The authors wish to thank the editors of Tissue Engineering for allowing images from a previous publication to be used in this report.

1. Whole-organ Macaque Lung Decellularization
<ol>
	<li>Preparation of Solutions
	<ol>
		<li>Autoclave 10-15 L of deionized water (dH2O) in 1-2 L bottles.</li>
		<li>Prepare &#34;Triton solution&#34; (0.1% Triton X-100 in dH2O) by mixing 1 ml Triton X-100 in 999 ml of dH2O while stirring on a magnetic stirrer. Filter the solution through a 0.22 &#181;m filter apparatus. Store at room temperature.</li>
		<li>Prepare &#34;SDC solution&#34; (2% SDC in dH2O) by slowly adding 20 g of sodium deoxycholate (SDC) to ~900 ml dH2O while stirring. Once SDC is dissolved transfer the solution to a graduated cylinder and bring the volume to 1 ml with dH2O. Filter the solution through a 0.22 &#181;m filter apparatus. Store at room temperature.</li>
		<li>Prepare &#34;NaCl solution&#34; by adding 58 g of NaCl to ~900 ml of dH2O and stir until completely dissolved. Add dH2O to a final volume of 1 L, filter, and store at room temperature.</li>
	</ol>
	</li>
	<li>Organ preparation
	<ol>
		<li>Utmost care must be taken to prevent infectious exposure incidents when working with macaque tissues. Put on the following personal protective equipment (PPE) before handling nonhuman primate tissues: surgical mask, face shield, surgical gloves, disposable gown, and a second layer of surgical gloves.</li>
		<li>Collect intact heart-lung blocs from animal necropsy in PBS containing 50 U/ml heparin to prevent coagulation of external blood.</li>
		<li>With the lungs lying flat in a dissecting tray, cannulate the pulmonary artery using a female Luer connector with an appropriately sized barb. Secure the cannula in place with an alcohol-sterilized zip tie.</li>
		<li>Slip a sterile zip tie around the trachea, insert a 0.250 in&#160;female Luer connector into the tracheal opening, and tighten the zip tie around the barb of the connector to hold the trachea in place around the barb.</li>
		<li>Remove trapped air from the lungs by instilling PBS containing 30 U/ml heparin and 5 &#181;g/ml sodium nitroprusside (SNP). Allow the solution to be expelled by natural recoil and repeat instillation twice more. After the third instillation, cap the tracheal Luer cannula with a Luer plug to hold the solution in the lungs.</li>
		<li>Cut off the apex of the heart and irrigate the internal ventricles with PBS-heparin-SNP to remove residual blood. Lacerate both atria to facilitate drainage of the pulmonary circuitry upon perfusion.</li>
		<li>Submerge the heart-lung bloc in dH2O. Fill a 60 ml syringe with the PBS-heparin-SNP solution. Using forceps, submerge the arterial cannula so that all air bubbles escape from the vascular opening. Attach the syringe and carefully remove the plunger and allow the liquid to flow into the vasculature.</li>
		<li>Remove the plug from the tracheal cannula and allow the fluid in the airway to be expelled by natural recoil. Continue to add PBS-heparin-SNP solution to the syringe connected to the vascular cannula in order to maintain ~15-20 cm H2O pressure above the artery. Continue perfusion until as much blood as possible is removed from the pulmonary vasculature.</li>
	</ol>
	</li>
	<li>Decellularization
	<ol>
		<li>Day 1: Submerge the heart-lung bloc in dH2O. Inflate and perfuse the lung with dH2O using 60 ml&#160;syringes attached to the submerged tracheal and arterial cannulae, respectively. Repeat the airway and vascular washes with dH2O 4x more for a total of five rinses.</li>
		<li>Remove the lungs from water and submerge the bloc in Triton solution. Inflate and perfuse the lungs with Triton solution as before. Repeat the instillation a second time, and incubate the submerged organs in Triton solution overnight at 4 &#176;C.</li>
		<li>DAY 2: After overnight incubation, remove the lung bloc from Triton solution, wash externally with dH2O, and then submerge the bloc in fresh dH2O. Repeat dH2O washes 5x as in step&#160;1.3.1.</li>
