Objective:
Chronic kidney disease (CKD) represents an increasing global health burden, with limited options for regenerative therapy. Kidney organoids have emerged as promising models for studying kidney disorders. However, their application remains limited by dependence on fetal bovine serum (FBS), insufficient structural maturation, and poor clinical translatability. To address these limitations, we developed a novel protocol integrating podocytes, key glomerular filtration cells, with renal progenitor cells in a co-culture system. Furthermore, FBS was replaced with human umbilical cord blood serum (HUCBS), an ethical and clinically relevant supplement, to enhance organoid functionality and translational potential.
Material and Methods:
Primary mouse mesenchymal-like (MLC) and epithelial-like (ELC) cells were isolated from FVBN mouse kidneys. We established a three-dimensional four-cell co-culture system using MLCs, ELCs, human umbilical vein endothelial cells (HUVECs), and human podocytes. An optimized cell ratio of 10:5:2:10 (MLC/ELC/HUVEC/Podocyte) with a total of 420,000 cells per well yielded the most compact and uniform aggregates. In a key innovation, the culture system was maintained in RPMI-based medium supplemented with 15% HUCBS, completely eliminating FBS and recombinant growth factors.
Results:
The optimized four-cell system cultured in HUCBS-supplemented medium generated significantly larger and more compact kidney organoids compared to podocyte-free controls. Quantitative real-time PCR (qRT-PCR) analysis at day 7 demonstrated significantly elevated expression of podocyte markers (NPHS1, PODXL, WT1), tubular markers (E-cadherin, Aquaporin-1), mesangial marker (PDGFR-β), endothelial marker (CD34), and collecting duct markers (GATA3, Aquaporin-2). Histological assessment using H&E staining confirmed high viability, dense cellular organization, and absence of central necrosis. Immunofluorescence further validated organized E-cadherin-positive epithelial structures.
Conclusion:
This study presents a cost-effective and translational approach for generating structurally mature kidney organoids. Incorporating podocytes into a vascularized co-culture enhanced glomerular and tubular marker expression, while HUCBS served as an ethical human-derived alternative to FBS and expensive growth factors, reducing immunogenicity and production costs. This all-human-supplemented chimeric platform provides strong potential for drug screening, nephrotoxicity testing, and future autologous kidney organoid engineering.
Keyword:
Kidney Organoid, Podocyte, Co-culture, Human Umbilical Cord Blood Serum (HUCBS), Tissue Engineering
Reza Moghadasali 1, 2, Nazanin Bazgir 1, 2
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Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
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Department of Developmental Biology, University of Science and Culture, Tehran, Iran