The authors would like to thank Jack Milwid for the design of the in vitro perfusion chamber. This work was supported by grants from the US NIH, R01DK59766 and R01DK084053 to M.Y., R00DK080942 to K.U., US NSF CBET- 0853569 to K.U. and the Shriners Hospitals for Children to B.E.U. (grant no. 8503). We also acknowledge support and the Shriners Hospitals for Children.

1. Liver Decellularization
<ol>
	<li>Harvest a rat liver with portal vein cannulation using and 18-gauge catheter. Leave the inferior and superior vena cava open. Keep the organ hydrated in phosphate buffered saline (PBS) in a 10-cm petri dish.</li>
	<li>Set up a perfusion system that consists of 8-liter reservoir, peristaltic pump and a bubble trap.</li>
	<li>Fill the perfusion system with phosphate buffered saline and keep it running for 10 minutes. Fill PBS into a 10-cm petri dish and reduce the flow rate of phosphate buffered saline to 1 mL/min.</li>
	<li>Carefully transfer the harvested liver to the phosphate buffered saline filled 10-cm petri dish.</li>
	<li>Continue with PBS perfusion overnight.</li>
	<li>Start perfusion with 0.01% (w/v) sodium dodecyl sulfate (SDS) in distilled water for 5 minutes.</li>
	<li>Perfuse with PBS for 1 hour.</li>
	<li>Repeat steps 1.6 and 1.7 three more times increasing the SDS perfusion time to 10, 15 and 20 minutes at each time.</li>
	<li>Continue perfusion with 0.01% (w/v) SDS for 24 hours.</li>
	<li>Continue perfusion with 0.1% (w/v) SDS for 24 hours.</li>
	<li>Perfuse with 0.2% (w/v) SDS for 3 hours.</li>
	<li>Perfuse with 0.5% (w/v) SDS for 3 hours.</li>
	<li>Perfuse with distilled water for 15 minutes.</li>
	<li>Perfuse with 1% (w/v) Triton X-100 in distilled water for 30 minutes to remove any bound nucleic acids.</li>
	<li>To wash the decellularized liver matrix (DLM), perfuse with PBS for 2 hours.</li>
	<li>Optional: Resect all lobes except the median lobe.</li>
	<li>Store the DLM in a clean and sealed Petri dish soaked in PBS at 4oC until ready to use (Figure 1).</li>
	<li>To sterilize the DLM: 1) Flush the DLM with sterile PBS containing 0.1% (v/v) peracetic acid and 4% (v/v) ethanol and incubate for 3 hours at 4oC. 2) Wash with sterile PBS 2 times. 3) Wash with sterile PBS containing 2% penicillin-streptomycin, 10ug/ mL gentamicin, and 2.5 ug/ mL amphotericin B. Store the decellularized liver in the same solution at 4oC until ready to use for recellularization experiments.</li>
</ol>
2. Recellularization of Decellularized Liver Matrix
<ol>
	<li>Set up a perfusion system that consists of a perfusion chamber, peristaltic pump and a bubble trap under sterile conditions. Fill the perfusion system with 200 mL of culture medium, e.g., high glucose DMEM (Sigma), 10% fetal bovine serum (Hyclone,), 100 U mL-1 penicillin, and 100 &mu;g mL-1 streptomycin (Invitrogen).</li>
	<li>Place the decellularized liver matrix in the perfusion chamber and connect the DLM to the perfusion system through the portal vein cannula while the pump is running at 5 mL/min to avoid formation of any air bubbles.</li>
	<li>Allow perfusion of the medium through the DLM for 30 minutes.</li>
	<li>Isolate primary hepatocytes from an adult rat with at least 90% viability.</li>
	<li>Stop the flow in the perfusion system and slowly inject 50 million hepatocytes (in 1-3 mL of culture medium) into perfusion system through the bubble trap.</li>
	<li>Start the flow at 10 mL/min and recirculate the medium for 10 minutes.</li>
	<li>Repeat steps 2.5 and 2.6 until a total of 200 million cells are introduced into the DLM (Figure 2)6.</li>
	<li>Once all the cells are perfused into the DLM, collect the perfusate into four 50-ml centrifuge tubes and centrifuge at 600 rpm for 10 minutes. Discard the supernatants and collect the pellets into a single tube. Determine the number of cells and viability via trypan blue exclusion to determine the seeding efficiency.</li>
</ol>
3. In Vitro Culture of the Recellularized Liver Graft
<ol>
	<li>Set up a perfusion system that consists of a perfusion chamber, peristaltic pump, oxygenator and a bubble trap under sterile conditions (Figure 3). Fill the perfusion system with 50 mL of culture medium, e.g., Williams' E (Sigma), 5% fetal bovine serum (Hyclone,),0.5 U mL-1 insulin (Eli Lilly), 20 ng mL-1 EGF (Invitrogen), 14 ng mL-1 glucagon(Bedford Laboratories),7.5 &mu;g mL-1 hydrocortisone (Pharmacia), 100 U mL-1 penicillin, and 100 &mu;g mL-1 streptomycin (Invitrogen).</li>