		<li>Remove the lungs from dH2O and submerge in SDC solution. Inflate and perfuse with SDC solution in the same manner as in step 1.3.2. Incubate submerged in SDC solution overnight at 4 &#176;C.</li>
		<li>DAY 3: Wash the tissue again with dH2O as in 1.3.1.</li>
		<li>Remove the tissue from dH2O and submerge in NaCl solution. Inflate and perfuse with NaCl solution in the same manner as in step 1.3.2. Incubate submerged in NaCl solution for 1 hr at room temperature.</li>
		<li>Wash the lungs clean of NaCl solution with dH2O as in step 1.3.1.</li>
		<li>Prepare fresh &#34;DNase solution&#34; (30 &#181;g/ml DNase, 1.3 mM MgSO4, 2.0 mM CaCl2 in dH2O) Bathe the lungs in this solution and instill into the airway and vasculature as in step 1.3.2. Incubate the lungs in DNase solution for 1 hr at room temperature.</li>
		<li>Prepare fresh &#34;PBS solution&#34; (1x PBS + 5x antibiotic/antimycotic). Remove the lungs from DNase solution and wash externally with PBS solution. Submerge the lungs in PBS solution and wash with this solution five times as in step 1.3.1.</li>
		<li>Store the lungs in PBS solution in a sealed container overnight at 4 &#176;C. On the next day (DAY 4), wash the lungs in fresh, ice-cold PBS solution five times as in step 1.3.1 and store in PBS solution in a sterile, sealed container at 4 &#176;C until use.</li>
	</ol>
	</li>
</ol>
2. Large-organ Bioreactor
<ol>
	<li>Assembly and Tissue Installation
	<ol>
		<li>Assemble breathing loop and vascular loop structures as well as tracheal and vascular cannulae adapters ahead of time (Figure 1). With the exception of sterile-packaged air filters and syringe ports, autoclave all bioreactor components.</li>
		<li>Assemble the bioreactor components under a laminar flow hood according to the schematic in Figure 2. Fill the main chamber with culture medium that has been equilibrated to the 5% CO2 atmosphere of a cell culture incubator immediately prior to use in the bioreactor.</li>
		<li>Apply the tracheal and vascular cannulae adapters to the lung cannulae and install the organs in the main bioreactor chamber by bathing the lungs in the culture medium and attaching the cannulae adapters to the appropriate ports in the modified lid. Once connected, affix the lid securely and tightly; the chamber will not be opened again for the duration of recellularization.</li>
		<li>Fill the tracheal reservoir approximately half-full with culture medium and affix the modified cap.</li>
		<li>Apply sterile 0.22 &#181;m syringe filters, Luer plugs, and syringe ports to all chambers as indicated.</li>
		<li>Connect the trachea reservoir to the main chamber via the breathing loop apparatus. Attach the breathing loop tubing as indicated.</li>
		<li>Aspirate air from the tubing using the syringe ports and a 60 ml syringe fitted with an 18 G needle; move the directionality of the 3-way stopcocks to direct the flow of liquid into the syringe.</li>
		<li>Move the sealed, contiguously connected bioreactor chambers with installed organs to a tissue culture incubator to equilibrate temperature and gas. Ventilate the lungs using a syringe pump attached to the main chamber at ~1 full &#34;breath&#34; every 2 min, and perfuse the vasculature via the peristaltic pump at approximately 10 ml/min for a total of ~30 min.</li>
	</ol>
	</li>
	<li>Airway seeding
	<ol>
		<li>Prepare suspensions of cells to be seeded into the airway at the desired cell density (volume will depend on organ size).</li>
		<li>Inflate the lungs with the cell suspension by gently injecting through the syringe port attached to the three-way stopcock in the breathing loop.</li>
		<li>Hold the lungs statically at 37 &#176;C, 5% CO2 overnight without airway or vascular perfusion to allow cells to attach to the decellularized lung matrix.</li>
		<li>After overnight incubation, reinitiate the standard airway ventilation program, and culture the organs for 3-7 days (or other desired duration).</li>
