Abstract: SA-PO453
Isolation of Primary Cell Types from the Human Kidney Cortex
Session Information
- Development and Regenerative Medicine
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 500 Development, Stem Cells, and Regenerative Medicine
Authors
- Shumansky, Kara, Novabiosis, RTP, North Carolina, United States
- Perin, Laura, Children's Hospital Los Angeles, Los Angeles, California, United States
- Da Sacco, Stefano, Children's Hospital Los Angeles, Los Angeles, California, United States
- Cossis, Casey, Novabiosis, RTP, North Carolina, United States
Background
In vitro studies can help elucidate kidney pathophysiology and nephrotoxicity. Cell lines and primary animal cells can be poor representatives of human cell biology. Primary human renal cells are essential tools for studying such mechanisms. Herein we describe isolation of primary cell types from the cortex of whole human kidneys.
Methods
Podocytes, Proximal Tubule Cells (PT), Mesangial, and Glomerular Endothelial Cells were isolated from non-transplantable human kidneys donated for research. The kidney cortex is surgically separated from the medulla, the tissue is minced, glomeruli are separated, and further digested using a collagenase based enzymatic blend to obtain a heterogenous single cell suspension. Cells are cultured and expanded in specialized media to enrich for the target cell populations. Homogenous cell isolates for a. proximal tubules (CD10/CD13) b. mesangial cells (PDGFRB) c. glomerular endothelial cells (CD31) d. podocytes (nephrin) are isolated by immunomagnetic sorting. Proximal tubular cells, mesangial cells and glomerular endothelial cells further expanded in specific media before undergoing a second round of selection to ensure purity. Podocytes are not expanded to avoid de-differentiation. Characterization of the isolated populations is performed by immunofluorescence and flow cytometry for cell-specific proteins.
Results
We successfully obtained pure human proximal tubular cells, podocytes, mesangial cells and glomerular endothelial cells using the outlined methodology. Each population was assessed for viability, attachment, proliferative ability. Purity and cell-specific marker expression were assessed by confocal fluorescent microscopy and flow cytometry; PT: AQ1, cytokeratin, Na/K-ATPase, and ENT1; Mesangial: PDGFRb, CD206, and Vimentin, GEC: EDH3, Ve Cadherin, and CD31; Podocytes: WT1, Synaptopodin, and Podocin. Proximal Tubular cells retained activity demonstrated by enzymatic GGT assay.
Conclusion
We have developed and optimized a streamlined method for isolating target populations from the human kidney. Our process yields highly pure homogenous cell populations that can proliferate in vitro and express population specific surface markers. Furthermore, isolated proximal tubule cells retained functionality and can be used in transporter assays.
Funding
- Commercial Support – Parent company, Promethera Biosciences, partially funding the Novabiosis division. Additional funding comes from revenue generation of life science products.