Experiment: AAV2/2 transduction efficiencies in kidney organoids cultured under flow determined by FACS
PI: Ryuji Morizane, MD, PhD
Description: Human kidney orgnaoids generated from human ES and iPS cells are used to evaluate tropism of AAV2 that expresses eGFP under CMV promoter. Organoids are exposed to AAVs at MOI 10^5 from differentiation day 22 to 23. Four culture conditions are tested: full flow on a chip, pulsed flow on a chip, no flow on a chip, U-well suspension culture. Flow cytometry is performed to quantify the transduction efficiency in LTL+ proximal tubules. U-well culture shows the highest transduction efficiency among those four conditions.
Delivery Assays:
- Calculated transduction efficiency by flow cytometry of GFP+ cells out of total cells
- Calculated transduction efficiency by flow cytometry of GFP+ cells out of LTL+ cells.
Parent Project: Vascularized kidney organoids on chip for efficacy and toxicity testing of somatic genome editing
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Other experiments in this project: 6
- Evaluation of DNA damage, cellular toxicity and inflammatory markers following saCas9 and gRNAs delivery by AAV2 in kidney organoids.
- Biomarker assays to evaluate toxicity and inflammation induced by AAVs in ES cell derived kidney organoids.
- AAV tropism in kidney organoids derived from human pluripotent stem cells.
- AAV tropism in kidney organoids cultured under flow
- Biomarker assays to evaluate toxicity induced by AAVs in iPS cell derived kidney organoids.
- Delivery of saCas9 and gRNAs to nephron epithelia by AAV2 in kidney organoids.
Download: Submitted files
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Results |
| Delivery Efficiency | ||||||||
|---|---|---|---|---|---|---|---|---|
| Condition | Sex | Model | Delivery | Vector | Dosage | %GFP+ / total cells | % GFP+ LTL+ / LTL+ cells | Image |
| High flow (AAV-) | Male | Human kidney organoid (iPSC-derived) | control | |||||
| High flow | Male | Human kidney organoid (iPSC-derived) | AAV | AAV2/2CMVeGFP | MOI=10^5 | |||
| Pulsed flow | Male | Human kidney organoid (iPSC-derived) | AAV | AAV2/2CMVeGFP | MOI=10^5 | |||
| No flow | Male | Human kidney organoid (iPSC-derived) | AAV | AAV2/2CMVeGFP | MOI=10^5 | |||
| U-well | Male | Human kidney organoid (iPSC-derived) | AAV | AAV2/2CMVeGFP | MOI=10^5 | |||
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AAV2/2 transduction efficiencies in kidney organoids cultured under flow (A) Flow cytometry of kidney organoids exposed to AAV2/2 at MOI 10^5 for 1 day in four culture conditions. (B) Graphs showing percentages of transduced cells by AAV2/2 in LTL+ proximal tubules. |
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