	</ol>
	</li>
	<li>Vascular seeding
	<ol>
		<li>Prepare suspensions of endothelial cell cultures as above for airway cell seeding.</li>
		<li>Transfer the cell suspension to the endothelial-seeding reservoir that contains a small magnetic stir bar.</li>
		<li>Change the directionality of the flow path from the main chamber to the endothelial seeding reservoir by rotating the valve on the stopcock positioned in the tubing connecting these two compartments, and seed endothelial cells gradually while gently stirring using the peristaltic pump.</li>
		<li>When seeding is complete (i.e.&#160;the volume of endothelial cell suspension is depleted in the endothelial seeding reservoir), stop perfusion and incubate statically for ~4-6 hr. Reinitiate vascular perfusion with main chamber medium at a rate of ~10 ml/min (or desired pressure if monitored).</li>
		<li>Culture for 3-7 days with continuous vascular perfusion.</li>
	</ol>
	</li>
</ol>
3. Troubleshooting and Alternative Approaches
<ol>
	<li>Tracheal cannulation: Sutures can be used in lieu of a zip tie to hold the Luer cannula in place if the trachea is too small. A &#188; in&#160;female Luer connection acts as a sufficient cannula for a wide range of trachea diameters.</li>
	<li>Removing blood by pulmonary artery perfusion: Since tissues are collected from cadaveric animals, microthrombi are common and prevent some areas of the pulmonary capillary bed from being completely cleared of residual blood. During decellularization, however, these small vessels become permeable, and the decellularization reagents efficiently lyse and degrade these clots. Predecellularization perfusion should be performed to clear as much blood as possible, but it is not necessary to remove all traces of blood from the parenchyma.</li>
	<li>Expulsion of fluids from the lungs: Macaque lungs retain strong natural recoil ex vivo, even when inflated with fluids. During decellularization, however, cells are lysed and release materials (including DNA) that increase the viscosity of the instilled fluids thereby making them more difficult to expel. Overnight incubation at 4 &#176;C while submerged in the instilled fluid is usually sufficient to allow the lungs to completely expel the viscous fluid. Alternatively, allow as much fluid as possible to be expelled from the lungs and then proceed with instillation of the subsequent solutions; addition of detergents and deionized water washes usually facilitates the removal of the viscous fluids and allows decellularization to proceed efficiently.</li>
	<li>Addition of antibiotics to detergent solutions: Antibiotics and antimycotics can be added to deionized water wash solutions as well as detergent solutions; however, penicillin/streptomycin precipitates in the presence of sodium deoxycholate, and these precipitates may interfere with the decellularization and washing processes. Moreover, the presence of antibiotics/antimycotics is not necessary during decellularization as the detergents used at these concentrations are able to lyse and kill microbes. After decellularization, rinsing and storage with PBS solution (containing 5x penicillin/streptomycin/amphotericin B) is sufficient to prevent most contamination.</li>
	<li>Removal of air from bioreactor tubing: Air is efficiently aspirated from the bioreactor tubing using a syringe with a needle inserted into the needle ports in the three-way stopcocks. Alternatively, 1x PBS or culture media can be flushed through the tubing before installing the organs using a syringe attached in the same manner.</li>
	<li>Ventilation and perfusion of lungs during and after recellularization: While the flow rates and times listed in this report were sufficient for cell attachment as well as airway liquid ventilation and vascular perfusion, these values should be determined empirically by the user. Culture times can also be adjusted based on the growth rates of the instilled cells.</li>
</ol>

