This work was funded in part through a services agreement under NIH 1R01HL140231-01A1.

The protocol follows the guidelines of the human research ethics committee of the University of New Haven.
1. Coating of the artificial lung circuit
NOTE: The artificial lung circuit was coated following a two-step zwitterionic DOPA-SBMA grafting approach. The fiber bundle/oxygenator details are proprietary information. In experiments where the effects of flow on the antifouling activity of coated lung was the focus, the oxygenator and tubing circuit (5/16&#34; Tygon tubing with conjugable polycarbonate connectors) was subjected to a 24 h phosphate-buffered saline (PBS; pH 7.34, temp. ~37 &#176;C) flow limited by our pump&#39;s maximum flow rate of 1.22 L/min. For context, the exact flow rates in oxygenators depend on the patient&#39;s needs and the specific clinical situation, but they generally range from 2-7 L/min in adult patients. PBS was used in this case to allow for a facile quantification of key blood coagulation marker fouling on coated fibers after flow. Any confounding effects from whole blood fouling, if whole blood flow were used to evaluate the effects of shear rate on the antifouling activity of coated fibers, would be eliminated by using PBS. The following steps were used to coat the artificial lung:
<ol>
	<li>Clean the lung circuit by recirculating 30% methanol in deionized (DI) water for 20 min, followed by recirculating 10% methanol in DI water and DI water for 20 min.</li>
	<li>Dry the circuit with filtered house air at low flow for 2 h prior to ultraviolet ozonolysis (UVO) plasma exposure.</li>
	<li>Place the cleaned lung circuit into a UVO plasma generator (UVO-Cleaner), close it, and turn on the UVO plasma generator instrument for plasma interaction with the device for 20 min.</li>
	<li>After plasma exposure, the device should proceed to the coating step without delay to limit surface chain rearrangement and hiding of reactive sites generated from plasma interaction.</li>
	<li>While the surfaces of the device are being modified, prepare a fresh coating solution. In 600 mL of TRIS buffer (pH 8.5), dissolve 1.2 g of Dopamine-HCl followed by dissolving sulfobetaine methacrylate (SBMA) monomer at 1:15 DOPA:SMBA ratio.</li>
	<li>Add 5 mM of sodium periodate (20 &#181;L) droplets to the coating solution and mix. Use a glass beaker, a magnetic stir bar, and a stir plate at 150 rpm to aid in mixing. 
	NOTE: Add the coating solution to the device no more than 20 min after preparation. Therefore, if multiple lungs are coated, a facile way of priming the devices- for example, the use of pumps for priming- can be helpful.</li>
	<li>Prime the lung device with the coating solution using a large volume syringe (60 cc) to draw and fill the circuit. With one end of the circuit clamped, prime it from the other, ensuring that air bubbles can be guided out of the circuit through a circuit access connector.</li>
	<li>When fully primed, place the device under a UV light source for 2 h and agitate the solution by re-orienting the ends of the primed device up and down every 10 min.</li>
	<li>After the UV light treatment, drain the circuit and gently rinse all samples with deionized (DI) water by priming with a 60 cc syringe and draining repeatedly. When the effluent of the DI rinse is clear, store the device primed with DI water in a 4 &#176;C fridge for autopsy and surface analyses.</li>
</ol>
2. Lung autopsy
<ol>
	<li>Drain and dry lung circuit with filtered and gentle airflow in a chemical hood.</li>
	<li>Perform device autopsy by fixing them in a Bench Vise w/ Swivel Base. Use a bandsaw to section the inlet/outlet connections to the device housing and the front and back face plates of the housing, carefully cutting along their peripheries using an industrial-grade X-acto knife and a hammer.</li>
