This model recapitulates both the pre-malignant and tumor microenvironments by incorporating physiologically relevant hydrogels with tunable stiffness and hepatocellular and stroma associated cell lines. This model is modular and cost-effective, can be prepared with basic equipment and readily available materials and can be used to study complex tumor-stroma interactions. This tumor-stroma interaction model can provide insight into the hepatocarcinogenesis.
And this is very important for both a mechanistic perspective and a treatment perspective. Demonstrating procedure with Carlemi Calitz will be Jenny Rosenquist, a doctoral student from the Angstrom laboratory. To prepare 10 milliliters of an 80 milligram per milliliter fibrinogen stock solution, first add 2.21 grams of calcium chloride and five milligrams of aprotinin to individual 20 milliliter volumes of distilled water.
Then add 2.051 milliliters of the aprotinin stock solution and 100 microliters of the calcium chloride stock solution to 7.849 milliliters of PBS. Next, incrementally add 800 milligrams of fibrinogen and 200 milligrams of sodium chloride to the solution without stirring or shaking and place the tube of fibrinogen stock solution horizontally on a shaker. After two to five hours of shaking at 300 revolutions per minute, filter the resulting solution to remove any clumps that formed during the preparation.
For cell culture insert collagen coating, in a biosafety cabinet, use sterilized tweezers to invert the inserts onto the lid of the cell culture plate. Next, mix 115 microliters of glacial acetic acid with 25 milliliters of distilled water before adjusting the solution with additional distilled water to a final volume of 100 milliliters before filtering. To prepare two milliliters of a 100 microgram per milliliter collagen solution, add 40 microliters of a five milligram per milliliter collagen solution to 1.9 milliliters of the 20 millimolar glacial acetic acid stock solution.
Add 100 microliters of the resulting collagen solution to the bottom of each insert. When the inserts have dried, place a custom 3D printed spacer over the inserts. After two to three hours of air drying, wash the inserts by briefly placing them, collagen coating side down, in individual wells of a 12-well plate containing one milliliter of fresh PBS per well per wash.
After the last wash, place the inserts back onto the plate lid for another one to two hours of air drying. Then cover the inverted inserts with the bottom of the plate and place the inserts into the cell culture incubator. Before seeding the cells, add 3.99 grams of sodium hydroxide to 100 milliliters of distilled water and filter the solution through a 0.22 micrometers syringe filter.
Next, wash T175 hepatic stellate and liver carcinoma cell cultures two times with 10 milliliters of PBS per wash before treating the cells with six milliliters of trypsin for four minutes at 37 degrees Celsius. When the cells have detached, inactivate the enzymes with six milliliters of 37 degrees Celsius 10%DMEM and transfer each cell culture to individual 15 milliliter tubes. Sediment the cells by centrifugation.
And resuspend the pellets in five milliliters of pre-warmed 10%DMEM per tube. After counting, dilute both cell populations to 10 to the sixth cells per milliliter of medium concentrations and collect the cells by centrifugation. After removing the supernatants, add the appropriate volume of pre-warmed 10%DMEM to the cells as indicated in the table and neutralize the appropriate volume of four degrees Celsius collagen with 10 microliters per milliliter of four degrees Celsius sodium hydroxide.
Add the chilled neutralized collagen to the cell suspension. The medium will turn yellow. After mixing the suspension thoroughly with a cut pipette tip, the medium will turn bright pink.
Add the appropriate volume of 37 degrees Celsius fibrinogen as indicated in the table and use a cut pipette tip to thoroughly mix the suspension. After mixing, add 0.1 kilo international units of thrombin for each 10 milligrams of fibrinogen to each tube and use a modified 200 microliter pipette tip to add 200 microliters of the cell suspension to the bottom of each insert. Allow the gels to cross-link for 15 minutes at room temperature before gently placing the bottom section of the plate over the gels, then place the inserts into the incubator for additional cross-linking at 37 degrees Celsius for 45 minutes.
At the end of the incubation, place the inserts into individual wells of a 12-well plate and add two milliliters of pre-warmed 10%DMEM to each well. For the seeding of endothelial cells onto the inserts, wash a T175 human vascular endothelial cell culture with 10 milliliters of PBS before detaching the cells with 37 degrees Celsius trypsin as demonstrated. Resuspend the cells in five milliliters of endothelial growth medium for counting and resuspend the cells to 10 to the fourth cells per milliliter of endothelial growth medium concentration.
Then add 500 microliters of cells to the top of each insert and place the plate in the cell culture incubator for 21 days. To measure the storage moduli of the gel formations, use a rheometer 60 minutes after seeding to perform frequency sweeps from 0.1 to 20 Hertz at 0.26%and 37 degrees Celsius with a constant axial force of 0.1 Newtons using an eight millimeter diameter parallel plate with stainless steel geometry. In this representative analysis, 10 formulations of the fibrinogen and collagen hydrogels combinations were prepared to determine which formulations could mimic liver stiffness similar to that observed during the development of a hepatocellular carcinoma.
The storage modulus of each concentration was then determined using a rheometer. From these 10 formulas, the two milligrams per milliliter of collagen type one to 10 milligrams per milliliter of fibrinogen, which corresponds to the liver stiffness values at the onset of fibrosis, two milligrams per milliliter of collagen type one to 30 milligrams per milliliter of fibrinogen, which corresponds to cirrhosis and two milligrams per milliliter of collagen type one and 40 milligrams per milliliter of fibrinogen which corresponds to hepatocellular carcinoma were selected. AlamarBlue analysis showed an overall reduced cell viability within 2D co-cultures that is lower than expected based on the known reported inhibitory concentration values compared to an untreated control.
In the 3D model, however, an increase in doxorubicin resistance was observed. Be sure to act quickly when seeding the hydrogels cell suspension onto the inserts as a streamlined workflow is essential to the success of the protocol. Optimizing RNA and protein isolation techniques will allow to further study the tumor-stroma interactions at a transcriptional and translational level.
