Published: April 13th, 2021
Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.
Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.
In recent years, a number of protocols to differentiate human pluripotent stem cells into kidney organoids have been developed1,2,3,4,5. Kidney organoids have provided an important tool to aid research into new regenerative medicine approaches, model kidney-related diseases, perform toxicity studies and therapeutic drug development. Despite their wide applicability, kidney organoids have limitations such as lack of maturation, limited long-term culture capacity in vitro, and a paucity of several ce....
All experiments using hPSCs were performed in compliance with institutional guidelines, and were carried out in a Class II biosafety hood with appropriate personal protective equipment. All reagents are cell culture-grade unless stated otherwise. All cultures are incubated at 37 °C, 5% CO2 air atmosphere. At all stages of the assay, embryoid bodies or kidney organoids can be collected, and fixed or prepared for analysis. The hPSC lines used to generate this data have been fully characterized and published.......
In this most recent version of our protocol, kidney organoid differentiation is initiated in a defined, low protein medium. The assays are performed entirely in suspension and rely on the innate ability of hPSCs differentiation and organization for initiation of tubulogenesis. A single assay originating from a 100 mm ~60% confluent hPSC culture plate routinely yields 500-1,000 kidney organoids, as shown in our previous publication5. Due to such high numbers of organoids generated, this protocol is.......
Previous studies have shown that the initial protocol steps are critical for intermediate mesoderm differentiation5,19,20 and, therefore, it is essential to implement a stringent medium composition at this stage. Removing undefined components such as serum, albumin, protein free hybridoma medium II from the first stage of the protocol may help to improve consistent differentiation efficiency between assays21
|Anti-adherence rinsing solution
|10 mM stock in DMSO
|Corning disposable spinner flasks
|Corning Ultra-Low Attachment 6-well plates
|Corning Slow-Speed Stirrers
|Multi plate magnetic stirrer for spinner flask culture
|Aliquot and freeze
|DMEM, low glucose, pyruvate, no glutamine, no phenol red
|DPBS 1x, no calcium, no magnesium
|Egg / Oval Stirring Bars
|Excelta General-Purpose Tweezers
|Keep sterile in the cell culture hood
|EZBio Single Use Media Bottle, 250mL
|Foxx Life Sciences
|Used to make PVA 10%
|Falcon Standard Tissue Culture Dishes (100 mm)
|Fisherbrand Sterile Aspirating Pipet 2mL
|Fisherbrand Cell Lifters - Cell lifter
|Fisherbrand Multi Function 3D Rotators
|Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix
|BME. Aliquot on ice and freeze. Another suitable matrix alternative is Matrigel or Cultrex.
|Gentle Cell Dissociation Reagent
|KnockOut Serum Replacement - Multi-Species
|KOSR. Aliquot and freeze
|Lipid Mixture 1, Chemically Defined
|MEM Non-Essential Amino Acids Solution
|MilliporeSigma Stericup Quick Release-GP Sterile Vacuum Filtration System 500mL
|MilliporeSigma Stericup Quick Release-GP Sterile Vacuum Filtration System 250mL
|MIXcontrol MTP / Variomag TELEcontrol MTP Control Unit
|VMF 90250 U
|MIXdrive 6 MTP / Variomag TELEdrive 6 MTP Microplate Stirring Drive
|MP Biomedicals 7X Cleaning Solution
|Tissue culture suitable detergent. Make a 5% solution in water
|hPSC medium.TeSR-E8, NutriStem XF, and mTeSR Plus medium have also been tested and are suitable alternatives.
|Nunc 50 mL Conical, Sterile Centrifuge Tubes
|Nunc 15mL Conical Sterile Centrifuge Tubes
|Aliquot and freeze
|Anti-mycoplasma reagent. Aliquot and freeze
|pluriStrainer® 200 µm
|pluriStrainer® 500 µm
|Poly(vinyl alcohol) 87-90% hydrolyzed (PVA)
|10% in DPBS stirring at 98 degrees C until disolves, make in 138-3211-FLS
|ROCK inhibitor Y-27632 (ROCKi)
|10 mM stock in DPBS
|Sterile Disposable Serological Pipets - 10mL
|Sterile Disposable Serological Pipets - 25mL
|Sterile Disposable Serological pipette - 5 mL
|Serum-free, low protein base medium for E5-ILP
|Variomag distriBOX 2 Distributor
|If you use more than one MIXdrive
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