	<li>After removing the face plates, fiber mat layers are accessible. Carefully section across fibers to access fiber matt samples at any location within the bundle (e.g., the surface and within). Handle samples with tweezers at their edges and transfer them into 60 mL tubes filled with deionized water to limit sample contamination.</li>
</ol>
3. Evaluation of protein fouling on coated lung circuit materials
<ol>
	<li>Fibrinogen adsorption test.
	<ol>
		<li>Incubate size (~1 cm x 1 cm) standardized fiber matt samples in 1 mL of 3 mg/mL fibrinogen in well plates for over 90 min at 37 &#176;C with agitation at 60 rpm.</li>
		<li>Wash (3x) samples with PBS buffer, transfer them into new wells, and add 1 mL of 1 mg/mL of bovine serum albumin (BSA) to each well. Incubate for another 90 min, then wash again with PBS buffer (3x) and transfer samples into new wells.</li>
		<li>In the new wells, add 1 mL of 1:1000 dilution of Horse radish peroxidase (HRP) conjugated fibrinogen antibody in PBS to each well and incubate for 30 min. Then, wash (3x) samples with PBS buffer and transfer them into new wells.</li>
		<li>In the new wells, add 500 &#181;L of 1 mg/mL o-Phenylenediamine (OPD) in 0.1 M citrate-phosphate buffer with 0.03% hydrogen peroxide, pH of 5.0 at 30 s intervals and incubate away from light for 30 min.</li>
		<li>Halt the peroxidase and OPD reaction by adding 500 &#181;L of 1 N HCL to each well.</li>
		<li>Remove and transfer supernatant from each well to a cuvette. Measure the absorbance of supernatant using a UV-visible spectrophotometer at 492 nm.</li>
	</ol>
	</li>
	<li>Platelet adhesion test.
	<ol>
		<li>Thaw the lactate dehydrogenase (LDH) assay kit for 20 min.</li>
		<li>While the thawing is underway, prepare pooled adult human plasma to obtain platlete rich plasma (PRP).</li>
		<li>To prepare PRP, centrifuge thawed human plasma sample tubes at a hard spin at 483 x g to separate the plasma into two regions, in which the lower one-third is PRP and the upper two-thirds of the tube will contain the platelet poor plasma (PPP).</li>
		<li>Remove platelet pellets formed at the bottom of the tube as well as the upper two-thirds at the centrifuged blood plasma and disperse the pellet into the upper two-thirds of plasma by gently shaking the tubes.</li>
		<li>Add calcium chloride (0.2 M) to the PRP (1:1 v/v) to reverse the effects of citrates before testing.</li>
		<li>Incubate the samples (~1 cm x 1 cm) in 500 &#181;L of PRP for 90 min at 37 &#176;C, then rinse three times with PBS buffer, and transfer them into new wells.</li>
		<li>Add 300 &#181;L of PBS and 10 &#181;L of 10x lysis buffer and incubate for 45 min.</li>
		<li>Add 50 &#181;L of the reaction mix and incubate for 30 min away from light.</li>
		<li>Add 50 &#181;L of HCL to stop well reactions.</li>
		<li>To detect the LDH activity of lysates from adsorbed platelets, measure the light absorbance by the developed well solutions at 490 nm and 680 nm wavelengths and subtract the 680 nm reading from the 490 nm to analyze platelet adhesion.</li>
	</ol>
	</li>
</ol>
4. Flow effects on antifouling activity
<ol>
	<li>Prime the artificial lung circuit with PBS and ensure no leakage. Then, affix the circuit to the roller pump and recirculate the PBS for 24 h. 
	NOTE: A flow rate of 1.22 L/min was the highest achievable.</li>
	<li>To simulate the normothermic temperature of the blood that would flow through the lung device, incubate the artificial lung in a 37 &#176;C water bath during the recirculation.</li>
	<li>Then, perform lung autopsy and protein fouling challenge studies as described above. 
	NOTE: One-way analysis of variance was used to determine if there were statistically significant differences among the means of the independent groups, and Tukey&#39;s HSD was used to determine which specific groups differed.</li>
</ol>