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We have nothing to disclose.

Tissues can be efficiently and effectively decellularized by a number of methods employing physical, chemical, and enzymatic agents12,20. The challenges of producing 3D biological matrices from large organs include the requirement for large volumes of decellularization solutions, expensive commercial equipment (i.e. bioreactors), and a dizzying amount of methodological perturbations required to achieve the final tissue-derived product. Our method provides a straightforward approach that minimizes phys...

Bioengineering of tissues and organs is an attractive addition to the field of regenerative medicine. The creation of &#34;lab-grown&#34; organs that are suitable for transplant into patients to replace functionality of diseased organs is highly desirable in order to meet the current and future demand for transplantation needs. The principles of tissue engineering center around the seeding of desired cell types, or progenitors thereof, into a scaffold that supports the shape of the engineered tissue while supplying the appropriate growth factors and culture conditions necessary to mimic developmental or regenerative processes. While synthetic scaffolds have been used for tissue engineering, and the natural extracellular matrix (ECM) may be the best source of organ-specific scaffolds for this purpose. Whole-organ decellularization is a process which allows the removal of cells while leaving the chemical and structural aspects of the native ECM intact. The resulting acellular matrix scaffold can be used as a platform onto which regenerative cells can be seeded and cultured in vitro1,2.
Several rodent models of lung decellularization and subsequent recellularization have been developed to study the feasibility of this technology3-6. While offering essential proof-of-concept, rodent models are not directly translatable to human clinical needs. A recent study pointed out that genomic responses to traumatic injury (and related inflammation) do not correlate well between mice and humans; these findings raise questions of the validity of using mice as models for such complex biochemical processes in humans7. Nonhuman primate (NHP) models offer the advantage of closely resembling the biology of humans at the genomic, anatomic, and physiologic levels and allow more flexible manipulation for greater extrapolation to human use. The rhesus macaque has been used in a variety of preclinical applications and is an excellent model in which to study tissue engineering8-11. We recently described the successful decellularization of rhesus macaque (Macacca mulatta) lungs utilizing a procedure that has minimum impact on the lung ECM12.&#160;Lung decellularization is accomplished by treating the tissue consecutively with four decellularization solutions composed of detergents, salts, and enzymes with intermittent washing with deionized water (dH2O) and PBS. We have optimized this procedure by modifying a protocol originally described by Price et al.4 A variety of histological and protein analytical techniques were used to characterize the components of resulting acellular matrices relative to native macaque lungs.
In this report, we demonstrate a detailed protocol for the decellularization of nonhuman primate lungs and the recellularization of the resulting acellular lung scaffolds in a large-organ bioreactor originally demonstrated by Calle et al.13&#160;in JoVE.&#160;By modifying their original protocol to accommodate the size, ventilation, and perfusion requirements for large-animal lungs, the technology was successfully moved from the rodent model to the rhesus macaque model. All studies presented in this report were performed in accordance with the Institutional Biosafety Committee (IBC) policies in place at the Tulane National Primate Research Center. Demonstration of this technique is essential because identification of anatomical structures and physical manipulation of larger organs is sometimes difficult without visualizing the steps of the protocol. The studies made possible by these methods provide a basis for essential preclinical studies in decellularized rhesus macaque lungs in which recellularization with rhesus mesenchymal lineage stem cells and rhesus microvascular endothelial cells can be assessed. Our version of this bioreactor simulates the developmental environment and applies forces of mechanical stretch and strain in large-animal lungs and allows the investigation of lung recellularization under conditions known to facilitate pulmonary and endothelial development13-15.

