Skip to main content

Lund University Publications

LUND UNIVERSITY LIBRARIES

Parsing Glomerular and Tubular Structure Variability in High-Throughput Kidney Organoid Culture

Uusi-Rauva, Kristiina ; Pirttiniemi, Anniina ; Hassinen, Antti ; Trokovic, Ras ; Lehtonen, Sanna ; Kallijärvi, Jukka LU ; Lehto, Markku LU ; Fellman, Vineta LU orcid and Groop, Per Henrik (2025) In Methods and Protocols 8(5).
Abstract

High variability in stem cell research is a well-known limiting phenomenon, with technical variation across experiments and laboratories often surpassing variation caused by genotypic effects of induced pluripotent stem cell (iPSC) lines. Evaluation of kidney organoid protocols and culture conditions across laboratories remains scarce in the literature. We used the original air-medium interface protocol to evaluate kidney organoid success rate and reproducibility with several human iPSC lines, including a novel patient-derived GRACILE syndrome iPSC line. Organoid morphology was assessed with light microscopy and immunofluorescence-stained maturing glomerular and tubular structures. The protocol was further adapted to four... (More)

High variability in stem cell research is a well-known limiting phenomenon, with technical variation across experiments and laboratories often surpassing variation caused by genotypic effects of induced pluripotent stem cell (iPSC) lines. Evaluation of kidney organoid protocols and culture conditions across laboratories remains scarce in the literature. We used the original air-medium interface protocol to evaluate kidney organoid success rate and reproducibility with several human iPSC lines, including a novel patient-derived GRACILE syndrome iPSC line. Organoid morphology was assessed with light microscopy and immunofluorescence-stained maturing glomerular and tubular structures. The protocol was further adapted to four microplate-based high-throughput approaches utilizing spheroid culture steps. Quantitative high-content screening analysis of the nephrin-positive podocytes and ECAD-positive tubular cells revealed that the choice of approach and culture conditions were significantly associated with structure development. The culture approach, iPSC line, experimental replication, and initial cell number explained 35–77% of the variability in the logit-transformed proportion of nephrin and ECAD-positive area, when fitted into multiple linear models. Our study highlights the benefits of high-throughput culture and multivariate techniques to better distinguish sources of technical and biological variation in morphological analysis of organoids. Our microplate-based high-throughput approach is easily adaptable for other laboratories to combat organoid size variability.

(Less)
Please use this url to cite or link to this publication:
author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
differentiation, GRACILE syndrome, high-throughput, induced pluripotent stem cell, kidney organoid, validation
in
Methods and Protocols
volume
8
issue
5
article number
125
publisher
MDPI AG
external identifiers
  • scopus:105019962989
  • pmid:41149808
ISSN
2409-9279
DOI
10.3390/mps8050125
language
English
LU publication?
yes
additional info
Publisher Copyright: © 2025 by the authors.
id
4b75c51f-09cc-4f02-bf25-88576e5a15a3
date added to LUP
2025-12-18 07:52:36
date last changed
2025-12-19 15:12:31
@article{4b75c51f-09cc-4f02-bf25-88576e5a15a3,
  abstract     = {{<p>High variability in stem cell research is a well-known limiting phenomenon, with technical variation across experiments and laboratories often surpassing variation caused by genotypic effects of induced pluripotent stem cell (iPSC) lines. Evaluation of kidney organoid protocols and culture conditions across laboratories remains scarce in the literature. We used the original air-medium interface protocol to evaluate kidney organoid success rate and reproducibility with several human iPSC lines, including a novel patient-derived GRACILE syndrome iPSC line. Organoid morphology was assessed with light microscopy and immunofluorescence-stained maturing glomerular and tubular structures. The protocol was further adapted to four microplate-based high-throughput approaches utilizing spheroid culture steps. Quantitative high-content screening analysis of the nephrin-positive podocytes and ECAD-positive tubular cells revealed that the choice of approach and culture conditions were significantly associated with structure development. The culture approach, iPSC line, experimental replication, and initial cell number explained 35–77% of the variability in the logit-transformed proportion of nephrin and ECAD-positive area, when fitted into multiple linear models. Our study highlights the benefits of high-throughput culture and multivariate techniques to better distinguish sources of technical and biological variation in morphological analysis of organoids. Our microplate-based high-throughput approach is easily adaptable for other laboratories to combat organoid size variability.</p>}},
  author       = {{Uusi-Rauva, Kristiina and Pirttiniemi, Anniina and Hassinen, Antti and Trokovic, Ras and Lehtonen, Sanna and Kallijärvi, Jukka and Lehto, Markku and Fellman, Vineta and Groop, Per Henrik}},
  issn         = {{2409-9279}},
  keywords     = {{differentiation; GRACILE syndrome; high-throughput; induced pluripotent stem cell; kidney organoid; validation}},
  language     = {{eng}},
  number       = {{5}},
  publisher    = {{MDPI AG}},
  series       = {{Methods and Protocols}},
  title        = {{Parsing Glomerular and Tubular Structure Variability in High-Throughput Kidney Organoid Culture}},
  url          = {{http://dx.doi.org/10.3390/mps8050125}},
  doi          = {{10.3390/mps8050125}},
  volume       = {{8}},
  year         = {{2025}},
}