Zwitterionic Polymer Grafts for Antifouling in Artificial Lung Fiber Bundles

The authors declare no competing financial interests. Dr. Keith Cook and Dr. David Skoog hold ownership equity in ART LLC.

The PDMS-coated polypropylene (PP) fibers in the artificial lung demonstrated a relationship between ozone exposure and fiber structure, establishing a sensitization limit for ultraviolet ozone plasma. This limit guides the exposure times needed to generate surface radicals for grafting coating materials, specifically polydopamine and polysulfobetaine methacrylate. When the fibers were exposed to ultraviolet ozone plasma for less than 20 min, no significant structural changes were observed. However, exposure times of 30 min or more resulted in noticeable macroscopic changes, such as curling, stiffening, and increased brittleness. After applying the antifouling zwitterionic grafting, X-ray photoelectron spectroscopy confirmed the presence of the polydopamine/poly sulfobetaine coating on the fibers.
Key conclusions about the antifouling activity of coated fibers directly supported by the study outcomes data are that 1) fiber surface protein absorption depended on the location of the fiber in the bundle where outer bundle fibers, compared to the inner bundle fibers, showed reduced fibrinogen fouling, and 2) fibrinogen adsorption level on each coated substrate was significantly lower than fouling on uncoated material control. Also, platelet adsorption significantly decreased due to polydopamine/polysulfobetaine coating on the inner bundle fibers but increased significantly on the outer bundle fibers compared to uncoated fibers. An interesting and unexpected result that was consistent in the fibrinogen fouling test by ELISA and fluorescein isothiocyanate (FITC)-conjugated antibody to human fibrinogen adsorption and platelet adhesion test was the near equalization of fouling between the outer and inner bundle fibers after the artificial lung was exposed to recirculatory flow for 24 h. This unexpected outcome may be due to fibrinogen converting to fibrin, which did not result in higher absorbance levels-not because there was less fibrinogen, but because it had transformed into fibrin. Since fibrinogen antibodies vary in their ability to distinguish between fibrin (mature) and fibrinogen (immature), this could have influenced the results. The observed decrease in platelet fouling on the outer bundle after 24 h of exposure to PBS could be related to changes in the surface density of polydopamine (pDOPA)40,41,42,43. The surface density of pDOPA plays a crucial role in modulating platelet adhesion. While an optimal pDOPA surface density can reduce platelet adhesion and improve biocompatibility, deviations from this optimal density (either too low or too high) can have the opposite effect. In the case of the coated lung, the outer layer fibers appeared darker due to higher amounts of pDOPA adhesion to their surfaces. This dense pDOPA formation may have undergone de-densification to improve platelet anti-adhesion levels. Careful control of pDOPA deposition parameters may, therefore, be essential when dopamine is used as linkers for coatings in the design of blood-contacting devices with improved anti-thrombotic activity.
The lack of uniformity of the coating appearance, we think, is attributable mainly to the virtually stagnant coating solution, which can promote settlement of the coating solution between reorientations of the primed circuit during coating. As PDA molecules get heavier with polymerization during the coating process, their increasing molecular weight can accelerate their settlement. Prior to priming, the devices were also not flushed with carbon dioxide (CO2), which has higher solubility in aqueous media than air, and therefore it is possible that not all trapped air pockets within the bundle were removed. As air pockets within lung devices can limit effective priming, commercial ECMO lungs are CO2 flushed before priming with saline to prepare the ECMO circuit for extracorporeal circulation.
Several commercially available ECMO devices utilize antifouling technologies, including 1) a system that uses a heparin coating to reduce clot formation and protein adhesion44,45,46,47, 2) another which is a heparin-based coating that reduces thrombogenicity and biofouling48,49,50,51, 3) a phosphorylcholine coating, which mimics the natural cell membrane, reducing protein adsorption and platelet activation, leading to better biocompatibility and reduced fouling, and 4) poly-2-methoxyethylacrylate, a hydrophilic polymer coating (poly-2-methoxyethylacrylate) that creates a water-attracting surface, reducing protein and platelet adhesion, and thus minimizing biofouling and thrombus formation52,53,54,55. The first two systems work by enhancing the activity of antithrombin III plasma enzyme to inhibit thrombin generation. They are not hydrophilic coatings like the zwitterionic pSBA brushes evaluated in this study. In addition, the antifouling activity of pSBA is only second to polycarboxybetaine (pCBA). Altogether, these coatings help extend the lifespan of ECMO devices, reduce the risk of complications, and improve patient outcomes by minimizing interactions between the device surfaces and biological elements.
