Abstract: SA-PO042
Protocol to Make Renal Tubuloids from Human Kidneys
Session Information
- Engineering-Based Approaches to Problems in Nephrology
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Bioengineering
- 300 Bioengineering
Authors
- Mori, Yutaro, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
- Ichimura, Takaharu, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
- Patel, Ankit B., Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
- Siedlecki, Andrew M., Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
- Bonventre, Joseph V., Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
Background
Kidney organoids derived from human induced pluripotent stem (hiPS) cells can be used to simulate a response to drugs in human kidneys. We have developed an alternative way to make more homogeneous epithelial-like structures from kidney tissue derived from multiple patients in a short period of time.
Methods
Human primary epithelial cell cultures were obtained from the non-tumor kidney tissue removed from patients with renal cell carcinoma. Human renal cortex was diced and then digested with collagenase. Tubules were seeded on matrigel-coated plates with serum-free media containing epidermal growth factor. After passage, primary cells and immortalized LLC-PK1 cells, for comparison, were cultured on ultra-low attachment plates for several days. Then cells were transferred into media containing matrigel, hepatocyte growth factor, fibroblast growth factor-2 and 5% fetal bovine serum.
Results
Primary human renal tubular epithelial cells (hRTECs) tubuloids were generated from dissected patients’ kidneys using epidermal growth factor, serum-free media and matrigel. We have generated a library of hRTECs derived from 15 patients. hRTECs showed phenotypes reflecting age and renal function of each original patient, especially in growth rate and in γH2AX expression, a marker for DNA damage response. We also generated tubuloids using a 3D culture technique both with hRTECs and with LLC-PK1 cells. Tubuloids had polarized expression of cell surface markers, LTL, KIM-1 (apical) and Na-K-ATPase (basolateral). The tubuloids endocytosed labeled oxidized LDL, which is observed in 2D KIM-1-expressing epithelial cell culture. It took only a week to establish hRTECs from patient kidneys and four weeks to form tubuloids from hRTECs.
Conclusion
We succeeded in making renal tubuloids using hRTECs derived from multiple patients. This strategy is potentially an excellent way to simulate pathological conditions and response of epithelial cells to toxins and therapeutic agents in a personalized fashion.
Funding
- NIDDK Support