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

Podsumowanie

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

Streszczenie

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.

Wprowadzenie

Many clinical procedures, such as cardiopulmonary bypass and renal dialysis, require the use of polymeric blood conduits and are often associated with post-procedural complications¹. When perfused with blood, these polymers illicit the foreign body reaction (FBR), resulting in adsorption of blood proteins and platelets, monocyte/macrophage adhesion, and the release of pro-inflammatory cytokines, all of which contribute to post-procedural complications and/or device failure^2,3. Thus, strategies to address this issue remain an important and ongoing area of biomaterials research. Investigators have attempted to address this issue by modifying blood contacting surfaces with bioactive or bioinert molecules^4-6. Research in our laboratory has focused on appending recombinant CD47 (recCD47) to polymeric biomaterials as a strategy to mitigate the FBR and increase the efficacy of these materials. CD47 is a ubiquitously expressed transmembrane protein with a known role in immune evasion, conferring “self” status upon expressing cells^7-10 and shows promise at conferring biocompatibility when appended to polymeric surfaces^11-13. Signal-regulatory protein alpha (SIRPα), the cognate receptor for CD47, and a member of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing family of transmembrane proteins, is expressed on cells of myeloid origin¹⁴. We have previously demonstrated that CD47, via SIRPα-mediated cell signaling, down-regulates the immune responses to polyurethane (PU) and polyvinylchloride (PVC) in in vitro, ex vivo, and in vivo models^11-13.

Central to our investigations is a relatively novel photoactivation chemistry, described herein, in which chemically reactive thiol groups are covalently appended to polymeric tubing by reacting the tubing with a multifunctional polymer (PDT-BzPh), composed of 2-pyridyldithio (PDT), the photoreactive benzophenone (BzPh) and a carboxy-modified polyallylamine^11-13. Reducing the covalently appended PDT groups with tris(2-carboxyethyl) phosphine hydrochloride (TCEP)¹¹ yields a thiolated surface that can be subsequently reacted with therapeutic moieties. Detailed herein and previously^12,13, recCD47, further modified with the addition of a C-terminal poly-lysine tail^12,13, is reacted with Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) for 1 hr to generate thiol-reactive groups, allowing for a monosulfide bond formation between the tubing and recCD47¹¹. The anti-inflammatory capacity of the CD47 functionalized surfaces was tested, ex vivo, using the Chandler Loop Apparatus with whole human blood, which was originally described in 1958 as an in vitro model of thrombotic coagulation¹⁵. The apparatus relies on a closed tube system partially filled with air and a rotary motor to circulate the blood through the tubing¹⁵. This experimental model provides the opportunity to examine the effect of blood exposure upon modified and unmodified surfaces as well as the effect of those surface modifications upon the physiology of cells of the blood.

recCD47 can be appended to a variety of polymeric surfaces using this photoactivation chemistry, and its anti-inflammatory capacity can be assessed by utilizing a clinically relevant ex vivo model mimicking blood perfusion over polymeric surfaces^11,12. Clinical grade blood conduits modified with recCD47 show significantly less platelet and inflammatory cell attachment as compared to unmodified polymers when exposed to human blood in the apparatus. A step-by-step description of this modification process is detailed below.

Protokół

1. Modifying Polymeric Surfaces with recCD47

NOTE: The protocol is summarized schematically in Figure 1. Figure 1A illustrates generation of thiol-reactive polymeric surfaces. Figure 1B illustrates generation of thiol-reactive recCD47.

