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 ...

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 coat...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

212 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. ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

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.