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>
	</li>
	<li>Day 2
	<ol>
		<li>Cut polymeric tubing into 40 cm long pieces (long enough to fit around rotating wheels).</li>
		<li>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.</li>
		<li>Acidify the solution of PDT-BzPh from Day 1 by adding 15% aqueous potassium phosphate monobasic (KH2PO4). Add 50 &#181;l KH2PO4 per ml of PDT-BzPh, forming a cloudy micellar solution.</li>
		<li>Soak the tubes in the acidified PDT-BzPh solution for 40 min at room temperature on a shaker or on apparatus.</li>
		<li>Rinse the tubes with dilute acetic acid (1:1,000) once.</li>
		<li>Expose the tubes to UV irradiation for a total duration of 60 min. Rotate tubes &#188; turn every 15 min to irradiate the entire surface area.</li>
		<li>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.</li>
		<li>Rinse the tubes with sterile water 3x and store at 4 &#176;C in sterile water.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Dissolve 5 mg Sulfo-SMCC in 200 &#181;l Dimethylformamide (DMF) (Caution:&#160;Perform in fume hood).</li>
		<li>Add 50 &#181;l 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.</li>
		<li>During 60 min stir, degas sterile Dulbecco&#8217;s Phosphate Buffered Saline (DPBS) and set aside.</li>
		<li>Purify Sulfo-SMCC reacted recCD47 using a 7K molecular weight cut-off spin desalting column per manufacturer&#8217;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.</li>
		<li>Rinse the tubes from day 2 with sterile, degassed DPBS 3x.</li>
		<li>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.</li>
		<li>Add the Sulfo-SMCC-reacted recCD47 to the tubes and incubate overnight at 4 &#176;C on a shaker or on apparatus.</li>
	</ol>
	</li>
</ol>
2. Immunoassay Quantification of recCD47 on Modified Surfaces
<ol>
	<li>Rinse modified tubes to be quantified from Day 3 with DPBS 3x. Store tubes not used for quantification in DPBS at 4 &#176;C.</li>
	<li>Use a 4 mm biopsy punch to make duplicate tube samples and place biopsy punches in wells of a 96-well plate.</li>
	<li>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.</li>
	<li>Block samples with 0.4% bovine serum albumin (BSA) in DPBS for 60 min at room temperature on a shaker.</li>
	<li>After blocking, aspirate BSA and rinse with tris-buffered saline plus 1% Tween-20 (TBST) 3x, 10 min each on a shaker.</li>
	<li>Prepare a working dilution of antibody in 0.4% BSA according to manufacturer&#8217;s recommended dilution for immunoassays. Dilute human CD47 Antibody B6H12-FITC 1:100 in 0.4% BSA.</li>
	<li>Add 200 &#181;l 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).</li>
	<li>Rinse with TBST 3x, 10 min each on a shaker. Aspirate the last TBST rinse and add 200 &#181;l DPBS to tube sample wells.</li>
	<li>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.</li>
	<li>Read FITC signal intensity using a microplate reader with FITC excitation (485 nm) and emission (538 nm) settings.</li>
	<li>Calculate recCD47 bound to polymeric surface based on standard values, accounting for auto-fluorescence and non-specific binding from the antibody control sample.</li>
</ol>
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.
<ol>
	<li>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 &#176;C and allow temperature to equilibrate.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Drain the blood into a waste beaker treated with a 10% bleach solution (or as required by institutional policy).</li>
	<li>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.</li>
	<li>Disinfect the apparatus and any blood contacting surfaces using a 10% bleach solution or according to institutional policy.</li>
	<li>Process samples for fluorescent microscopy or scanning electron microscopy as described below.</li>
</ol>
4. Fluorescent Microscopy and Cell Counting
<ol>
	<li>Prepare a 4% paraformaldehyde (PFA) solution (Caution:&#160;Perform in fume hood).</li>
	<li>Incubate tubing in a 4% PFA solution overnight at 4 &#176;C.</li>
	<li>After overnight incubation, remove 4% PFA and rinse films very gently with DPBS.</li>
	<li>Cut the tubing into sections to expose the lumen surface for staining.</li>
	<li>Stain a section of the tubing with a few drops of mounting media with 4&#39;,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.</li>
	<li>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.</li>
	<li>Record cell counts per field of view and perform appropriate statistical analyses to determine statistical significance of results.</li>
</ol>
5. Scanning Electron Microscopy
<ol>
	<li>Prepare a 2% glutaraldehyde solution (Caution:&#160;Perform in fume hood).</li>
	<li>Incubate tubing in a 2% glutaraldehyde solution overnight at 4 &#176;C.</li>
	<li>After overnight incubation, gently rinse the tubing 3x with DPBS.</li>
	<li>Prepare a 1% solution of osmium tetroxide (Caution:&#160;Severe inhalation hazard! Use in fume hood).</li>
	<li>Incubate tubing in a 1% solution of osmium tetroxide for 15 min at room temperature.</li>
	<li>Gently rinse the tubing 3x with DPBS.</li>
	<li>Prepare a series of ethanol concentrations (25%, 50%, 75%, 95%, and 100% ethanol).</li>
	<li>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.</li>
	<li>Supercritical point dry the tubing with CO2 for 45 min to remove all moisture.</li>
	<li>Mount tube sections on a specimen stub with silver paste or graphite.</li>
	<li>Coat tube sections with 12.5 nm of gold-palladium.</li>
	<li>Examine in a scanning electron microscope. &#160;&#160;</li>
</ol>