1. Day 1
   1. Prepare a solution of PDT-BzPh (1 mg/ml) and potassium bicarbonate (KHCO₃) (0.7 mg/ml) in sterile water. Stir overnight at 4 °C (protect from light).
2. Day 2
   1. Cut polymeric tubing into 40 cm long pieces (long enough to fit around rotating wheels).
   2. Soak tubes in a 0.1% aqueous solution of hexacylpyridinium for 90 min at room temperature on a shaker or on Chandler Loop Apparatus. Rinse the tubes with sterile water 3x after the 90 min soak.
   3. Acidify the solution of PDT-BzPh from Day 1 by adding 15% aqueous potassium phosphate monobasic (KH₂PO₄). Add 50 µl KH₂PO₄ per ml of PDT-BzPh, forming a cloudy micellar solution.
   4. Soak the tubes in the acidified PDT-BzPh solution for 40 min at room temperature on a shaker or on apparatus.
   5. Rinse the tubes with dilute acetic acid (1:1,000) once.
   6. Expose the tubes to UV irradiation for a total duration of 60 min. Rotate tubes ¼ turn every 15 min to irradiate the entire surface area.
   7. Soak the tubes in a solution of 20 mg/ml potassium bicarbonate (KHCO₃) for 20 min at room temperature on a shaker or on apparatus.
   8. Rinse the tubes with sterile water 3x and store at 4 °C in sterile water.
3. Day 3
   1. Dissolve 5 mg Sulfo-SMCC in 200 µl Dimethylformamide (DMF) (Caution: Perform in fume hood).
   2. Add 50 µl of the Sulfo-SMCC solution prepared in step 1 to 0.1 mg/ml of recCD47 poly-lysine solution, and stir for 60 min at room temperature.
   3. During 60 min stir, degas sterile Dulbecco’s Phosphate Buffered Saline (DPBS) and set aside.
   4. Purify Sulfo-SMCC reacted recCD47 using a 7K molecular weight cut-off spin desalting column per manufacturer’s instructions. Collect final flow-through (high-quality thiol-reactive recCD47) and dilute to a volume necessary to coat the interior of the tube with DPBS.
   5. Rinse the tubes from day 2 with sterile, degassed DPBS 3x.
   6. React the modified surface of the tubes with a solution of 20 mg/ml with tris(2-carboxyethyl) phosphine hydrochloride (TCEP) in degassed DPBS for less than 2 min. Rinse the tubes with sterile degassed DPBS 4x.
   7. Add the Sulfo-SMCC-reacted recCD47 to the tubes and incubate overnight at 4 °C on a shaker or on apparatus.

2. Immunoassay Quantification of recCD47 on Modified Surfaces

1. Rinse modified tubes to be quantified from Day 3 with DPBS 3x. Store tubes not used for quantification in DPBS at 4 °C.
2. Use a 4 mm biopsy punch to make duplicate tube samples and place biopsy punches in wells of a 96-well plate.
3. Prepare a negative control consisting of an unmodified tube sample not treated with the detection antibody. Prepare an antibody control consisting of an unmodified tube sample treated with the detection antibody. Prepare the modified tubing test samples treated with the detection antibody.
4. Block samples with 0.4% bovine serum albumin (BSA) in DPBS for 60 min at room temperature on a shaker.
5. After blocking, aspirate BSA and rinse with tris-buffered saline plus 1% Tween-20 (TBST) 3x, 10 min each on a shaker.
6. Prepare a working dilution of antibody in 0.4% BSA according to manufacturer’s recommended dilution for immunoassays. Dilute human CD47 Antibody B6H12-FITC 1:100 in 0.4% BSA.
7. Add 200 µl of antibody dilution to appropriate wells. Incubate negative controls with 0.4% BSA only. Incubate at room temperature for 60 min on a shaker (protect from light).
8. Rinse with TBST 3x, 10 min each on a shaker. Aspirate the last TBST rinse and add 200 µl DPBS to tube sample wells.
9. Collect the final flow-through, which is high-quality thiol-reactive recombinant CD47 and dilute to a volume necessary to coat the interior of the tubes with DPBS.
10. Read FITC signal intensity using a microplate reader with FITC excitation (485 nm) and emission (538 nm) settings.
11. Calculate recCD47 bound to polymeric surface based on standard values, accounting for auto-fluorescence and non-specific binding from the antibody control sample.

3. Chandler Loop Apparatus Protocol

Seek Institutional Review Board (IRB) approval of blood collection protocol and informed consent paperwork prior to initiation of collection of human blood samples. Obtain informed consent from a human blood donor.
NOTE: A diagram depicting the apparatus is shown in Figure 2.

1. Fill water bath with distilled water until approximately 1/2 of the diameter wheels are submerged. Add enough bleach to the water bath to make a 10% bleach solution. Set water bath to 37 °C and allow temperature to equilibrate.
2. Gather metal adapters (shown in Figure 1B), unmodified tubing and modified tubing, and assemble around apparatus wheels, as shown in Figure 1C. Ensure that tubing fits snuggly around the wheel with the metal adapter in place.
3. Obtain 30 ml blood sample using an IRB-approved protocol, in a vial prefilled with 2 ml of citrate or other anticoagulant, and mix by inversion to prevent clotting once the specimen has been collected.
4. Add approximately 10 ml of blood to each tube using the metal valve and syringe, leaving some air in the tube. Secure the valve cap and rotate for 3 hr.
5. Drain the blood into a waste beaker treated with a 10% bleach solution (or as required by institutional policy).
6. Gently rinse tubing interior with DPBS to remove all traces of blood. Collect flow-through in the waste beaker. Dispose of blood in accordance with institutional policy.
7. Disinfect the apparatus and any blood contacting surfaces using a 10% bleach solution or according to institutional policy.
8. Process samples for fluorescent microscopy or scanning electron microscopy as described below.

