Isolation of Primary Human Proximal Tubule Epithelial Cells and Their Use in Creating a Microphysiological Model of the Renal Proximal Tubule
May 9th, 2025
We present an optimized protocol for processing whole human kidneys to isolate and culture primary renal proximal tubule epithelial cells and the application of these cells in a three-dimensional, microfluidic, microphysiological platform to recapitulate the renal proximal tubule.
Our research focuses on the development and application of a microphysiological system that mimics a human renal proximal tubule, enabling a more predictive and physiologically relevant in vitro assessment of drug transport, metabolism, and nephrotoxicity. The current challenges we face with our experiments is maintaining cell viability and function over extended culture time of six months to study chronic drug exposure. Our research is addressing the need for an accurate in vitro model to accurately predict human kidney-specific responses to drugs, particularly in the context of drug transport, metabolism, and nephrotoxicity.
To begin, place the kidney on a round 15-centimeter culture dish. Aspirate and discard the remaining media from the dish. Using sterile razor blades, remove the surrounding fat and the renal capsule.
Gently score the renal capsule to create a slit down the center. Using fingers, pull off the capsule. Then, with razor blades, slice off any fat attached to the kidney before discarding both the capsule and fat into another culture dish.
Separate the cortex from the medulla and discard the medulla. Using a razor blade, mince the tissue into pieces smaller than one cubic centimeter until a slurry-like appearance is achieved. Add a small amount of kidney digestion buffer to the dish.
Transfer the slurry into the first set of 50-milliliter tubes containing 35 milliliters of the kidney digestion solution. Distribute evenly and ensure the total volume in each tube does not exceed 45 milliliters. Place the tubes onto a 37-degrees Celsius orbital shaker and incubate for 30 minutes at the highest speed that keeps the tubes secure.
Spray the tubes with 70%ethanol before returning them to the biosafety cabinet. Invert the tubes to mix and allow the larger tissue pieces to settle to the bottom. Transfer the supernatant to new 50-milliliter conical tubes containing 10 milliliters of fetal bovine serum or FBS without transferring the intact tissue.
Centrifuge the tubes at 200 g for seven minutes. Now carefully aspirate out the supernatant from each tube. Add 10 milliliters of proximal tubule epithelial cells, or PTEC media, into each tube and resuspend the pellets.
Strain the resulting cell suspensions through 100-micrometer cell strainers into new 50-milliliter conical tubes. Centrifuge the cell filtrates at 300 g for five minutes. Next, wash the pellets with five milliliters of DPBS and resuspend them using a P1000 tip.
After centrifuging the tubes again for five minutes, aspirate off the supernatant and repeat this step for two more washes. Then resuspend the cell pellets with 15 milliliters of PTEC media and plate cells into sterile T75 cell culture flasks. Label the flasks appropriately and place them in a sterile incubator set at 37 degrees Celsius with 5%carbon dioxide to allow the primary tubular epithelial cells to grow undisturbed for 48 hours before the first media change.
Aspirate media from the flask. Add five milliliters of prewarmed 0.05%Trypsin-EDTA into each T25 flask and incubate at 37 degrees Celsius for one to two minutes for Trypsin digestion. Once cells are detached, neutralize Trypsin by adding five milliliters of prewarmed defined trypsin inhibitor solution.
Resuspend the mixture five times to dislodge any remaining attached cells, then transfer the cell suspension into 15-milliliter conical tubes before centrifuging at 400 g for five minutes. After centrifugation, aspirate the supernatant from the centrifuge tubes. Resuspend the cell pellets with 14 milliliters of PTEC media per tube and transfer the suspensions into T75 flasks.
Perform a T shake to evenly distribute cells across the flask. For cryopreserving PTECs, resuspend the cell pellets in two milliliters of 10%dimethyl sulfoxide and 90%low-glucose PTEC media per 15-milliliter tube. Transfer one milliliter into each cryo vial.
Transfer the cryo vials to a cell-freezing container for temperature-controlled cooling and place the container in a minus 80 degrees Celsius freezer for 24 hours. Gently flick the PTEC-containing tube to resuspend the cells. After filling the syringe with the resuspended cells, insert the needle into the injection port farthest from the screw valve ports.
Carefully press the plunger of the syringe to inject the cells. The morphology and confluency of isolated PTECs over time are shown in this figure. Small patches of PTECs with cobblestone-like morphology emerged by Day 6, interspersed with residual blood cells, and formed more defined colonies by Day 8 with reduced blood cell presence.