The effectiveness of the hydrophilic pSBMA coating on porous polypropylene (PP) fiber materials used in commercially available lungs also needs to be addressed. The potential of increased wetting of the porous PP fibers by hydrophilic coatings and the resulting impact on lung function remain underexplored. Plasma leakage is a concern in PP fibers that feature porosities. Although the PP material is hydrophobic, its pores get wet over time, which can lead to blood plasma seeping into the air space. This leakage is a mass transport problem in that water, proteins, and cells flow through those pores from blood, and the transport process is bound by pore size, effective void fraction in the PP fiber, size of transport elements, and other parameters such as blood flow rate and temperature56,57,58. Therefore, optimization of the PP fiber drawing process to reduce pore size and maintain the pore void fraction needed for efficient gas transfer could impact the leakage. At small (e.g., nanoscale) pore sizes it is possible that the zwitterionic polymer brushes and their strongly bound and stable water molecules themselves can serve as an additional physical barrier that limits plasma transport while improving wetting, antifouling properties, reducing the risk of thrombosis and improving biocompatibility.
The utility of this method of coating lungs could have broader impacts. The SARS-co-V2 pandemic, which has recently been categorized as an endemic disease, was a healthcare crisis that required accelerated technology development and emergency use authorization. ECMO was one of the technologies that afforded patients with severe cases of COVID-19 better prognostic outcomes, although its thrombogenic materials that warrant systemic anticoagulation associated with bleeding risks remained a significant concern59,60,61. In such healthcare crises, blood-contacting life support devices could be modified with our method to enhance care outcomes. Additionally, specific patient populations who are critically ill and require bridging to transplantation on ECMO or the use of thrombogenic blood contacting devices can benefit from enhanced device surface-focused anticoagulation.
The in vitro tests conducted were fibrinogen adsorption by conventional ELISA and by FITC-conjugated antibody to human fibrinogen fouling onto coated fibers sectioned from the outer or inner locations of the fiber bundle and platelet adsorption on these fibers. Together, they evaluate the ability of coatings on the artificial lung fibers to resist fibrinogen and platelet adhesion as these blood coagulation factors play integral roles in the contact, extrinsic, and common phases of the blood coagulation pathway. Optimization studies are now warranted to enhance the antifouling activity of coatings on the fiber bundle. Future work to assess the evenness of zwitterionic polysulfobetaine coating on the rest of the circuit is also vital.
This study has limitations in its methods that need improvement. The 1 x 1 cm samples that were evaluated are small compared to the large gas exchange surface area of the fibers. As such, larger surface area inspections and evaluations can improve the error rate associated with small area samples. Another limitation is that performing fouling assays on an artificial lung post-autopsy utilizes stagnant platelets which are incubated with hollow fiber samples. These platelets do not experience the turbulent nature of flow through a hollow fiber device. Therefore, the patterns of deposition (outer vs inner fibers) may differ when blood products are ultimately flowed through these devices. Using a blood flow model containing labeled platelets and fibrinogen may improve the utility of the presented model. For the evaluation of adhered fibrinogen, it is possible that fibrin formation was induced, mimicking or bypassing the thrombin pathway. Such formed fibrin will be less sensitive to fibrinogen antibodies62 utilized in the fibrinogen ELISA assay and can underestimate the detected amount of adsorbed fibrinogen. Additionally, the coating uniformity of the ECMO circuit tubing needs to be evaluated as they make up significant surface areas and present blood flow pattern disruption points where connectors or changes in tubing diameter exist. Also, the entire assembled lung circuit was placed in the UVO plasma generator as it was expected that although the strength of the top-down UV light interaction with parts of the circuit can be different across the depth UVO plasma chamber, sub-optimum UVO plasma interaction can catalyze surface activation. We interpreted parts of the system operation instructions of the UVO-cleaner model stating, &#34;For optimum results, the surface of the substrate should be situated approximately 0.20&#34; [5 mm] below the grid lamp&#34; to mean so. However, studying the interaction of UVO plasma with lung fibers placed at different distances from the energy source can yield definite answers. The orientation of the lung was such that the largest cross-sectional area of the bundle faced the UV grip lamp. For the non-commercial lung studied, this orientation resulted in the thinnest device profile from the lamp position to support uniformity in UVO plasma and fiber bundle interaction. The UVO plasma generator operation settings were 6500 V peak-to-peak, 50 mA, 20 min UVO plasma generation, and 3 min exhaust time. We note that the 24 h flow experiment used to evaluate the potential long-term stability and antifouling performance of coatings in the artificial lung is limiting. Patients can be supported on ECMO for longer durations depending on their diagnosis and care being provided. It is typically continued until the cardiac or lung problem improves or resolves in 5 days. However, an ECMO course could be continued for up to 25 days to 30 days. Test durations during the evaluation of methods for enhanced ECMO can provide more detailed and complete data if extended. Furthermore, the time course of fouling dynamics was not fully evaluated by this study; it only captured fouling data at time zero and at 24 h. Additional time points for fouling assessment may provide insights on coating stability and performance for optimization.