<table border="0" cellpadding="0" cellspacing="0" width="587">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Sodium nitroprusside</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:145px;">71778-25G</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Heparin sodium salt</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:145px;">BP2425</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Triton X-100</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:145px;">BP151-100</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Sodium deoxycholate</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:145px;">BP349-100</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Sodium chloride</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:145px;">7647-14-5</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Bovine pancreatic DNase</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:145px;">DN25</td>
			<td style="width:132px;">Prepare stock, aliquot, and freeze</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Magnesium sulfate</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:145px;">10034-99-8</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Calcium chloride</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:145px;">C614-500</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">PBS (no Ca/Mg)</td>
			<td style="width:135px;">Gibco, Life Technologies</td>
			<td style="width:145px;">10010-031</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Antibiotic-Antimycotic</td>
			<td style="width:135px;">Gibco Life Technologies</td>
			<td style="width:145px;">15240062</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td></td>
			<td></td>
			<td></td>
			<td>Cell Culture Media</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Alpha MEM</td>
			<td style="width:135px;">Gibco Life Technologies</td>
			<td style="width:145px;">12561-072</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Medium 199</td>
			<td style="width:135px;">Gibco Life Technologies</td>
			<td style="width:145px;">11150067</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Premium Fetal Bovine Serum</td>
			<td style="width:135px;">Atlanta Biologicals</td>
			<td style="width:145px;">S11150</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">L-Glutamine 100x</td>
			<td style="width:135px;">Gibco Life Technologies</td>
			<td style="width:145px;">25030-081</td>
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			<td height="36" style="height:36px;width:175px;">Endothelial Cell Growth Supplement (ECGS)</td>
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			<td style="width:145px;">1052</td>
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		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Antibiotic-Antimycotic</td>
			<td style="width:135px;">Gibco Life Technologies</td>
			<td style="width:145px;">15240062</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td></td>
			<td></td>
			<td></td>
			<td>Cell Seeding and Bioreactor Culture</td>
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			<td height="36" style="height:36px;width:175px;">Check valves</td>
			<td style="width:135px;">Cole-Parmer</td>
			<td style="width:145px;">EW-98553-20</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Y-connectors</td>
			<td style="width:135px;">Cole-Parmer</td>
			<td style="width:145px;">ED-30614-08</td>
			<td style="width:132px;"></td>
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		<tr height="36">
			<td height="36" style="height:36px;width:175px;">3-Way stopcocks</td>
			<td style="width:135px;">Harvard Apparatus</td>
			<td style="width:145px;">721664</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">MasterFlex L/S 14 tubing</td>
			<td style="width:135px;">Cole-Parmer</td>
			<td style="width:145px;">96420-14</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">MasterFlex L/S 16 tubing</td>
			<td style="width:135px;">Cole-Parmer</td>
			<td style="width:145px;">96420-16</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Male lock Luer 1/8 in</td>
			<td style="width:135px;">Cole-Parmer</td>
			<td style="width:145px;">EW-45505-04</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Female Luer 1/8 in</td>
			<td style="width:135px;">Cole-Parmer</td>
			<td style="width:145px;">SI-45502-04</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Male Luer lock plug</td>
			<td style="width:135px;">Cole-Parmer</td>
			<td style="width:145px;">EW-45505-56</td>
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		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Injection ports</td>
			<td style="width:135px;">Medi-Dose EPS</td>
			<td style="width:145px;">IV2004</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Latex tubing</td>
			<td style="width:135px;">St. Louis Medical Suppy</td>
			<td style="width:145px;">HN10910</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Hose clamp</td>
			<td style="width:135px;">Cole-Parmer</td>
			<td style="width:145px;">EW-06832-02</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">2 L Wide-mouth jar</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:145px;">05-719-276&#160;</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">60 ml Syringes</td>
			<td style="width:135px;">Fisher</td>
			<td style="width:145px;">NC9035364</td>
			<td style="width:132px;"></td>
		</tr>
	</tbody>
</table>

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.

Results shown below represent separate experiments in which either airway or vascular compartments were seeded with rhesus macaque bone marrow-derived mesenchymal stem cells or rhesus lung-derived microvascular endothelial cells, respectively, that were isolated and characterized as previously described16-18.
Throughout the decellularization process, macaque lungs displayed a progressive whitening culminating in a translucent appearance at the end of the process; however, the lungs ...

Watch this Scientific Journal Video about Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor at JoVE.com

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

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 ...

nonhuman-primate-lung-decellularization-recellularization-using

Research

JoVE Journal

Bioengineering

9.7K ビュー.  Tulane University School of Medicine. 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.

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

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

全肝臓の細胞化とRecellularization

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

生物工学

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

拍動バイオリアクターを用いた工学生物学ベースの人工血管

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

ベンチトップリアクターの操作

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

使い捨て空気圧バイオリアクターシステムを使用して、哺乳動物細胞の培養

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

Decellularization とひと伏在静脈の Recellularization 方法

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

逆方向に加圧ポーチ内全体の人間の心の decellularization

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Isolation and Decellularization of a Whole Porcine Pancreas