4. Fluorescent Microscopy and Cell Counting

1. Prepare a 4% paraformaldehyde (PFA) solution (Caution: Perform in fume hood).
2. Incubate tubing in a 4% PFA solution overnight at 4 °C.
3. After overnight incubation, remove 4% PFA and rinse films very gently with DPBS.
4. Cut the tubing into sections to expose the lumen surface for staining.
5. Stain a section of the tubing with a few drops of mounting media with 4',6-diamidino-2-phenylindole (DAPI) for 30 min at room temperature (protect from light). After staining, rinse the tubing very gently with DPBS to reduce background DAPI signal.
6. Image using a fluorescent microscope equipped with a DAPI filter and digital camera to count the number of cells in 9 blindly selected fields of view under 200X magnification.
7. Record cell counts per field of view and perform appropriate statistical analyses to determine statistical significance of results.

5. Scanning Electron Microscopy

1. Prepare a 2% glutaraldehyde solution (Caution: Perform in fume hood).
2. Incubate tubing in a 2% glutaraldehyde solution overnight at 4 °C.
3. After overnight incubation, gently rinse the tubing 3x with DPBS.
4. Prepare a 1% solution of osmium tetroxide (Caution: Severe inhalation hazard! Use in fume hood).
5. Incubate tubing in a 1% solution of osmium tetroxide for 15 min at room temperature.
6. Gently rinse the tubing 3x with DPBS.
7. Prepare a series of ethanol concentrations (25%, 50%, 75%, 95%, and 100% ethanol).
8. Dehydrate the tubing by incubation in the series of ethanol concentrations. Incubate in 25%, 50%, 75%, and 95% ethanol for 20 min each. Incubate in 100% ethanol for 30 min.
9. Supercritical point dry the tubing with CO₂ for 45 min to remove all moisture.
10. Mount tube sections on a specimen stub with silver paste or graphite.
11. Coat tube sections with 12.5 nm of gold-palladium.
12. Examine in a scanning electron microscope.   

Wyniki

Generating thiol-reactive polymeric surfaces through the use of PDT-BzPh and TCEP along with thiol-reactive recCD47 poly-lysine using SMCC allows for the attachment of recCD47 to polymeric surfaces. The modification process is summarized schematically in Figure 1. The convenience of this modification process is that it can be applied to many different proteins and many different polymeric surfaces, assuming the protein can be modified with sufficient chemically reactive groups such as amine-containing ly...

Dyskusje

The photoactivation chemistry (summarized in Figure 1) allows for the modification of virtually any polymer surface that has sufficient hydrocarbons to facilitate PDT-BzPh attachment and subsequent UV irradiation to photo-activate the PDT-BzPh. Functionalizing the polymeric surface with reactive thiol groups allows for the subsequent attachment of a range of testable molecules of interest. In our particular studies we chose recombinant CD47^11-13. The particular conjugation chemistry that we us...

Materiały

Name	Company	Catalog Number	Comments
16% Paraformaldehyde (PFA)	Thermo Scientific	58906	Caution! Use in fume hood
25% Glutaraldehyde	VWR	AAA17876-AP	Caution! Use in fume hood
2-pyridyldithio,benzophenone (PDT-BzPH)	Synthesized in lab	N/A
Bovine Serum Albumin (BSA)	Sigma	A3059-100G
Citrate	Sigma	S5770-50ML
Digital Camera	Leica	DC500	Out of production
Dimethylformamide (DMF)	Sigma	270547-100ML	Caution! Use in fume hood
Dulbecco’s Phosphate Buffered Saline (DPBS)	Gibco/Life Technologies	14190-136
Fluorescent Microscope	Nikon	TE300
Glacial Acetic Acid	Fisher Scientific	A38-212	Caution! Use in fume hood
Human CD47 (B6H12) – FITC Antibody	Santa Cruz Biotechnology	SC-12730
Osmium Tetroxide	Acros Organics	197450050	Caution! Use in fume hood
Potassium Bicarbonate (KHCO3)	Sigma	237205-100G
Potassium Phosphate Monobasic (KH2PO4)	Sigma	P5655-100G
PVC Tubing (Cardiovascular Procedure Kit)	Terumo Cardiovascular Systems	60050	Most clinical-grade tubing will work
Scanning Electron Microscope	JEOL	JSM-T330A
Sodium Chloride (NaCl)	Fisher Scientific	BP358-212
Microplate Reader	Molecular Devices	Spectramax Gemini EM
Sulfo-SMCC	Sigma	M6035-10MG	Moisture Sensitive!
tris (2-carboxyethyl) phosphine (TCEP-HCl)	Thermo Scientific	20491
Tween-20	Bio-Rad	170-6531
Vectashield with DAPI	Fisher Scientific	H-1200	Light sensitive!
Zeba Spin Desalt Columns – 7 K MWCO	Thermo Scientific	89891