Artificial lung fibers, also known as hollow fiber membranes, are essential materials for fabricating extracorporeal membrane oxygenation (ECMO) devices that provide respiratory support to critically ill patients. Multiple layers of these fibers make up a dense bundle that serves as the gas exchange unit. The polymeric fiber surface, however, activates the blood coagulation cascade -leading to clot formation (thrombosis). Thrombosis on artificial surfaces is primarily driven by the activation of the coagulation cascade, a complex series of enzymatic reactions that lead to the formation of a blood clot. When blood comes into contact with foreign materials, such as those in medical devices (e.g., artificial lungs, stents, catheters), the coagulation cascade is triggered1,2. This process begins with the exposure of blood to the surfaces of the artificial material, which activates the intrinsic pathway of the cascade. This activation leads to the generation of thrombin, a key enzyme that converts fibrinogen to fibrin, forming the structural basis of a clot. Simultaneously, platelets are activated and aggregate at the site, further reinforcing the clot. The result is thrombosis, which can obstruct blood flow and lead to serious complications such as stroke or myocardial infarction.
To prevent thrombosis on artificial surfaces, traditional anticoagulants, such as heparin, warfarin, and newer direct oral anticoagulants (DOACs), are commonly used3,4. These medications work by interfering with various steps of the coagulation cascade. For example, heparin enhances the activity of antithrombin III, a natural inhibitor of thrombin, while warfarin inhibits the synthesis of vitamin K-dependent clotting factors. However, the use of anticoagulants presents several challenges. First, they increase the risk of bleeding, which can be life-threatening in certain situations. Second, the effectiveness of anticoagulants can be variable, requiring regular monitoring and dose adjustments, particularly with warfarin. Additionally, long-term anticoagulant use is associated with adverse effects such as osteoporosis and skin necrosis. The need for systemic anticoagulation also limits the use of medical devices in patients who are at high risk for bleeding.
Because thrombosis can impede gas exchange across the hollow fiber membrane, antifouling coatings have been applied to the lung fibers using various methods, such as dip coating and electrospinning, to prevent biofouling5,6. Artificial lung manufacturers typically process hollow fibers that are commercially obtained from fiber manufacturers and assemble them into lungs through steps including bundling of the fibers around a solid core, potting (gluing) bundle ends, incorporating potted bundles in a housing capsule featuring gas and blood flow channels, and post assembly cleaning. While the coating of fibers that have not been implanted in the lung can be more flexible, a surface modification at the pre-bundling stage will be subjected to several manufacturing steps that necessitate mechanical and chemical interactions between the surface coating and the downstream process environments, which can lead to denuded fibers in a device where high coating coverage is essential for limiting thrombosis. Alternatively, the coating can be applied to the potted bundle. An advantage of the ability to coat finished lungs is that it is a practical and facile modification approach to surface engineering the artificial lung device and many other devices. But in general, the method of applying coatings, whether through spray or dip coating, is less critical to thrombosis prevention than the effectiveness of the coating itself. For instance, hollow fibers used in medical devices can be dip-coated during extrusion, then knitted into mats, wound into bundles, and incorporated into a finished artificial lung device. Alternatively, coatings can be applied after the device has been manufactured. The key factors, however, are&#160;the effective application, durability, and efficacy of the anti-thrombotic coating5. This is because, in the absence of systemic anticoagulation, the function of these coatings is an essential piece of the puzzle for preventing clot formation, necessitating the need for a highly efficient and long-lasting antifouling property to ensure effective thrombosis prevention.
Despite the application of antithrombogenic coatings and the simultaneous low-dose administration of anticoagulants to date, the artificial lung module&#160;must be replaced only after a relatively short period of use, ranging from days to 3 weeks7,8, because of thrombosis. The gas exchange efficiency of their fiber membranes deteriorates after a relatively short time because of fouling by a membranous blood clot structure (composed of fibrin, single cells, and cell clusters) that covers large areas of the fibers,&#160;increasing their gas diffusion barrier9. In general, the type of coating and application method10,11,12,13,14,15,16,17,18 used depends on the desired properties, such as biocompatibility and durability. Several examples of antifouling coatings have been used on artificial lung fibers. They include silicone which is widely used due to its biocompatibility19, durability, and resistance to biofouling; polyurethane (PU) due to its biocompatibility and resistance to biofouling20; chitosan due to its biocompatible and antimicrobial properties21,22, heparin that inactivates thrombin23,8, and hydrophilic24 polymer-based coatings including poly(ethylene glycol)25,26, poly (2-methoxyethyl acrylate)27, and phosphorylcholine28,29.
Zwitterionic coatings represent a promising strategy for reducing thrombosis on artificial surfaces without the need for systemic anticoagulation5,6. These coatings are composed of molecules with both positive and negative charges, which balance each other out and result in a highly hydrophilic, non-fouling surface. The zwitterionic nature of these coatings reduces protein adsorption and platelet adhesion, both of which are critical steps in the initiation of the coagulation cascade. By preventing the initial interaction between blood proteins and the artificial surface, zwitterionic coatings effectively inhibit the activation of the coagulation cascade and reduce the risk of thrombosis. This approach not only minimizes the need for systemic anticoagulants but also offers a more biocompatible solution for the long-term use of medical devices.