<ol><li>Bruck, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Medical+applications+of+polymeric+materials%5BTitle%5D)+AND+%22Med.+Prog.+Technol%22%5BJournal%5D)">Medical applications of polymeric materials.</a> Med. Prog. Technol. 9 (1), 1-16 (1982).</li>
<li>Anderson, J. M., Rodriguez, A., Chang, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Foreign+body+reaction+to+biomaterials%5BTitle%5D)+AND+%22Semin.+Immunol%22%5BJournal%5D)">Foreign body reaction to biomaterials.</a> Semin. Immunol. 20 (2), 86-100 (2008).</li>
<li>Levy, J. H., Tanaka, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+response+to+cardiopulmonary+bypass%5BTitle%5D)+AND+%22Ann.+Thorac.+Surg%22%5BJournal%5D)">Inflammatory response to cardiopulmonary bypass.</a> Ann. Thorac. Surg. 75, S715-S720 (2003).</li>
<li>Sperling, C., Maitz, M. F., Talkenberger, S., Gouzy, M. F., Groth, T., Werner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+blood+reactivity+to+hydroxylated+and+non-hydroxylated+polymer+surfaces%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((In+vitro+hemocompatibility+of+self-assembled+monolayers+displaying+various+functional+groups%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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. S. Improved biomaterials for tissue engineering applications: surface modification of polymers. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aVasita%2C+R+%22Curr.+Top.+Med.+Chem%22">Curr. Top. Med. Chem</a>. 8, 341-353 (2008).</li>
<li>Subramanian, S., Parthasarathy, R., Sen, S., Boder, E. T., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Species-+and+cell+type-specific+interactions+between+CD47+and+human+SIRPalpha%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Species- and cell type-specific interactions between CD47 and human SIRPalpha.</a> Blood. 107 (6), 2548-2556 (2006).</li>
<li>Tsai, R. K., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+%27self%27+engulfment+through+deactivation+of+myosin-II+at+the+phagocytic+synapse+between+human+cells%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Innate+immune+%27self%27+recognition%3A+a+role+for+CD47-SIRPalpha+interactions+in+hematopoietic+stem+cell+transplantation%5BTitle%5D)+AND+%22Trends+Immunol%22%5BJournal%5D)">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/pubmed?term=((Role+of+CD47+as+a+marker+of+self+on+red+blood+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((The+effect+of+CD47+modified+polymer+surfaces+on+inflammatory+cell+attachment+and+activation%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Diminished+adhesion+and+activation+of+platelets+and+neutrophils+with+CD47+functionalized+blood+contacting+surfaces%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Intracellular+signaling+mechanisms+associated+with+CD47+modified+surfaces%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Immune+inhibitory+receptors%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Immune inhibitory receptors.</a> Science. 290, 84-89 (2000).</li>
<li>Chandler, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+thrombotic+coagulation+of+blood%3A+a+method+for+producing+a+thrombus%5BTitle%5D)+AND+%22Lab+Invest%22%5BJournal%5D)">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. T., Holmsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bubble-induced+aggregation+of+platelets%3A+effects+of+gas+species%2C+proteins%2C+and+decompression%5BTitle%5D)+AND+%22Undersea+Hyperb+Med%22%5BJournal%5D)">Bubble-induced aggregation of platelets: effects of gas species, proteins, and decompression.</a> Undersea Hyperb Med. 20 (2), 101-119 (1993).</li>
<li>Ritz-Timme, S., Eckelt, N., Schmidtke, E., Thomsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genesis+and+diagnostic+value+of+leukocyte+and+platlet+accumulations+around+%E2%80%9Cair+bubbles%E2%80%9D+in+blood+after+venous+air+embolism%5BTitle%5D)+AND+%22Intl+J+of+Legal+Med%22%5BJournal%5D)">Genesis and diagnostic value of leukocyte and platlet accumulations around “air bubbles” in blood after venous air embolism.</a> Intl J of Legal Med. 111 (1), 22-26 (1998).</li>
<li>Miller, R., Fainerman, V. B., Wüstneck, R., Krägel, J., Trukhin, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+the+initial+period+of+protein+adsorption+by+dynamic+surface+tension+measurements+using+different+drop+techniques%5BTitle%5D)+AND+%22Colloids+and+Surfaces+A%22%5BJournal%5D)">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. F., de Hart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+modified+chandler%2C+roller+pump%2C+and+ball+valve+circulation+models+for+in+vitro+testing+in+high+blood+flow+conditions%3A+application+in+thrombogenicity+testing+of+different+materials+for+vascular+applications%5BTitle%5D)+AND+%22Int+J+Biomater%22%5BJournal%5D)">Comparison of modified chandler, roller pump, and ball valve circulation models for in vitro testing in high blood flow conditions: application in thrombogenicity testing of different materials for vascular applications.</a> Int J Biomater. 2012, (2012).</li>
</ol>