By Day 10, cell confluency reached approximately 50 to 60%with expanded epithelial colonies, and, by Day 15, full confluency was achieved across the culture surface. After injection into the MPS device, PTECs were observed flowing into the circular lumen structure from the injection port, confirming successful cell delivery.
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Overview
This study presents an optimized protocol for isolating and culturing primary renal proximal tubule epithelial cells from human kidneys. These cells are utilized in a microfluidic, microphysiological platform to mimic the renal proximal tubule for drug transport and nephrotoxicity assessments.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Pharmacology
Background
- Human renal proximal tubule cells are crucial for studying kidney function.
- Current in vitro models often fail to accurately predict human kidney responses.
- Maintaining cell viability over extended culture periods is a significant challenge.
- Microphysiological systems can provide more relevant drug testing environments.
Purpose of Study
- To develop a reliable in vitro model for assessing drug transport and nephrotoxicity.
- To enhance the understanding of chronic drug exposure effects on kidney cells.
- To improve the predictive capabilities of kidney-specific responses to drugs.
Methods Used
- Isolation of renal proximal tubule epithelial cells from human kidneys.
- Culture of cells in a three-dimensional microfluidic platform.
- Assessment of drug transport and metabolism in the cultured cells.
- Long-term culture maintenance for chronic exposure studies.
Main Results
- Successfully isolated and cultured primary renal proximal tubule cells.
- Developed a microphysiological system that mimics kidney function.
- Demonstrated improved cell viability over extended culture periods.
- Provided insights into nephrotoxic effects of drugs in vitro.
Conclusions
- The optimized protocol allows for better modeling of kidney responses to drugs.
- This approach can enhance drug development and safety assessments.
- Future studies can build on this model to explore various nephrotoxic agents.
Frequently Asked Questions
Executive Industry Relevance
Human primary proximal tubule epithelial cells (PTECs) cultured in microphysiological systems (MPS) provide a predictive, physiologically relevant platform for assessing renal drug transport, metabolism, and nephrotoxicity. This capability addresses a critical gap in early drug discovery by enabling chronic exposure studies and supporting translational confidence in kidney safety assessments. Integrating such models into the pipeline enhances risk-adjusted decision-making and portfolio prioritization for nephrotoxic liability.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of renal drug transport mechanisms and transporter function in a human-relevant system.
- Supports biological de-risking by modeling chronic drug exposure and kidney-specific responses.
- Facilitates functional target validation for nephrotoxicity and clearance pathways.
- Improves predictive confidence for triaging compounds with renal liabilities.
Screening & Assay Development
- Provides a validated, reproducible platform for nephrotoxicity and drug interaction screening.
- Delivers quantitative outputs on cell viability, morphology, and transporter polarization over extended culture periods.
- Enables assay standardization and scalability for compound evaluation in renal contexts.
- Supports downstream workflows by maintaining physiological morphology and function for up to six months.
Translational & Preclinical Research
- Aligns in vitro findings with in vivo renal physiology for improved translational relevance.
- Enables long-term studies of chronic kidney disease mechanisms and drug-induced nephrotoxicity.
- Supports continuity from early discovery through preclinical safety assessment for renal endpoints.
- Provides mechanistic insights into human-specific renal responses to xenobiotics.
Pipeline & Workflow Integration
This protocol positions primary human PTEC-based MPS models at the intersection of early discovery, lead identification, and preclinical nephrotoxicity assessment.
- Discovery Biology: Supports hypothesis testing for renal drug transport and toxicity mechanisms.
- Screening: Delivers reproducible, quantitative readouts for compound nephrotoxicity and transporter activity.
- Analytics: Enables measurement of cell viability, confluency, and functional polarization over time.
- Translational Research: Bridges in vitro and in vivo renal responses for biomarker alignment and risk assessment.
- Enterprise Reuse: Establishes a reusable platform for ongoing nephrotoxicity and renal clearance studies across programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in renal safety assessment.
- Operational Value: Standardizes long-term culture and assay conditions for reproducibility and scalability.
- Strategic Value: Enables informed go/no-go decisions and capital-efficient portfolio management for nephrotoxic risk.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of compounds with renal liabilities.
Implementation Considerations
- Requires expertise in primary cell isolation, culture, and microphysiological system operation.
- Demands access to specialized instrumentation for cell culture, microfluidics, and analytical readouts.
- Necessitates cross-team standardization of protocols for reproducibility and data comparability.
- May require adaptation for different donor sources or disease-relevant model systems.
- Long-term culture maintenance and functional validation are essential for reliable outputs.