In this study, we evaluated the effectiveness of priming the artificial lung with an ultra-low fouling zwitterionic poly(sulfobetaine methacrylate) (pSBMA) coating combined with a surface adhesive polydopamine (pDOPA) layer. After priming the device, it was positioned side-to-side every 10 min for 2 h during the coating process. To assess potential variations in coating across the fiber bundle, we measured fibrinogen and platelet fouling on fibers located at the surface and within the bundle. Additionally, we analyzed the impact of flow on the antifouling performance by comparing fouling data from lungs before and after exposure to flow. For long-term antibiofouling applications involving complex media flow, zwitterionic coatings must not only inhibit fouling from whole blood -- a challenging task -- but also maintain their effectiveness under hemodynamic stress throughout the application period. These coatings need to provide strong steric repulsion against non-specific protein adsorption and achieve a suitable surface packing density to form a hydration film barrier between the substrate and the complex media. Moreover, they must remain securely attached to the surface without detachment of the linkers anchoring the coating to the substrate30. The protocol described here is designed to ensure the application of coatings that meet these critical requirements for effective and durable surface protection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Beakers</td>
			<td>Thermo Fisher Scientific</td>
			<td>https://www.thermofisher.com/search/browse/category/us/en/90094065</td>
			<td>Used in experiments</td>
		</tr>
		<tr>
			<td>Beckman Coulter Allegra X-30R centrifuge</td>
			<td>Beckman Coulter</td>
			<td>https://www.mybeckman.in/centrifuges/general-purpose/allegra-x-30</td>
			<td>For centrifugations</td>
		</tr>
		<tr>
			<td>Biochemguard BSL2 safety hood</td>
			<td>Biochemguard</td>
			<td>https://bakerco.com/images/uploads/assets/BiochemGARD_220v_Web_0.pdf</td>
			<td>Used for UV light source in graft coating</td>
		</tr>
		<tr>
			<td>Bovine albumin serum (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>https://www.sigmaaldrich.com/US/en/substance/bovineserumalbumin123459048468</td>
			<td>Fibrinogen assay materials</td>
		</tr>
		<tr>
			<td>Citrated pooled male blood plasma</td>
			<td>ZenBio</td>
			<td>https://www.zen-bio.com/products/serum/human-blood-products.php</td>
			<td>Used for experiments</td>
		</tr>
		<tr>
			<td>Citrate-phosphate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>https://www.sigmaaldrich.com/US/en/search/citrate-phosphate-buffer?focus=products&#38;page=1&#38;perpage=30&#38;sort=relevance&#38;term=citrate-phosphate%20buffer&#38;type=product</td>
			<td>Fibrinogen assay materials</td>
		</tr>
		<tr>
			<td>Dopamine-hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>https://www.sigmaaldrich.com/US/en/product/aldrich/h60255</td>
			<td>For coating</td>
		</tr>
		<tr>
			<td>Dopamine-hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>N/A</td>
			<td>Fibrinogen assay materials</td>
		</tr>
		<tr>
			<td>Fluorescein conjugated Goat Immunoglobulin G (IGG)</td>
			<td>Sigma Aldrich</td>
			<td>https://www.sigmaaldrich.com/US/en/product/mm/aq303f</td>
			<td>For Fluorescence Light Intensity measurements</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase-conjugated anti-fibrinogen antibody</td>
			<td>Sigma-Aldrich</td>
			<td>https://www.sigmaaldrich.com/US/en/search/horseradish-peroxidase-conjugated-anti-fibrinogen?focus=products&#38;page=1&#38;perpage=30&#38;sort=relevance&#38;term=horseradish%20peroxidase%20conjugated%20anti-fibrinogen&#38;type=product</td>
			<td>Fibrinogen assay materials</td>
		</tr>
		<tr>
			<td>Hot Plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>https://www.thermofisher.com/in/en/home/life-science/lab-equipment/hot-plates-stirrers/lab-hot-plates.html</td>
			<td>Used in experiments</td>
		</tr>
		<tr>
			<td>Human fibrinogen powder</td>
			<td>Sigma-Aldrich</td>
			<td>https://www.sigmaaldrich.com/US/en/search/human-fibrinogen?focus=products&#38;page=1&#38;perpage=30&#38;sort=relevance&#38;term=human%20fibrinogen&#38;type=product</td>
			<td>Fibrinogen assay materials</td>
		</tr>
		<tr>
			<td>Jelight UVO-Cleaner model 144AX</td>
			<td>Jelight</td>
			<td>https://www.jelight.com/uvo-cleaner/</td>
			<td>Used for plasma treatment of medical device materials</td>
		</tr>
		<tr>
			<td>LDH assay kit</td>
			<td>ABCAM</td>
			<td>https://www.abcam.com/en-us/products/assay-kits/ldh-assay-kit-lactate-dehydrogenase-assay-kit-colorimetric-ab102526</td>
			<td>For LDH assay</td>
		</tr>
		<tr>
			<td>O-phenylenediamine (OPD)</td>
			<td>Sigma-Aldrich</td>
			<td>https://www.sigmaaldrich.com/US/en/product/sigma/p9029</td>
			<td>Fibrinogen assay materials</td>
		</tr>
		<tr>
			<td>PDMS coated polypropylene fibers</td>
			<td>ART LLC</td>
			<td>N/A</td>
			<td>Part of artificial lung materials</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>https://www.sigmaaldrich.com/US/en/product/sigma/p4417</td>
			<td>Fibrinogen assay materials</td>
		</tr>
		<tr>
			<td>Plate Reader (BioTek)</td>
			<td>BioTek</td>
			<td>https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/plate-reader-metabolic-assays</td>
			<td>For reading Fluorescence Light Intensity</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>ART LLC</td>
			<td>N/A</td>
			<td>Part of artificial lung materials</td>
		</tr>
		<tr>
			<td>Sodium periodate (NaIO4)</td>
			<td>Sigma-Aldrich</td>
			<td>https://www.sigmaaldrich.com/US/en/substance/sodiummetaperiodate213897790285</td>
			<td>For coating</td>
		</tr>
		<tr>
			<td>Stockert Shiley multiflow roller pump</td>
			<td>Sorin Biomedical</td>
			<td>N/A</td>
			<td>For flow experiments</td>
		</tr>
		<tr>
			<td>Sulfobetaine methacrylate (SBMA)</td>
			<td>Sigma-Aldrich</td>
			<td>https://www.sigmaaldrich.com/US/en/search/sulfobetaine-methacrylate-(sbma)</td>
			<td>For coating</td>
		</tr>
		<tr>
			<td>TRIS-buffered saline (pH 8.5)</td>
			<td>Sigma-Aldrich</td>
			<td>https://www.sigmaaldrich.com/US/en/product/sigma/t8793</td>
			<td>Prepared in the lab from TRIS sachets</td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>ART LLC</td>
			<td>N/A</td>
			<td>Part of artificial lung materials</td>
		</tr>
	</tbody>
</table>

