Name: nonhuman primate lung decellularization and recellularization using a specialized large-organ bioreactor
Uploaded: 2013-12-15T20:00:00
Description: Watch this Scientific Journal Video about Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor at JoVE.com

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 ad...

<ol>
	<li>Ott, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat. Med. 14, 213-221 (2008).</li><li>Badylak, S. F., Taylor, D., Uygun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-organ+tissue+engineering:+decellularization+and+recellularization+of+three-dimensional+matrix+scaffolds.">Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds.</a> Annu. Rev. Biomed. Eng. 13, 27-53 (2011).</li><li>Ott, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung.">Regeneration and orthotopic transplantation of a bioartificial lung.</a> Nat. Med. 16, 927-933 (2010).</li><li>Price, A. P., England, K. A., Matson, A. M., Blazar, B. R., Panoskaltsis-Mortari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+decellularized+lung+bioreactor+system+for+bioengineering+the+lung:+the+matrix+reloaded.">Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.</a> Tissue Eng. Part A. 16, 2581-2591 (2010).</li><li>Petersen, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+lungs+for+in+vivo+implantation.">Tissue-engineered lungs for in vivo implantation.</a> Science. 329, 538-541 (2010).</li><li>Song, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+in+vivo+function+of+bioartificial+lungs+in+rats.">Enhanced in vivo function of bioartificial lungs in rats.</a> Ann. Thorac. Surg. 92, 998-1005 (2011).</li><li>Seok, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+responses+in+mouse+models+poorly+mimic+human+inflammatory+diseases.">Genomic responses in mouse models poorly mimic human inflammatory diseases.</a> Proc. Natl. Acad. Sci. U.S.A. , (2013).</li><li>Joag, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primate+models+of+AIDS.">Primate models of AIDS.</a> Microbes Infect. 2, 223-229 (2000).</li><li>Hu, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-human+primate+models+for+AIDS+vaccine+research.">Non-human primate models for AIDS vaccine research.</a> Curr. Drug. Targets Infect. Disord. 5, 193-201 (2005).</li><li>Borda, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+immunopathologic+alterations+in+rhesus+macaques+affected+with+globoid+cell+leukodystrophy.">Clinical and immunopathologic alterations in rhesus macaques affected with globoid cell leukodystrophy.</a> Am. J. Pathol. 172, 98-111 (2008).</li><li>Fabbrini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+response+to+high-carbohydrate+and+low-carbohydrate+meals+in+a+non-human+primate+model.">Metabolic response to high-carbohydrate and low-carbohydrate meals in a non-human primate model.</a> Am. J. Physiol. Endocrinol. Metab. , (2012).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32, 3233-3243 (2011).</li><li>Calle, E. A., Petersen, T. H., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+Lung+Engineering.">Procedure for Lung Engineering.</a> J. Vis. Exp. (49), e2651 (2011).</li><li>Arold, S. P., Wong, J. Y., Suki, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+new+stretching+apparatus+and+the+effects+of+cyclic+strain+and+substratum+on+mouse+lung+epithelial-12+cells.">Design of a new stretching apparatus and the effects of cyclic strain and substratum on mouse lung epithelial-12 cells.</a> Ann. Biomed. Eng. 35, 1156-1164 (2007).</li><li>Huang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+from+embryonic+stem+cells+to+vascular+wall+cells+under+in+vitro+pulsatile+flow+loading.">Differentiation from embryonic stem cells to vascular wall cells under in vitro pulsatile flow loading.</a> J. Artif. Organs. 8, 110-118 (2005).</li><li>Izadpanah, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+multipotent+mesenchymal+stem+cells+from+the+bone+marrow+of+rhesus+macaques.">Characterization of multipotent mesenchymal stem cells from the bone marrow of rhesus macaques.</a> Stem Cells Dev. 14, 440-451 (2005).</li><li>Craig, L. E., Nealen, M. L., Strandberg, J. D., Zink, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+replication+of+ovine+lentivirus+in+endothelial+cells+cultured+from+different+tissues.">Differential replication of ovine lentivirus in endothelial cells cultured from different tissues.</a> Virology. 238, 316-326 (1006).</li><li>Craig, L. E., Spelman, J. P., Strandberg, J. D., Zink, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+from+diverse+tissues+exhibit+differences+in+growth+and+morphology.">Endothelial cells from diverse tissues exhibit differences in growth and morphology.</a> Microvasc. Res. 55, 65-76 (1998).</li><li>Bonvillain, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Nonhuman+Primate+Model+of+Lung+Regeneration:+Detergent-Mediated+Decellularization+and+Initial+In+Vitro+Recellularization+with+Mesenchymal+Stem+Cells.">A Nonhuman Primate Model of Lung Regeneration: Detergent-Mediated Decellularization and Initial In Vitro Recellularization with Mesenchymal Stem Cells.</a> Tissue Eng. Part A. 18, 2437-2452 (2012).</li><li>Badylak, S. F., Taylor, D., Uygun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Organ+Tissue+Engineering:+Decellularization+and+Recellularization+of+Three-Dimensional+Matrix+Scaffolds.">Whole-Organ Tissue Engineering: Decellularization and Recellularization of Three-Dimensional Matrix Scaffolds.</a> Annu. Rev. Biomed. Eng. , (2010).</li></ol>

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.

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			<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>
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			<td height="36" style="height:36px;width:175px;">Antibiotic-Antimycotic</td>
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			<td style="width:145px;">15240062</td>
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			<td>Cell Culture Media</td>
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			<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>
			<td style="width:132px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:175px;">Endothelial Cell Growth Supplement (ECGS)</td>
			<td style="width:135px;">ScienCell</td>
			<td style="width:145px;">1052</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 Seeding and Bioreactor Culture</td>
		</tr>
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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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			<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>
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		<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>
			<td style="width:132px;"></td>
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			<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>
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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

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.

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 ...

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.

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.

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

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.
Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

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.

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.

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

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.

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.

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.

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