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an allogeneic or xenogeneic source with human cells. A demonstration of methods for the efficient dissection, decellularization, and recellularization of porcine pancreas might benefit the field. Akin to human pancreases, porcine pancreases have a special anatomical arrangement with three lobes (splenic, duodenal, and connection) rounded by the duodenum and small intestine. The duodenal lobe of the pancreas connects to the duodenum by several small blood vessels. Tissue engineering of the pancreas is complicated because of its exocrine and endocrine nature. In this paper, we show a detailed protocol to dissect the whole porcine pancreas and decellularize it with detergents while saving its structure and some extracellular matrix components. To achieve complete perfusion, the aorta is chosen as inlet and the portal vein as outlet. The other blood vessels (hepatic artery, splenic vein, splenic artery,&#160;mesenteric artery and vein tree) and bile duct are ligated. To prevent the formation of thrombus, the pig is heparinized and, immediately after dissection, the organ is flushed with cold heparin. To inhibit the action of exocrine enzymes, the pancreas decellularization is set at 4 &#176;C. The decellularization is performed by perfusion of Triton X-100, sodium deoxycholate, and deoxyribonuclease, with an intermittent and final extensive washing. With a successful decellularization, the pancreas appears white, and a histological evaluation with hematoxylin and eosin shows an absence of nuclei with a preserved extracellular matrix structure. Thus, the proposed method can be used to successfully dissect and decellularize whole porcine pancreas.

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

分離と全体ブタ膵臓の Decellularization

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an ...

A MRI-Based Toolbox for Neurosurgical Planning in Nonhuman Primates

In this paper, we outline a method for surgical preparation that allows for the practical planning of a variety of neurosurgeries in NHPs solely using data extracted from magnetic resonance imaging (MRI). This protocol allows for the generation of 3D printed anatomically accurate physical models of the brain and skull, as well as an agarose gel model of the brain modeling some of the mechanical properties of the brain. These models can be extracted from MRI using brain extraction software for the model of the brain, and custom code for the model of the skull. The preparation protocol takes advantage of state-of-the-art 3D printing technology to make interfacing brains, skulls, and molds for gel brain models. The skull and brain models can be used to visualize brain tissue inside the skull with the addition of a craniotomy in the custom code, allowing for better preparation for surgeries directly involving the brain. The applications of these methods are designed for surgeries involved in neurological stimulation and recording as well as injection, but the versatility of the system allows for future expansion of the protocol, extraction techniques, and models to a wider scope of surgeries.

The method outlined below aims to provide a comprehensive protocol for the preparation of nonhuman primate (NHP) neurosurgery using a novel combination of three-dimensional (3D) printing methods and MRI data extraction.

非ヒト霊長類の脳神経外科計画のためのMRIベースのツールボックス

In this paper, we outline a method for surgical preparation that allows for the practical planning of a variety of neurosurgeries in NHPs solely using ...

Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict regional aerosol delivery. Leveraging 3D printing to generate patient-specific lung models, we outline the design of a high-throughput, in vitro experimental setup for quantifying lobular pulmonary deposition. This system is made with a combination of commercially available and 3D printed components and allows the flow rate through each lobe of the lung to be independently controlled. Delivery of fluorescent aerosols to each lobe is measured using fluorescence microscopy. This protocol has the potential to promote the growth of personalized medicine for respiratory diseases through its ability to model a wide range of patient demographics and disease states. Both the geometry of the 3D printed lung model and the air flow profile setting can be easily modulated to reflect clinical data for patients with varying age, race, and gender. Clinically relevant drug delivery devices, such as the endotracheal tube shown here, can be incorporated into the testing setup to more accurately predict a device&#8217;s capacity to target therapeutic delivery to a diseased region of the lung. The versatility of this experimental setup allows it to be customized to reflect a multitude of inhalation conditions, enhancing the rigor of preclinical therapeutic testing.

We present a high-throughput, in vitro method for quantifying regional pulmonary deposition at the lobe level using CT scan-derived, 3D printed lung models with tunable air flow profiles.

患者固有の3Dプリント肺モデルを用いた局所肺堆積の評価

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict ...

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces.

滑膜関節バイオメカニクス、メカノバイオロジー、物理調節の研究のための摩擦試験バイオリアクター装置

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...