	<li>Place the recellularized liver graft in the perfusion chamber and connect the DLM to the perfusion system through the portal vein cannula while the pump is running at 5 mL/min to avoid formation of any air bubbles.</li>
	<li>Aseptically close the perfusion chamber and seal tightly to avoid any leakage during culture.</li>
	<li>Transfer the perfusion system to an incubator that is 37oC and has 10% CO2 and increase the perfusion flow rate to 15 mL/min.</li>
	<li>Connect the oxygenator to a 95% O2 and 5% CO2 gas mixture tank and set the gas flow rate to 0.5 liters/min. This should achieve an oxygen partial pressure of approximately 400 mmHg.</li>
	<li>The culture may continue up to 10 days with daily changes of culture medium. The culture medium may be sampled daily for monitoring of liver functions of the graft such as albumin, urea and total bile acid secretion. At the end of the culture period, the recellularized liver graft may be sampled for molecular and histological analysis.</li>
</ol>
4. Representative Results:
The complete decellularization of a rat liver takes about 72 hours using the described protocol. The resulting matrix retains 100% of the fibrillar collagen, 50% of the glycosaminoglycans and only 5% of the DNA of the native liver (Table 1)6. The vascular structure of the matrix is preserved as evidenced by corrosion casting and scanning electron microscopy analysis (Figure 4)6. The presence of the vascular microarchitecture within the DLM facilitates its repopulation with cells with an efficiency of 96% and its subsequent perfusion for in vitro culture. The recellularized liver graft may be cultured up to 10 days in vitro and displays proper liver functions as confirmed via albumin, urea and total bile acid secretion (Figure 5)6.
<img src="/files/ftp_upload/2394/2394fig1.jpg" alt="Figure 1" /> 
Figure 1. Decellularized liver matrix at the end of the decellularization process. (a) the whole liver (b) the median lobe after resection.
<img src="/files/ftp_upload/2394/2394fig2.jpg" alt="Figure 2" /> 
Figure 2. Schematic representation of the recellularization of the DLM.
<img src="/files/ftp_upload/2394/2394fig3.jpg" alt="Figure 3" /> 
Figure 3. Perfusion system setup for in vitro culture of the recellularized liver graft.
<img src="/files/ftp_upload/2394/2394fig4.jpg" alt="Figure 4" /> 
Figure 4. The microvascular structure is retained in the decellularized liver matrix. Corrosion cast images of a) a normal liver b) a decellularized liver, portal (red) and venous (blue) vasculature. Scanning electron microscopy images of the DLM c) a vessel, d) a section featuring bile duct-like small vessels (arrows), Scale bars (a,b) 5 mm (c,d) 20 &mu;m.
<img src="/files/ftp_upload/2394/2394fig5.jpg" alt="Figure 5" /> 
Figure 5. Liver specific functions of the recellularized liver graft during the in vitro perfusion culture. a) albumin secretion (p = 0.5249), b) urea production (p = 0.5271) and c) total bile acid secretion (p = 0.0114). Statistical analysis of the difference between the experiment and the control was done over the 10 d culture period by Friedman's test at a = 0.01. Error bars represent s.e.m. (n = 3).
<table border="1">
<tbody>
 <tr>
 <td>&nbsp;</td>
 <td>Fresh livera</td>
 <td>Decellularized liver matrixa</td>
 <td rowspan="2">p-values</td>
 <td rowspan="2">% of fresh liver</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>n = 4</td>
 <td>n = 8</td>
 </tr>
 <tr>
 <td>Collagen</td>
 <td rowspan="2">0.07 &plusmn; 0.01</td>
 <td rowspan="2">0.08 &plusmn; 0.03</td>
 <td rowspan="2">0.56</td>
 <td rowspan="2">114%</td>
 </tr>
 <tr>
 <td>(mg per g liver)</td>
 </tr>
 <tr>
 <td>Glycosaminoglycans</td>
 <td rowspan="2">73.1 &plusmn; 6.7</td>
 <td rowspan="2">34.2 &plusmn; 2.9</td>
 <td rowspan="2">0.004</td>
 <td rowspan="2">47%</td>
 </tr>
 <tr>
 <td>(mg per g liver)</td>
 </tr>
 <tr>
 <td>DNA</td>
 <td rowspan="2">14.9 &plusmn; 5.6</td>
 <td rowspan="2">0.44 &plusmn; 0.08</td>
 <td rowspan="2">3.3 10-5</td>
 <td rowspan="2">2.9%</td>
 </tr>
 <tr>
 <td>(mg per g liver)</td>
 </tr>
 </tbody>
</table>
Table 1. The biochemical composition of the decellularized liver matrix compared to the native liver. 
aValues are represented as mean &plusmn; s.e.m.

