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.
<ol>
	<li>Day 1
	<ol>
		<li>Prepare a solution of PDT-BzPh (1 mg/ml) and potassium bicarbonate (KHCO3) (0.7 mg/ml) in sterile water. Stir overnight at 4 &#176;C (protect from light).</li>
	...

<ol>
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F., Groth, T., Werner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+blood+reactivity+to+hydroxylated+and+non-hydroxylated+polymer+surfaces.">In vitro blood reactivity to hydroxylated and non-hydroxylated polymer surfaces.</a> Biomaterials. 28, 3617-3625 (2007).</li><li>Sperling, C., Schweiss, R. B., Streller, U., Werner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+hemocompatibility+of+self-assembled+monolayers+displaying+various+functional+groups.">In vitro hemocompatibility of self-assembled monolayers displaying various functional groups.</a> Biomaterials. 26, 6547-6457 (2005).</li><li>Vasita, R., Shanmugam, I. K., Katt, D. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+'self'+engulfment+through+deactivation+of+myosin-II+at+the+phagocytic+synapse+between+human+cells.">Inhibition of 'self' engulfment through deactivation of myosin-II at the phagocytic synapse between human cells.</a> J Cell Biol. 180 (5), 989-1003 (2008).</li><li>Berg, T. K., vander Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+'self'+recognition:+a+role+for+CD47-SIRPalpha+interactions+in+hematopoietic+stem+cell+transplantation.">Innate immune 'self' recognition: a role for CD47-SIRPalpha interactions in hematopoietic stem cell transplantation.</a> Trends Immunol. 29 (5), 203-206 (2008).</li><li>Oldenborg, P. A., Zheleznyak, A., Fang, Y. F., Lagenaur, C. F., Gresham, H. D., Lindberg, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CD47+as+a+marker+of+self+on+red+blood+cells.">Role of CD47 as a marker of self on red blood cells.</a> Science. 288 (5473), 2051-2054 (2000).</li><li>Stachelek, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+CD47+modified+polymer+surfaces+on+inflammatory+cell+attachment+and+activation.">The effect of CD47 modified polymer surfaces on inflammatory cell attachment and activation.</a> Biomaterials. 32 (19), 4317-4326 (2001).</li><li>Finley, M. J., Rauva, L., Alferiev, I. S., Weisel, J. W., Levy, R. J., Stachelek, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diminished+adhesion+and+activation+of+platelets+and+neutrophils+with+CD47+functionalized+blood+contacting+surfaces.">Diminished adhesion and activation of platelets and neutrophils with CD47 functionalized blood contacting surfaces.</a> Biomaterials. 33, 5803-5811 (2012).</li><li>Finley, M. J., Clark, K. A., Alferiev, I. S., Levy, R. J., Stachelek, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+signaling+mechanisms+associated+with+CD47+modified+surfaces.">Intracellular signaling mechanisms associated with CD47 modified surfaces.</a> Biomaterials. 34, 8640-8649 (2013).</li><li>Ravetch, J. V., Lanier, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+inhibitory+receptors.">Immune inhibitory receptors.</a> Science. 290, 84-89 (2000).</li><li>Chandler, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+thrombotic+coagulation+of+blood:+a+method+for+producing+a+thrombus.">In vitro thrombotic coagulation of blood: a method for producing a thrombus.</a> Lab Invest. 7, 110-114 (1958).</li><li>Thorsen, T., Klausen, H., Lie, R. 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B., W&#252;stneck, R., Kr&#228;gel, J., Trukhin, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+initial+period+of+protein+adsorption+by+dynamic+surface+tension+measurements+using+different+drop+techniques.">Characterization of the initial period of protein adsorption by dynamic surface tension measurements using different drop techniques.</a> Colloids and Surfaces A. 131 (1-3), 225-230 (1998).</li><li>Oeveren, W. V., Tielliu, I. 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Research reported in this publication was supported by National Institute of Biomedical Imaging and Bioengineering, under award number R21 EB015612 (SJS), and the National Heart, Lung, and Blood Institute, under award number T32 HL007915 (JBS and RJL), of the National Institute of Health.

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 CD4711-13. The particular conjugation chemistry that we us...

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 failure2,3. 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 molecules4-6. 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 &#8220;self&#8221; status upon expressing cells7-10 and shows promise at conferring biocompatibility when appended to polymeric surfaces11-13. Signal-regulatory protein alpha (SIRP&#945;), 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 SIRP&#945;-mediated cell signaling, down-regulates the immune responses to polyurethane (PU) and polyvinylchloride (PVC) in in vitro, ex vivo, and in vivo models11-13.
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 polyallylamine11-13. 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 previously12,13, recCD47, further modified with the addition of a C-terminal poly-lysine tail12,13, 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 surfaces11,12. 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.

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Name</td>
			<td style="width:175px;">Company</td>
			<td style="width:107px;">Catalog Number</td>
			<td style="width:251px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">16% Paraformaldehyde (PFA)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">58906</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">25% Glutaraldehyde</td>
			<td style="width:175px;">VWR</td>
			<td style="width:107px;">AAA17876-AP&#160;</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">2-pyridyldithio,benzophenone (PDT-BzPH)</td>
			<td style="width:175px;">Synthesized in lab</td>
			<td style="width:107px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">A3059-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Citrate</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">S5770-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Digital Camera</td>
			<td style="width:175px;">Leica</td>
			<td style="width:107px;">DC500</td>
			<td>Out of production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dimethylformamide (DMF)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">270547-100ML</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:175px;">Gibco/Life Technologies</td>
			<td style="width:107px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fluorescent Microscope</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:107px;">TE300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glacial Acetic Acid</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">A38-212</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Human CD47 (B6H12) &#8211; FITC Antibody</td>
			<td style="width:175px;">Santa Cruz Biotechnology</td>
			<td style="width:107px;">SC-12730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Osmium Tetroxide</td>
			<td style="width:175px;">Acros Organics</td>
			<td style="width:107px;">197450050</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Bicarbonate (KHCO3)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">237205-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Phosphate Monobasic (KH2PO4)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">P5655-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PVC Tubing (Cardiovascular Procedure Kit)</td>
			<td style="width:175px;">Terumo Cardiovascular Systems</td>
			<td style="width:107px;">60050</td>
			<td>Most clinical-grade tubing will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>JSM-T330A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Sodium Chloride (NaCl)</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:269px;">Microplate Reader</td>
			<td style="width:175px;">Molecular Devices</td>
			<td style="width:107px;">Spectramax Gemini EM&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sulfo-SMCC</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">M6035-10MG</td>
			<td>Moisture Sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">tris (2-carboxyethyl) phosphine (TCEP-HCl)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">20491</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tris Base</td>
			<td style="width:175px;">Sigma</td>
			<td>T1503-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tween-20</td>
			<td>Bio-Rad</td>
			<td>170-6531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Vectashield with DAPI</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">H-1200</td>
			<td>Light sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Zeba Spin Desalt Columns &#8211; 7K MWCO</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">89891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of the ex vivo chandler loop apparatus to assess the biocompatibility of modified polymeric blood conduits

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.

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...

Watch this Scientific Journal Video about The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits at JoVE.com

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

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.

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

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

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11.5K Görüntüleme.  The Children's Hospital of Philadelphia. 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. 