Bioengineering
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Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells
Chapters
Summary November 4th, 2022
Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.
Transcript
The isogenic glomerulus chip will enable patient specific disease modeling and nephrotoxicity testing and advanced precision medicine applications. Glomerular epithelium and endothelium are derived from a single population of human-induced pluripotent stem cells, or IPSCs. IPSCs possess unlimited self-renewal and can differentiate into almost any cell type.
This technique can be used to assess pathological cellular phenotypes and disease. The patient-specific organ chip can also serve as a platform for drug screening and mechanistic studies. This model can be applied to the development of a body on a chip system personalized to a specific patient.
This system has the potential to predict patient-specific responses prior to clinical testing. Begin by gently adding 25 microliters of basement membrane matrix 2 solution to the urinary channel and 20 microliters into the capillary channel of the chip. Take two sterile 15 milliliter conical tube caps and fill them with 500 microliters of sterile distilled water.
Place the cap in the Petri dish to prevent drying and place the lid on the dish. Incubate it at 37 degrees Celsius overnight. Proceeding, aspirate the medium from T75 flasks containing a 15 day culture of vascular endothelial cells at 90%confluency.
Add five milliliters of cell detachment buffer and incubate at 37 degrees Celsius for five to seven minutes. Then transfer the cells to a 15 milliliter conical tube and add five milliliters of DMEM/F-12. Centrifuge at 200 times G for five minutes.
Remove the supernatant and resuspend the cells in 300 microliters of endothelial maintenance medium. Count the cells with a hemocytometer to obtain approximately 2 million cells. Using a P200 barrier tip, flush both the top and bottom channels of the microfluidic chip with 200 microliters of DMEM/F-12 while simultaneously aspirating from the periphery of the outlet.
Hold the chip firmly with the P200 barrier tip attached to the aspirator away from the outlet of the bottom channel. Firmly inject 20 microliters of the vascular endothelial cell suspension into the capillary channel of the chip and carefully aspirate the medium from the periphery of the outlet. Check under the microscope for bubbles or an uneven seeding density.
Gently invert the chip so that the cells can adhere to the basal side of the flexible PDMS membrane. Place the chip into the holder cartridge and add three milliliters of PBS into the cartridge to prevent the membrane from drying. Incubate the chip at 37 degrees Celsius for three hours.
Check the bottom channel under the microscope for a confluent layer of cells attached to the flexible PDMS membrane. Wash the unattached cells by adding 200 microliters of endothelial maintenance medium on the inlet of the bottom channel while aspirating from the periphery of the capillary channel outlet. Place the chip back into the holder cartridge and incubate at 37 degrees Celsius overnight.
On the next day, gently flush the capillary channel with 200 microliters of endothelial maintenance medium. Flush the urinary channel with 200 microliters of DMEM/F-12 and drop 50 microliters of DMEM/F-12 on the inlet and outlet ports. Proceeding intermediate mesoderm cells, replace the intermediate mesoderm induction medium from each well of the 12 well plate with one milliliter of Trypsin EDTA and incubate at 37 degrees Celsius for five minutes.
Gently scrape the cells using a cell lifter and dissociate the cells using pipetting. Add two milliliters of Trypsin neutralization solution to each well and transfer the cells to a 50 milliliter conical tube. Make up the volume of the cell suspension to 50 milliliters with DMEM/F-12 and centrifuge at 200 times G for five minutes.
Aspirate the supernatant and resuspend the cells in 500 microliters of intermediate mesoderm induction medium to obtain approximately 3 million cells. Count the cells with a hemocytometer. Hold the chip and firmly inject 25 microliters of cell suspension into the urinary channel of the chip while carefully aspirating the medium from the periphery of the outlet.
Check for bubbles or uneven cell seeding density. Add three milliliters of PBS into the chip holder cartridge and incubate at 37 degrees Celsius for three hours. After incubation, flush both channels with 200 microliters of their respective cell culture medium.
Attach empty P200 barrier tips into both outlets of the urinary and capillary channels. Pipette 200 microliters of endothelial maintenance medium and inject half of it into the capillary channel inlet. Release the pipette tip inside the inlet such that both the inlet and outlet of the channel are attached to pipette tips filled with medium.
Repeat this procedure for the urinary channel inlet with 200 microliters of intermediate mesoderm maintenance medium. Incubate the chips with tips embedded at 37 degrees Celsius overnight. To connect the chips to organ chip bioreactor, first, remove P200 tips from the channels.
Add droplets of respective media to the inlet and outlet of the urinary and capillary channels to prevent drying. Add three milliliters of warm podocyte induction medium to the urinary inlet reservoir and three milliliters of warm endothelial maintenance medium to the capillary inlet reservoir. Add 300 microliters of the respective media to the outlet reservoirs directly over the outlet port.
Slide the pods onto the tray and into the organ chip bioreactor. Use the rotary dial to select and start the prime cycle for two minutes. Visually inspect the underside of the pod for small droplets at all four fluidic ports.
To achieve fluid to fluid contact between the pod underside and microfluidic chip ports, gently slide the chip carrier into the pod. Gently press the chip carrier tab in and up. Aspirate excess medium from the chip's surface.
Set the organ chip bioreactor flow rate to 60 microliters per hour. Set the cyclic strain to 10%at 0.4 hertz. Use the rotary dial on the organ chip bioreactor to select the regulate cycle and run for two hours.
Visually inspect the outlet reservoirs for an increase in the level of medium. Use the rotary dial on the organ chip bioreactor to select the regulate cycle. After day 17 of culture, daily aspirate the medium from the urinary channel outlet reservoirs diagonally away from the port but keep some medium in the reservoir.
Replenish the urinary channel inlet reservoir with up to three milliliters of podocyte induction medium every two days for five days. After five days, aspirate the medium from the urinary channel but keep some medium in the reservoir. Replenish the urinary channel inlet reservoir daily with three milliliters of podocyte maintenance medium.
Similarly, aspirate the medium from the capillary channel outlet and replenish the inlet reservoirs daily with endothelial maintenance medium. Using this protocol human induced pluripotent stem cells were used to develop a functional in vitro model of the glomerulus by propagating vascular endothelial cells and podocytes. On day 21 after podocyte induction and vascular endothelium propagation, the cells within the chips expressed lineage identification markers such as Podocin and Nephrine for podocytes, VE Cadherin and PECAM-1 for vascular endothelial cells.
Both the podocyte and vascular endothelial cell layers expressed Collagen IV which is the most abundant GBM protein in the kidney glomerulus. The glomerulus chips selectively filtered small molecules such as inulin from the capillary into the urinary channel, while preventing large proteins like albumin from leaving the capillary channel, showing a selective molecular filtration function. During endothelial induction on days four to seven, an increase in the cell number may result in a secondary layer of cells but over seeding may cause aggregation which can impede differentiation.
Unexpected fluid cross flow between the urinary and capillary channels may be observed in case of inadequate bonding of the PDMS chip components, blockage in the fluid flow path, or compromised filtration barrier. When seeding endothelium and epithelium and maintaining the chips from days 15 onward, it is important to visually inspect for air bubbles in the channels at every step. In the isogenic glomerulus chip, clinically relevant drug candidates can be administered to test their therapeutic potential or toxicity levels.
This technique now opens up opportunities to understand the genetic basis of human kidney disease and develop personalized therapeutics.
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