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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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