Novel Blood Contact Model System, Glass Coverslip Coating, and Sterilization
Creation of the resin 3D-printed chambers utilized Autodesk’s Fusion 360 and Photon Workshop V2.1.24 software to design and slice the model for printing. Set up of the ANYCUBIC Photon S SLA 3D resin printer required the leveling of the build plate and vigorous shaking of the Sculpt High Temperature Resin from Siraya Tech for five minutes. Approximately 150 mL of resin was poured into the resin vat before beginning the print.
Once printing was completed, the chambers were left to soak in ethanol overnight. The next day, the chambers were hand dried and placed into a dehydrator for 30 minutes to remove excess ethanol. During the last five minutes of drying the chambers, a glass container was filled with enough water to fully submerge the print. The water was heated until it was warm to the touch. Once the drying process concluded, the chambers were exposed to UV light on all sides in two 15-minute increments for a total of 30 minutes, completely curing the prints.
After curing, the prints were placed back into the dehydrator for 30 minutes to remove the excess volatile organic compounds (VOCs) within the resin. A glass coverslip (Thomas Scientific, Swedesboro, NJ) was added into each well as the substrate to seed the cells onto. The glass coverslip was bound to the base of the well using Loctite Clear Silicone Waterproof Sealant and Loctite Professional Super Glue placed with an 18-gauge needle. Finally, the chamber was dehydrated for a final 15 minutes to completely remove any excess VOCs left after the addition of glue.
Sterilization of the chambers was done by soaking them in 70% (w/v) ethanol for 30 minutes followed by two 10-minute washes with double distilled water (Growcells, Irvine, CA). Before seeding the cells on the glass coverslip, the chambers were coated with 500 µL of a 50 µg/mL poly-D-lysine solution overnight with phenol red (v/v 1%). The next morning, glass coverslips were rinsed with 500 µL of double distilled water containing phenol red (v/v 1%) (Sigma, St. Louis, MO) and left to dry for two hours before seeding.
pH Tests
During the sterilization process, washes with double distilled water with 1% (v/v) phenol red was used to observe color changes to ensure the ethanol was effectively removed. Insignificant color change showed that the majority of the ethanol was removed. Following sterilization, the glass coverslips were coated with poly-D-lysine with phenol red added overnight, and the next morning no color change was observed. The addition of 1.86 g of sodium bicarbonate was supplemented into the media to balance the pH within the system.
Blood Extraction and Storage
Blood storage buffer was created using the standard concentrations for the citrate-phosphate-dextrose with the addition of adenine (CPDA-1) buffer. On top of the CPDA-1 buffer, additive solution three (AS-3) was combined with the blood to provide extra nutrients(26). Briefly, a mixture of sodium citrate tribasic dehydrate, citric acid, dextrose, adenine, sodium phosphate monobasic, and sodium chloride were dissolved in double distilled water to create the buffer solution (Sigma, St. Louis, MO). Due to the small volume of buffer required, the reagents were massed and dissolved in 1000 µL of double distilled water. The necessary volume of the reagent was then combined and brought to 5 mL with excess double distilled water.
All animal protocols were approved by the Wayne State University Institution of Animal Care and Use Committee (IACUC). Extraction of blood from a C57BL/6 male mice was performed using a full body massage, similar to a retro-orbital massage, technique following euthanasia via carbon dioxide and consequent decapitation. Microcentrifuge tubes, filled with 122.8 µL of citrate buffer, were used to collect the blood. Each tube was filled with 877.2 µL of blood to obtain a 1 mL final volume. After extraction, the blood was immediately placed on ice until added to the cell culture media. For long term storage, the tubes of blood were kept at 4°C.
Cell Culturing and Catheter Insertion
Mouse primary astrocytes, Type III clone from the brain and cerebellum, were used for these experiments (ATCC C8-D30). The recommended cell culture media (ATCC) was a mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) with supplemental 50 mL of fetal bovine serum. The addition of 5 mL of penicillin-streptomycin solution was also added into the media. Stabilization of the pH within the culture was accomplished by adding 1.86 grams of sodium bicarbonate was added into the media. Cells were allowed to grow in vented T75 Tissue Culture Treated Flasks (Falcon) for two weeks with media changes every three days. Cell removal was done using 2 mL of Trypsin-EDTA (v/v 0.25%) with phenol red (Gibco, Waltham, MA) incubated at 37°C with a CO2 level of 5% for five minutes. Following the incubation, 4 mL of media was added into the flask to assist with cell dislodgement and stop the enzymatic activity of the trypsin. The cells were collected in a 15 mL falcon tube and centrifuged at 125 G for seven minutes. After centrifugation, the supernatant was removed, and 1 mL of media was added to homogenize the cells.
