The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

The overall goal of the following experiment is to assess the biocompatibility of polymers modified with recombinant CD 47 by using the Chandler Loop model of blood profusion. This is achieved by first modifying polymers with recombinant CD 47. Using photo activation chemistry as a second step, modified polymers are exposed to human blood in the Chandler Loop to mimic blood perfusion over implanted materials.

Next, attached cells are fixed and stained to count the number of adhered cells as a measure of biocompatibility. The results show that recombinant CD 47 promotes the biocompatibility of polymers and decreases the foreign body rejection based on the observation that significantly less cells adhere to recombinant CD 47 modified polymers compared to unmodified polymers. This method can answer key questions in the biomaterials field, such as how to prevent rejection of implanted medical devices.

The implications of this technique extend towards preventing implantable device failure because recombinant CD 47 shows promise at promoting biocompatibility and preventing the foreign body response To generate thiol reactive polymeric surfaces and thiol reactive recombinant CD 47 first cut polymeric tubing into 40 centimeter long pieces, which is long enough to fit around the rotating wheels soak tubes in a 0.1%aqueous solution of hexa seal perineum for 90 minutes. At room temperature on the Chandler loop apparatus, rinse the tubes with sterile water three times after the 90 minute soak acidify a previously prepared solution of alial ditol benzone phon by adding 15%aqueous potassium phosphate mono basic. Add 50 microliters of potassium phosphate mono basic per milliliter of the prepared solution, forming a cloudy mis celler solution.

Soak the tubes in the acidified solution for 40 minutes at room temperature on the apparatus. Then rinse the tubes with dilute acetic acid. Once next, expose the tubes to ultraviolet irradiation for a total duration of 60 minutes.

Rotate the tubes one quarter, turn every 15 minutes to irradiate the entire surface area following UV irradiation. Soak the tubes in a solution of 20 milligrams per milliliter potassium bicarbonate for 20 minutes at room temperature on the apparatus. Rinse the tubes with sterile water three times and store it four degrees Celsius and sterile water.

The next day dissolve five milligrams of sulfa SMCC and 200 microliters of add 50 microliters of the sulfa SMCC solution to 0.1 milligrams per milliliter of recombinant CD 47 polylysine solution and stir for 60 minutes at room temperature during the 60 minute stir DGAs sterile ECCOs phosphate buffer saline or DPBS and set it aside. Purify SFO SMCC reacted recombinant CD 47. Using a 7, 000 molecular weight cutoff spin desalting column per the manufacturer's instructions, collect the final flow through, which is the high quality TH reactive recombinant CD 47, and dilute to a volume necessary to coat the interior of the tubes with DPBS.

Then rinse the tubes with sterile DGAs DPB S3 times. React the modified surface of the tubes with a solution of 20 milligrams per milliliter of TCEP in DGAs DPBS for less than two minutes. After rinsing the tubes with sterile DGAs DPBS four times add the sulfa SMCC reacted recombinant CD 47 to the tubes and incubate overnight at four degrees Celsius on the apparatus for amino assay quantification.

First rinse modified tubes to be quantified with DPB S3 times. Use a four millimeter biopsy punch to make duplicate tube samples and place the biopsy punches. In the wells of a 96 well plate designate a negative control consisting of an unmodified tube sample that will not be treated with a detection antibody.

Also prepare an antibody control consisting of an unmodified tube sample that will be treated with a detection antibody. Finally, prepare the modified tubing test samples to be treated with a detection antibody. Next block the samples with 0.4%bovine serum albumin or BSA in DPBS for 60 minutes at room temperature on a shaker after blocking aspirate BSA and rinse with tris buffered saline plus 1%tween 20 or TBST three times for 10 minutes each.

On a shaker, prepare a working dilution of antibody in 0.4%BSA according to the manufacturer's recommended dilution for immunoassays in this assay. Human CD 47 antibody B six H 12 FE is diluted one to 100 and 0.4%BSA Add 200 microliters of antibody dilution to the appropriate wells and incubate negative controls with 0.4%BSA only incubate at room temperature for 60 minutes on a shaker protected from light. After rinsing the samples with TBST three times for 10 minutes each on a shaker aspirate the TBST rinse and add 200 microliters of DPBS to the sample wells.

Next, prepare a standard curve of antibody and DPBS as detailed in the text protocol, including A-D-P-B-S blank. Then add 200 microliters of the prepared standards to wells of a 96 well plate in duplicate. Read Fitz signal intensity using a MICROPLATE reader with Fitz excitation and emission settings.

Finally, calculate the recombinant CD 47 bound to polymeric surfaces based on standard values accounting for autofluorescence and non-specific binding from the antibody control sample. Fill the Chandler Loop apparatus water bath with distilled water until approximately one half of the diameter of the wheels are submerged. Add enough bleach to the water bath to make a 10%bleach solution.

Set the water bath to 37 degrees Celsius and allow the temperature to equilibrate, gather metal adapters, unmodified tubing, and modified tubing, and assemble around the apparatus wheels. Ensure that the tubing fits snugly around the wheel with the metal adapter in place. Obtain a 30 milliliter blood sample using an institutional review board approved protocol in a vial prefilled with two milliliters of citrate or other anticoagulant mixed by inversion to prevent clotting.

Once the specimen has been collected at approximately 10 milliliters of blood to each tube using the metal valve and syringe, leaving some air in the tube, secure the valve cap and rotate for three hours. After three hours, drain the blood into a waste beaker treated with a 10%bleach solution. Gently rinse the tubing interior with DPBS to remove all traces of blood collect flow through in the waste beaker and dispose of blood in accordance with institutional policy, disinfect the apparatus and any blood contacting surfaces using a 10%bleach solution or according to institutional policy.

Finally, process samples for fluorescent microscopy or scanning electron microscopy as described in the text protocol and antibodies directed against the external IG domain of CD 47, were used to quantify the amount of recombinant CD 47 bound to polyurethane films. Significant fit e staining is visible for recombinant CD 47 using a fit e conjugated antibody to the extracellular IG domain of CD 47, which is localized specifically to the film as shown by differential interference contrast imaging. These results demonstrate the recombinant CD 47 can be covalently attached to polyurethane films.

Cell attachment to the tubing was assessed via DPI staining. Cell counts obtained through DPI staining demonstrate that appended recombinant CD 47 significantly inhibit cell attachment as compared to unmodified surfaces. Scanning electron microscopy demonstrated similar levels of cell attachment to unmodified and recombinant CD 47 modified surfaces as determined by DPI cell counts.

These data indicate that appendage of recombinant CD 47 using the protocol described herein significantly inhibits inflammatory cell attachment in an ex vivo model of blood profusion After its development. This technique paved the way for researchers in the field of biomaterials to explore the biocompatibility of implanted polymers in the context of human blood Profusion. After watching this video, you should have a good understanding of how to assess the biocompatibility of polymers using the channel loop apparatus.