artificial lung device priming for in situ fiber bundle surface grafting

Although the high surface area-to-volume ratio of the artificial lung fiber bundle enhances gas exchange, the large surface area, dense arrangement, and surface chemistries of the fibers are major contributors to thrombosis. To mitigate this, it is essential to uniformly modify the surface chemistries to effectively reduce non-specific protein fouling, which can help limit thrombosis and lower the risk of thromboembolism or bleeding caused by systemic anticoagulants.
In this study, we explored the application and antifouling properties of zwitterionic polymer grafts on polypropylene fiber bundles. The grafting process involved priming the artificial lung device with zwitterionic polysulfobetaine molecules and polydopamine linkers for in situ coating. The antifouling performance was evaluated using standard fibrinogen enzyme-linked immunosorbent assay (ELISA) and platelet lactate dehydrogenase fouling assays. X-ray Photoelectron Spectroscopy confirmed the surface coating, and significant reductions in fouling were observed on coated fibers compared to uncoated ones, demonstrating the utility of the grafting process and the promise of its antifouling effects.
However, differences in the appearance of the coating on fibers within the bundle were noted with the coating by priming process, which could affect the overall antifouling performance. Addressing this issue could further enhance the antifouling efficiency of lung fiber bundles modified through in situ grafting.

A protocol for zwitterionic polymer grafting of artificial lung circuit by priming, disassembly of the device for coated fibers sample collection, and antifouling evaluation of sectioned fibers is presented. In Figure 1, the surface modification of the artificial lung circuit approach is illustrated. Lungs were exposed to UVO plasma for the interaction of radical oxygen singlet with fibers, which was followed by steps of zwitterionic pSBMA coating of the artificial lungs by priming with coating solution and 2 h of grafting under a UV light source. Fibers previously treated with ultraviolet ozone plasma are modified with zwitterionic polydopamine/polysulfobetaine methacrylate (pDOPA-pSBMA), where pSBMA is proposed to anchor onto the fiber surface on top of adhered polydopamine via hydrogen bonding and cation-&#960;-interactions. The lung autopsy process of the coated artificial lung for accessing the coated fiber bundle is shown in Figure 2. In Figure 3, a representative surface evaluation of zwitterionic polysulfobetaine-coated artificial lung fibers using X-ray photoelectron spectroscopy wide spectra scans is presented. In Figure 4 and Figure 5, fibrinogen and platelet fouling on coated fibers before and after recirculatory flows are presented.
The fibrinogen and platelet fouling levels were reported in comparison to fouling on uncoated surfaces. This method does not report the actual amounts of adsorbed fibrinogen or platelets like other methods for surface fouling quantification would. For example, ellipsometry is an example of such a method. However, it generally is compatible with flat samples. In addition, the surface plasmon resonance (SPR) technique can detect the presence of fouling, although it does not provide detailed information about the composition of the fouling layer. While fouling is reported as a percent of controls, pSBMA coatings in other studies, however, do show super low fouling (&#60;10 ng/cm2 or &#60;1% of protein adsorption in 100% blood plasma)6,31 indicating greater antifouling activity in the artificial lungs is possible. Figure 4C and Figure 5C were plotted without including uncoated control data from Figure 4A and Figure 5A because that could lead to misleading conclusions. The uncoated control fibers were not exposed to 24 h of PBS flow because (i) it was expected that flow would make no difference in the antifouling activity of uncoated fibers, and (ii) the project period had concluded with no additional devices available for evaluation. While the effect of PBS flow in uncoated fibers on fouling would be interesting to investigate, we point out that Figure 4B and Figure 5B represent coated samples that were not exposed to PBS flow.
The coating has been specifically designed with strong adhesion properties of pDOPA, the key component of the mussel foot protein, which allows strong adhesion to ship hauls. The durability of pDOPA adhesion is also enhanced through chemical cross-linking forming pDOPA, which stabilizes the adhesive network and resists degradation32. pDOPA forms strong covalent and non-covalent bonds with substrates through a variety of mechanisms, including hydrogen bonding, &#960;-&#960; interactions, and metal chelation. This results in a strong initial adhesion to surfaces like metals, oxides, polymers, and even biological tissues32,33,34,35,36. DOPA-based coatings also exhibit good resistance to mechanical stress, such as shear and tensile forces, due to the combination of strong covalent bonding and the formation of a dense, cross-linked network. One of the notable strengths of pDOPA-based adhesion is its performance in wet environments. The coating remains strongly adhered even in aqueous conditions, making it particularly suitable for biomedical applications where surfaces are often exposed to bodily fluids.
However, its adhesion can be influenced by environmental factors. Under acidic or basic conditions, the catechol groups in DOPA can undergo oxidation or reduction, which may weaken the adhesive bonds. Under oxidative conditions, the catechol groups in DOPA can oxidize to quinones. This oxidation can reduce the adhesive strength, although cross-linking reactions can sometimes counterbalance this effect by forming additional bonds. While the coating is expected to perform well under high flow, a systematic evaluation of its durability is critical to characterizing its long-term anti-fouling activity maintenance. However, for the scope of this work, the shear rate around the fibers was estimated by
