | 16 | |
10 k | 41 | |
20 k | 43 | |
50 k | 42 | |
100 k | 78 | |
200 k | 65 | |
mean (min, max) | mean (min, max) | |
Nephrin proportion | 0.146 (0.000, 0.686) | 0.125 (0.000, 0.531) |
ECAD proportion | 0.127 (0.000, 0.466) | 0.171 (0.000, 0.672) |
S+AMI (1) and S (4) (Organoids n = 149) | ML+S+AMI (2) and ML+S (3) (Organoids n = 143) | |||
---|---|---|---|---|
Nephrin 488 | ECAD 568 | Nephrin 488 | ECAD 568 | |
Approach: S+AMI (1) | 0.921 *** (0.223) | 1.120 *** (0.209) | ||
Approach: ML+S+AMI (2) | 0.893 *** (0.158) | 1.481 *** (0.215) | ||
Cell Line: HEL61.2 | 0.014 (0.167) | −0.265 (0.156) | 2.486 *** (0.129) | 1.206 *** (0.176) |
Experiment: B | −1.223 *** (0.170) | −1.677 *** (0.160) | 0.691 *** (0.134) | 0.577 ** (0.182) |
Cell no. | 0.023 *** (0.005) | 0.015 ** (0.005) | 0.001 (0.001) | −0.001 (0.002) |
r2 | 0.371 | 0.489 | 0.771 | 0.451 |
Adjusted r2 | 0.353 | 0.475 | 0.764 | 0.435 |
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Uusi-Rauva, K.; Pirttiniemi, A.; Hassinen, A.; Trokovic, R.; Lehtonen, S.; Kallijärvi, J.; Lehto, M.; Fellman, V.; Groop, P.-H. Parsing Glomerular and Tubular Structure Variability in High-Throughput Kidney Organoid Culture. Methods Protoc. 2025, 8, 125. https://doi.org/10.3390/mps8050125
Uusi-Rauva K, Pirttiniemi A, Hassinen A, Trokovic R, Lehtonen S, Kallijärvi J, Lehto M, Fellman V, Groop P-H. Parsing Glomerular and Tubular Structure Variability in High-Throughput Kidney Organoid Culture. Methods and Protocols. 2025; 8(5):125. https://doi.org/10.3390/mps8050125
Chicago/Turabian StyleUusi-Rauva, Kristiina, Anniina Pirttiniemi, Antti Hassinen, Ras Trokovic, Sanna Lehtonen, Jukka Kallijärvi, Markku Lehto, Vineta Fellman, and Per-Henrik Groop. 2025. "Parsing Glomerular and Tubular Structure Variability in High-Throughput Kidney Organoid Culture" Methods and Protocols 8, no. 5: 125. https://doi.org/10.3390/mps8050125
APA StyleUusi-Rauva, K., Pirttiniemi, A., Hassinen, A., Trokovic, R., Lehtonen, S., Kallijärvi, J., Lehto, M., Fellman, V., & Groop, P.-H. (2025). Parsing Glomerular and Tubular Structure Variability in High-Throughput Kidney Organoid Culture. Methods and Protocols, 8(5), 125. https://doi.org/10.3390/mps8050125