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><tbody><tr><td>16% Paraformaldehyde (PFA)</td><td>Thermo Scientific</td><td>58906</td><td>Caution! Use in fume hood</td></tr><tr><td>25% Glutaraldehyde</td><td>VWR</td><td>AAA17876-AP&amp;nbsp;</td><td>Caution! Use in fume hood</td></tr><tr><td>2-pyridyldithio,benzophenone (PDT-BzPH)</td><td>Synthesized in lab</td><td>N/A</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A3059-100G</td><td></td></tr><tr><td>Citrate</td><td>Sigma</td><td>S5770-50ML</td><td></td></tr><tr><td>Digital Camera</td><td>Leica</td><td>DC500</td><td>Out of production</td></tr><tr><td>Dimethylformamide (DMF)</td><td>Sigma</td><td>270547-100ML</td><td>Caution! Use in fume hood</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (DPBS)</td><td>Gibco/Life Technologies</td><td>14190-136</td><td></td></tr><tr><td>Fluorescent Microscope</td><td>Nikon</td><td>TE300</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Fisher Scientific</td><td>A38-212</td><td>Caution! Use in fume hood</td></tr><tr><td>Human CD47 (B6H12) &amp;ndash; FITC Antibody</td><td>Santa Cruz Biotechnology</td><td>SC-12730</td><td></td></tr><tr><td>Osmium Tetroxide</td><td>Acros Organics</td><td>197450050</td><td>Caution! Use in fume hood</td></tr><tr><td>Potassium Bicarbonate (KHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma</td><td>237205-100G</td><td></td></tr><tr><td>Potassium Phosphate Monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>P5655-100G</td><td></td></tr><tr><td>PVC Tubing (Cardiovascular Procedure Kit)</td><td>Terumo Cardiovascular Systems</td><td>60050</td><td>Most clinical-grade tubing will work</td></tr><tr><td>Scanning Electron Microscope</td><td>JEOL</td><td>JSM-T330A</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Microplate Reader</td><td>Molecular Devices</td><td>Spectramax Gemini EM&amp;nbsp;</td><td></td></tr><tr><td>Sulfo-SMCC</td><td>Sigma</td><td>M6035-10MG</td><td>Moisture Sensitive!</td></tr><tr><td>tris (2-carboxyethyl) phosphine (TCEP-HCl)</td><td>Thermo Scientific</td><td>20491</td><td></td></tr><tr><td>Tween-20</td><td>Bio-Rad</td><td>170-6531</td><td></td></tr><tr><td>Vectashield with DAPI</td><td>Fisher Scientific</td><td>H-1200</td><td>Light sensitive!</td></tr><tr><td>Zeba Spin Desalt Columns &amp;ndash; 7 K MWCO</td><td>Thermo Scientific</td><td>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 ...

the-use-ex-vivo-chandler-loop-apparatus-to-assess-biocompatibility

Research

JoVE Journal

Bioengineering

11.6K Views.  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. 

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

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

Developmental Biology

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

Engineering

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

3D Angiography for Venous Compliance Assessment in Sheep

Quantifying Inferior Vena Cava Compliance and Distensibility in an In Vivo Ovine Model Using 3D Angiography

Synthetic vascular grafts overcome some challenges of allografts, autografts, and xenografts but are often more rigid and less compliant than the native vessel into which they are implanted. Compliance matching with the native vessel is emerging as a key property for graft success. The current gold standard for assessing vessel compliance involves the vessel's excision and ex vivo biaxial mechanical testing. We developed an in vivo method to assess venous compliance and distensibility that better reflects natural physiology and takes into consideration the impact of a pressure change caused by flowing blood and by any morphologic changes present. 
This method is designed as a survival procedure, facilitating longitudinal studies while potentially reducing the need for animal use. Our method involves injecting a 20 mL/kg saline bolus into the venous vasculature, followed by the acquisition of pre and post bolus 3D angiograms to observe alterations induced by the bolus, concurrently with intravascular pressure measurements in target regions. We are then able to measure the circumference and the cross-sectional area of the vessel pre and post bolus. 
With these data and the intravascular pressure, we are able to calculate the compliance and distensibility with specific equations. This method was used to compare the inferior vena cava's compliance and distensibility in native unoperated sheep to the conduit of sheep implanted with a long-term expanded polytetrafluorethylene (PTFE) graft. The native vessel was found to be more compliant and distensible than the PTFE graft at all measured locations. We conclude that this method safely provides in vivo measurements of vein compliance and distensibility.

Author Spotlight: Improved Imaging for Neovascular Development in Congenital Heart Disease Research

This protocol allows for the in vivo quantification of venous compliance and distensibility using catheterization and 3D angiography as a survival procedure allowing for a variety of potential applications.

Synthetic vascular grafts overcome some challenges of allografts, autografts, and xenografts but are often more rigid and less compliant than the native ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

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

Samenvatting

Meer video's verkennen