<img alt="Equation 1" src="/files/ftp_upload/67271/67271eq01.jpg" style="margin: auto;" />
Where:
Q = Volumetric flow rate (m&#179;/s)
R = Radius of the tube (m) = fiber diameter/2
For the maximum flow rate, Q = 1.22 L/min, which equals 2.03 x 10-5 m3/s, and fiber diameter, d = 200 &#181;m and thus R = 100 &#181;m, the calculated shear rate was
<img alt="Equation 2" src="/files/ftp_upload/67271/67271eq02.jpg" style="margin: auto;" />
Actual flow in an artificial lung is more complex, involving not just simple laminar flow but also regions of recirculation and turbulence, especially at high flow rates37,38,39. The above method gives an estimate but does not capture these complexities. Also, blood is a non-Newtonian fluid, meaning its viscosity changes with shear rate. This can affect the exact shear rate experienced in the device. In practice, empirical data or computational fluid dynamics (CFD) simulations are often used to provide more accurate estimates of shear rates in specific oxygenator designs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67271/67271fig01.jpg" /> 
Figure 1: Washing through the coating of the oxygenator with zwitterionic polymer. Steps of zwitterionic polysulfobetaine methacrylate (pSBMA) coating of the artificial lungs. (A) The device coating steps include 20 min ultraviolet oxygen plasma exposure to the oxygenator followed by priming with dopamine (DOPA)/SBMA coating solution, allowing 2 h of grafting under a UV light source. The device is then rinsed with DI water. (B) Zwitterionic polydopamine/polysulfobetaine methacrylate (pDOPA-pSBMA) surface-modification of oxygenator fibers previously treated with ultraviolet ozone plasma. pSBMA is proposed to anchor onto fiber surface adhered (covalent bonding and physisorption) polydopamine via mechanisms including hydrogen bonding and cation-&#960;-interactions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67271/67271fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67271/67271fig02.jpg" /> 
Figure 2: Lung autopsy process of coated artificial lung accessing coated fiber bundle. From left to right: fixing the device in vice, separating housing and face plates from the device, and zwitterionic coating appearance across fiber bundle thickness. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67271/67271fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67271/67271fig03.jpg" /> 
Figure 3: Surface evaluation of zwitterionic polysulfobetaine coated artificial lung fibers. (A) autopsied coated artificial lung showing gradation in zwitterionic coating appearance across fiber bundle thickness, and (B) uncoated and (C) polysulfobetaine methacrylate coated Polypropylene Polydimethylsiloxane skin fibers&#39; XPS wide spectra scans. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67271/67271fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67271/67271fig04.jpg" /> 
Figure 4:&#160;Fibrinogen fouling of artificial lung&#39;s polydimethylsiloxane-coated polypropylene (PP-PDMS) fibers evaluated after polysulfobetaine methacrylate coating of the lung and lung autopsy. (A) Fibrinogen fouling on uncoated fibers in comparison to fouling on zwitterionic polysulfobetaine coated fibers in the outer and inner layers of the artificial lung&#39;s fiber bundle. (B) Fibers in the outer layers of the fiber bundle showed reduced fouling in comparison to those in the inner layers. (C) Fouling on polysulfobetaine methacrylate-coated fibers sectioned from artificial lung perfused with phosphate-buffered saline (37 &#176;C, 24 h). Three stars p &#60; 0.001. Outer fibers were sections from the third outermost fiber mat, and innerfibers were sectioned from midway of the fiber bundle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67271/67271fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67271/67271fig05.jpg" /> 
Figure 5: Platelet fouling of artificial lung&#39;s polydimethylsiloxane-coated polypropylene (PP-PDMS) fibers evaluated after polysulfobetaine methacrylate coating of the lung and lung autopsy. (A) Platelet fouling on uncoated fibers in comparison to fouling on zwitterionic polysulfobetaine coated fibers in the outer and inner layers of the artificial lung&#39;s fiber bundle, (B) fibers in the outer layers of the fiber bundle showed increased fouling in comparison to those in the inner layers, and (C) fouling on poly sulfobetaine methacrylate coated fibers sectioned from artificial lung perfused with phosphate-buffered saline (37 &#176;C, 24 h). Three stars p &#60; 0.001. Outer fibers were sections from the third outermost fiber mat, and innerfibers were sectioned from midway of the fiber bundle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67271/67271fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Artificial Lung Device Priming for In Situ Fiber Bundle Surface Grafting

This study investigated the antifouling activity of artificial lung hollow fibers coated by priming the lung device. While this surface modification of fibers approach is practical, the effectiveness of the coating process depends on the graft coverage across fiber mat layers within the bundle.

Although the high surface area-to-volume ratio of the artificial lung fiber bundle enhances gas exchange, the large surface area, dense arrangement, and ...

artificial-lung-device-priming-for-situ-fiber-bundle-surface

Nanyang Technological University

Research

JoVE Journal

Bioengineering

204 Views.  University of New Haven. This study investigated the antifouling activity of artificial lung hollow fibers coated by priming the lung device. While this surface modification of fibers approach is practical, the effectiveness of the coating process depends on the graft coverage across fiber mat layers within the bundle. 

Video: Artificial Lung Device Priming for In Situ Fiber Bundle Surface Grafting

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.