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Experiment: AAV tropism in kidney organoids cultured under flow

PI:   Ryuji Morizane, MD, PhD 

Description: Human kidney organoids generated from human ES and iPS cells are used to evaluate tropism of AAV2, AAV8, and AAV9 that express eGFP under CMV promoter. Organoids are exposed to AAVs at MOI 10^5 from differentiation day 21 to 28. Four culture conditions were tested: U-well suspension culture, static culture on gelbrin, low flow on a chip coated with gelbrin, and high flow on a chip coated with gelbrin. Immunohistochemistry was performed to visualize transduced cells with staining for podocytes (PODXL) and proximal tubules (LTL). AAV2/2 shows the highest transduction efficiency among three serotypes evaluated, and the GFP expression is highest in the static condition.
Delivery Assays:
  • Calculated total transduction efficiency by counting fluorescent cells (GFP) compared to DAPI stained cells.
  • Calculated transduction efficiency by counting fluorescent cells (GFP) compared to DAPI and podocytes (PODXL) stained cells.
  • Calculated transduction efficiency by counting fluorescent cells (GFP) compared to DAPI and proximal tubules (LTL) stained cells.
Download: Submitted files
Filter Results ..
Delivery Systems
 AAV
Models
 Human kidney organoid (stem cell-derived)
  Vectors
 AAV2/2CMVeGFP
 AAV2/8CMVeGFP
 AAV2/9CMVeGFP
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Results
AAV_tropism_in_kidney_organoids_cultured_under_flow
Delivery Efficiency
Record Id Condition Model Delivery Vector Dosage Delivery Efficiency %GFP+ Area Delivery Efficiency %GFP+ PODXL+ / PODXL+ cells Delivery Efficiency %GFP+ LTL+ / LTL+ cells Image
15000003403 Control, U-well Suspension Human kidney organoid (stem cell-derived) control
15000003404 AAV2/2, U-well Suspension Human kidney organoid (stem cell-derived) AAV AAV2/2CMVeGFP MOI=10^5
15000003405 AAV2/8, U-well Suspension Human kidney organoid (stem cell-derived) AAV AAV2/8CMVeGFP MOI=10^5
15000003406 AAV2/9, U-well Suspension Human kidney organoid (stem cell-derived) AAV AAV2/9CMVeGFP MOI=10^5
15000003407 Control, No Flow on Chip Human kidney organoid (stem cell-derived) control
15000003408 AAV2/2, No Flow on Chip Human kidney organoid (stem cell-derived) AAV AAV2/2CMVeGFP MOI=10^5
15000003409 AAV2/8, No Flow on Chip Human kidney organoid (stem cell-derived) AAV AAV2/8CMVeGFP MOI=10^5
15000003410 AAV2/9, No Flow on Chip Human kidney organoid (stem cell-derived) AAV AAV2/9CMVeGFP MOI=10^5
15000003411 Control, Low Flow on Chip Human kidney organoid (stem cell-derived) control
15000003412 AAV2/2, Low Flow on Chip Human kidney organoid (stem cell-derived) AAV AAV2/2CMVeGFP MOI=10^5
15000003413 AAV2/8, Low Flow on Chip Human kidney organoid (stem cell-derived) AAV AAV2/8CMVeGFP MOI=10^5
15000003414 AAV2/9, Low Flow on Chip Human kidney organoid (stem cell-derived) AAV AAV2/9CMVeGFP MOI=10^5
15000003415 Control, High Flow on Chip Human kidney organoid (stem cell-derived) control
15000003416 AAV2/2, High Flow on Chip Human kidney organoid (stem cell-derived) AAV AAV2/2CMVeGFP MOI=10^5
15000003417 AAV2/8, High Flow on Chip Human kidney organoid (stem cell-derived) AAV AAV2/8CMVeGFP MOI=10^5
15000003418 AAV2/9, High Flow on Chip Human kidney organoid (stem cell-derived) AAV AAV2/9CMVeGFP MOI=10^5

AAV tropism in kidney organoids cultured under flow (A) Immunostaining images of kidney organoids exposed to AAV2/2, 2/8, or 2/9 at MOI 10^5 for 1 week in four culture conditions. (B) Graphs showing percentages of transduced cells by AAV2/2, 2/8, or 2/9. (C) Immunocytochemistry for LTL and GFP in organoids treated with AAV2/2. (D, E) GFP+ cells in podocytes or proximal tubules in organoids treated with AAV2/2.