<ol>
	<li>Kulig, K. M., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+tissue+engineering.">Hepatic tissue engineering.</a> Transpl Immunol. 12, 303-310 (2004).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nature biotechnology. 23, 47-55 (2005).</li><li>Dahl, S. L., Koh, J., Prabhakar, V., 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=Decellularized+native+and+engineered+arterial+scaffolds+for+transplantation.">Decellularized native and engineered arterial scaffolds for transplantation.</a> Cell Transplant. 12, 659-666 (2003).</li><li>Schechner, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+a+vascularized+human+skin+equivalent.">Engraftment of a vascularized human skin equivalent.</a> FASEB J. 17, 2250-2256 (2003).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F., F, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+tissues+and+organs.">Decellularization of tissues and organs.</a> Biomaterials. 27, 3675-3683 (2006).</li><li>Uygun, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat Med. , (2010).</li></ol>

No conflicts of interest declared.

The perfusion decellularization method described here produces a whole liver scaffold that has the same gross structure and the vascular microarchitecture of the native liver. The scaffold has an extracellular matrix composition similar to the native liver. The recellularization method achieves repopulation of the scaffold with cells at high efficiency and the cells remain viable and functional during the in vitro culture period tested. With the addition of nonparenchymal cells into the recellularized liver graf...

A correction was made to <a href="http://www.jove.com/Details.php?ID=2394">Decellularization and Recellularization of Whole Livers</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Nima Saeidi
instead of:
Nima Saedi.

Erratum: Decellularization and Recellularization of Whole Livers

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sodium dodecyl sulfate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L4390</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Masterflex L/S Digital Drive</td>
<td>&nbsp;</td>
<td>Cole-Parmer</td>
<td>EW-07523-80</td>
<td>&nbsp;</td>
<tr>
<td>Masterflex L/S Standard pump head</td>
<td>&nbsp;</td>
<td>Cole-Parmer</td>
<td>EW- 07013-81</td>
<td>&nbsp;</td>
<tr>
<td>Bubble trap</td>
<td>&nbsp;</td>
<td>Radnoti</td>
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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.

Watch this Scientific Journal Video about Decellularization and Recellularization of Whole Livers at JoVE.com

Decellularization and Recellularization of Whole Livers

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 ...

decellularization-and-recellularization-of-whole-livers

Research

JoVE Journal

Bioengineering

21.9K ビュー.  Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children. 灌流細胞化は、臓器の細胞外マトリックスの組成とマイクロアーキテクチャを保持する全体の肝臓の足場を生成する新規な手法です。ここで、肝細胞で灌流細胞化およびその後の再投入を使って臓器全体の足場を準備する方法について説明する。機能性と移植肝移植は、このテクニックを使用して生成することができます。

ビデオ: Decellularization and Recellularization of Whole Livers

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

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, ...

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 ...

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

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

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

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

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

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 ...

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.

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.

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

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...