Using a 50% (v/v) trypan blue (Gibco, Waltham, MA) concentration, the cells were counted using a hemacytometer, and the cells were brought to a concentration of one million cells per 1 mL of media. They were then seeded by pipetting a 100 µL drop of the cell and media mixture on the center of the glass coverslip. This droplet of cells was left for one hour in a 37°C incubator with a 5% CO2 concentration to allow for the cells to attach on the coverslip. Afterwards, an additional 1000 µL of media was added to the culture. A media change was done after 24 hours and then changed every three days.
These cells were allowed to grow for one week before of the 20 mm catheter samples were added into the chamber. Using tweezers, the sterilized catheter segments were placed into two indentations on either side of the well. A mark was made on the side of the catheter without holes to position the sample. The three rows of holes punched into the catheter were oriented with the middle set of holes in direct contact with the cell layer. Labeling of the row of holes was done based on their orientation within the chamber.
At the same time as catheter insertion, the blood was added into the media. A concentration of 15 µL of blood per 1 mL of media was used. This concentration of blood followed the rationale tested in experiments performed by Castaneyra-Ruiz (14),(27). In a separate microcentrifuge tube, 100 µL of blood was mixed with CaCl2 (Sigma, St. Louis, MO) to obtain a final concentration of 20 mM concentration to reverse the anticoagulation. This solution was then homogenized and added into 5 mL of media. In the blood exposed samples, 1000 µL of the blood and media mixture was pipetted in the each well. The control group used the same chamber system, and the catheter was inserted in the same fashion as described above for the sample with blood. The media used for the control samples was the same as the blood exposed samples without the addition of blood. Media was changed every three days by exchanging spent media with 1000 µL fresh media.
Staining
Samples ran for a total of two weeks before fixing them with two washes of 0.01 M phosphate buffer solution (PBS) (Sigma, St. Louis, MO). The volume used for the following solutions was 500 µL per sample, except for PBS washes which were at 1000 µL. The cells were then exposed to a 4% paraformaldehyde (Sigma, St. Louis, MO) solution for seven minutes, followed by two rinses with a 0.01 M PBS. Permeabilization of the cells was done by incubating them in sodium borohydride (Sigma, St. Louis, MO) and a citrate buffer (Sigma, St. Louis, MO) for 30 minutes each. The citrate buffer was heated to 100°C before adding it on the samples, and then heated at 80°C during the incubation period. Samples were then incubated in BlockAid blocking solution (ThermoFisher, Waltham, MA) for 30 minutes.
Staining the astrocytes was done by using the primary antibody GFAP Polyclonal Antibody (ThermoFisher PA1-10004) and the secondary antibody Goat anti-Chicken IgY 555 ThermoFisher A-21437) for 24 hours each. The antibodies were diluted, using 0.4% Triton solution in 0.01 M PBS, to 1:1000 for the primaries and 5:1000 for the secondaries. After incubating the samples with the secondary antibodies, the samples were washed with 0.01 M PBS. The nuclei were stained using the DAPI Solution (ThermoFisher, Product No.62248) with a 1:1000 ratio in 0.01 M PBS for 30 minutes.
Imaging and Analysis
Before imaging, the top portion of the chamber was broken off to allow imaging. Using a Caliber I.D. RS-G4 Ribbon Scanning Confocal microscope with lasers set to 405 and 561 wavelength, images of the catheter were taken from above still in the chamber. Images were taken, once the catheter was removed, for the row of holes in direct contact with the cells. Analysis of these 3D images was done using Imaris software to obtain cell counts of each of the three sets of eight holes. Images taken from above the catheter while still in the chamber were taken to count the cells around the sides of the catheter. The image of the row of holes that came into direct contact with the cells were counted using the image taken below the catheter after removal. Counts for DAPI and GFAP were taken individually with a consistent volume. A box was positioned to obtain a cell count around the holes. The holes along the side of the sample used the box dimensions 1800x17000x125 and 2000x17000x75 around the holes in direct contact with the cells.
Statistical Analysis
Data collected from the cell counts were analyzed to test for normalcy by using the Anderson Darling equation. Parametric results from the normal distribution test were analyzed by using the Welch test with ⍺=0.05. Utilization of the Mann-Whitney U test was done with ⍺=0.05 for non-parametric results.
Experimental Timeline