Name: artificial lung device priming for in situ fiber bundle surface grafting
Uploaded: 2025-03-28T12:50:00.000+00:00
Duration: 8 min 53 s
Description: Although the high surface area-to-volume ratio of the artificial lung fiber bundle enhances gas exchange, the large surface area, dense arrangement, and ...

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><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&amp;amp;page=1&amp;amp;perpage=30&amp;amp;sort=relevance&amp;amp;term=citrate-phosphate%20buffer&amp;amp;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&amp;amp;page=1&amp;amp;perpage=30&amp;amp;sort=relevance&amp;amp;term=horseradish%20peroxidase%20conjugated%20anti-fibrinogen&amp;amp;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&amp;amp;page=1&amp;amp;perpage=30&amp;amp;sort=relevance&amp;amp;term=human%20fibrinogen&amp;amp;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 (NaIO&lt;sub&gt;4&lt;/sub&gt;)</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

Research

JoVE Journal

Bioengineering

253 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

Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.

The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

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 ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

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

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

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

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

Engineering

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Medicine

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and posttransplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. 
The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering.

This paper describes how to create bioengineered mouse lungs using decellularization and recellularization methods. It also details subsequent orthotopic lung transplantation.

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor ...

Artificial Lung Device Priming for In Situ Fiber Bundle Surface Grafting

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Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

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

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Engineered Lung Tissues Prepared from Decellularized Lung Slices

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells