This study was supported by a Biomedical Research Institute Grant from Pusan National University Hospital.

This study was approved by the administration of Pusan National University of Medicine and was conducted in accordance with ethical guidelines for the use and care of animals. (certificate no. 2017-119). Prior to any animal studies, institutional approval should be obtained. 
NOTE: All surgical and anesthetic instruments/equipment and reagents recommended for successful surgical presentation and imaging of abdominal organs are detailed in Table 1.
1. Preparation procedures for harvesting of rat kidneys
<ol>
	<li>In preparation for surgery, place 8-week-old Sprague-Dawley rats (weighing 200&#8211;250 g) on a warming pad. Place a rectal thermometer probe in the rectum to monitor core temperature.</li>
	<li>Anesthetize the rat with a 5% mixture of isoflurane gas (induction: 5%, maintenance: 3%).</li>
	<li>To start the operation, place the rat in a supine position after administration of anesthesia. Mount the four limbs of the rat on the operation table with tape.</li>
	<li>Shave and clean the abdomen of the donor rat with germicidal soap. Apply 2% betadine for at least 1&#8211;2 min, and wipe with a 70% ethanol solution. Repeat this sequence three times.</li>
	<li>Cover the operative field with a sterile fenestrated drape.</li>
	<li>Make a vertical abdominal incision and expose the left kidney, ureter, abdominal aorta, and inferior vena cava.</li>
	<li>Visualize and dissect the left kidney, ureter, abdominal aorta, and inferior vena cava just before cutting the pedicle.</li>
</ol>
2. Transcardial perfusion
<ol>
	<li>Before surgery, prepare the perfusion solution.
	<ol>
		<li>Make 50 mL of perfusion solution per rat.</li>
		<li>Mix 1x PBS with approximately 10 U/mL heparin (1 25 kU vial will make 2.5 L of PBS+Hep).</li>
		<li>Mix equal volumes of 8% paraformaldehyde with 1x PBS to make the 4% PFA/1xPBS solution. 
		NOTE: 8% PFA made in water can be stored at 4 &#176;C for up to 2 months. However, 4% PFA diluted in PBS is only stable for 1 week at 4 &#176;C. Make the dilution fresh.</li>
	</ol>
	</li>
	<li>Extend the vertical abdominal incision cranially. Be sure to draw the scissors away from the organs when cutting to avoid damaging the internal organs.</li>
	<li>Continue the incision through the rib cage, and then cut through the diaphragm by lifting the sternum.</li>
	<li>Pin the loose flap of skin out of the way, and free the heart by tearing any connective tissue with the forceps. 
	CAUTION: Do not use the scissors to free the heart and this could result in unwanted bleeding.</li>
	<li>Open the phosphate-buffered saline (PBS) line and ensure that the line is flowing before placing the needle into the left ventricle. Hold the heart gently with blunt forceps, and use a hemostat to control the needle. The needle should be inserted no more than 1/4 inch. 
	NOTE: Insertion greater than 1/4 inch may result in perforation to the other side of the tissue.</li>
	<li>While supporting the heart with the needle and hemostat, locate the right atrium and snip through it with iridectomy scissors. Rest the hemostat on the rat&#8217;s body, and make sure the needle is still positioned inside the heart. 
	NOTE: If the cut is sufficient, there should be blood in the body cavity as the pressure from the PBS flowing into the rat is relieved.</li>
	<li>Carefully unpin the front feet and skin flap.</li>
	<li>Continue perfusing PBS for 4 min or longer if there is still blood visible in the kidney and liver.</li>
</ol>
3. Kidney harvesting and decellularization
<ol>
	<li>Harvest the left kidney with the abdominal aorta and inferior vena cava.</li>
	<li>Ligate the ureter, thoracic aorta, superior vena cava, and branches of the abdominal aorta.</li>
	<li>Keep the organ hydrated in Dulbecco&#8217;s PBS (DPBS) in a 10 cm Petri dish.</li>
	<li>Cannulate the abdominal aorta and inferior vena cava with a 23 Gauge catheter. To remove residual blood, connect the cannula with a peristaltic pump, and wash with DPBS (500 mL) and 16 U/mL heparin for 90 min at a rate of 5 rpm at 37 &#176;C.</li>
	<li>To decellularize the kidney, perfuse the kidney with 1% Triton X-100 (1 L) for 3 h and then with 0.75% sodium dodecyl sulfate (SDS) solution (2 L) for 6 h at a constant pressure of 40 mmHg. 
	NOTE: The kidney will become transparent after 8 h.</li>
	<li>To remove residual SDS, perfuse the sample with 1% penicillin in distilled water (6 L) for 18 h (overnight) and then with sterile DPBS (500 mL) and 16 U/mL heparin for 90 min.</li>
</ol>

<ol>
	<li>Kawai, 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=Brief+report:+HLA-mismatched+renal+transplantation+without+maintenance+immunosuppression.">Brief report: HLA-mismatched renal transplantation without maintenance immunosuppression.</a> New England Journal of Medicine. 358 (4), 353-361 (2008).</li><li>Rana, D., Zreiqat, H., Benkirane-Jessel, N., Ramakrishna, S., Ramalingam, M. <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+decellularized+scaffolds+for+stem+cell-driven+tissue+engineering.">Development of decellularized scaffolds for stem cell-driven tissue engineering.</a> Journal of Tissue Engineering and Regenerative Medicine. 11 (4), 942-965 (2017).</li><li>Fu, R. 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=Decellularization+and+recellularization+technologies+in+tissue+engineering.">Decellularization and recellularization technologies in tissue engineering.</a> Cell Transplantation. 23 (4-5), 621-630 (2014).</li><li>Hopkinson, A., 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=Optimization+of+amniotic+membrane+(AM)+denuding+for+tissue+engineering.">Optimization of amniotic membrane (AM) denuding for tissue engineering.</a> Tissue Engineering. Part C, Methods. 14 (4), 371-381 (2008).</li><li>Shupe, T., Williams, M., Brown, A., Willenberg, B., Petersen, 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=Method+for+the+decellularization+of+intact+rat+liver.">Method for the decellularization of intact rat liver.</a> Organogenesis. 6 (2), 134-136 (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 (5991), 538-541 (2010).</li><li>Fernandez-Perez, J., Ahearne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+decellularization+methods+on+extracellular+matrix+derived+hydrogels.">The impact of decellularization methods on extracellular matrix derived hydrogels.</a> Scientific Reports. 9 (1), 14933 (2019).</li><li>Naik, A., Griffin, M., Szarko, M., Butler, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+decellularization+process+of+an+upper+limb+skeletal+muscle;+implications+for+muscle+tissue+engineering.">Optimizing the decellularization process of an upper limb skeletal muscle; implications for muscle tissue engineering.</a> Artificial Organs. 44 (2), 178-183 (2020).</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 (12), 3233-3243 (2011).</li><li>Mason, C., Dunnill, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+definition+of+regenerative+medicine.">A brief definition of regenerative medicine.</a> Regenerative Medicine. 3 (1), 1-5 (2008).</li><li>Guyette, J. P., 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+decellularization+of+whole+organs.">Perfusion decellularization of whole organs.</a> Nature Protocols. 9 (6), 1451-1468 (2014).</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=Regeneration+and+experimental+orthotopic+transplantation+of+a+bioengineered+kidney.">Regeneration and experimental orthotopic transplantation of a bioengineered kidney.</a> Nature Medicine. 19 (5), 646-651 (2013).</li></ol>

The authors have no conflicts of interest to disclose.

Various protocols have been used for decellularization of organs and other tissues. The optimal decellularization protocol should preserve the three-dimensional architecture of the extracellular matrix (ECM). In general, such protocols consist of lysing the cell membrane by physical processing or ionic solutions, dissociating the cytoplasm and nucleus from the ECM by enzymatic processing or detergents, and then removing cellular debris from the tissue3. Physical processes include scraping, solution agitation, pressure gradients, snap-freezing, nonthermal permanent electroporation, and supercritical fluids2. Cells on the external surface of a tissue or organ, such as the skin or small intestine, can be efficiently removed by mechanical processes combined with enzymes4. Ionic or nonionic detergents dissolve DNA/protein interactions, lipids, and lipoproteins, but can damage the ECM structure5. Enzymes remove the dissociated cytoplasm and nuclear material, but leave these materials in the ECM, which can cause an immune response6. The optimal agents for decellularization are determined by tissue thickness and density or the clinical use of the decellularized tissue.
For decellularization, we used a combination of nonionic and ionic detergents: Triton X-100 and SDS. Triton X-100, as a nonionic detergent, effectively disrupts lipid/lipid and lipid/protein interactions. However, Triton X-100 may also destroy the ECM ultrastructure owing to loss of glycosaminoglycan (GAG), laminin, and fibronectin contents. SDS, as an ionic detergent, effectively removes nuclear remnants and cytoplasmic proteins, but also disrupts the ECM ultrastructure by loss of GAG and collagen3. Although these agents destroy the microstructure of the ECM, SDS and Triton X-100 successfully remove all DNA contents7,8. This is essential because remaining DNA content within a scaffold can cause immune rejection. In tissue that has been properly decellularized, the DNA content should be less than 50 ng/mg9,10.
In the method, the pressure of the decellularization perfusion was 40 mmHg. Pressure control is required for decellularization perfusion. The optimal perfusion pressure varies from organ to organ, and 60 mmHg is the optimal pressure for human and porcine kidney or heart decellularization11. In rats, 40 mmHg is considered sufficient for decellularization perfusion12.
One promising treatment for replacing allograft transplantation is transplantation of stem cells using a vascularized scaffold. We hope that this protocol for organ decellularization may provide a foundation for future tissue engineering studies.

Cell culture techniques are applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Allogenic organ transplantation is currently the most common treatment for irreversible organ damage; however, this approach requires the use of immunosuppression to prevent rejection of the transplanted organ. Moreover, despite advances in transplant immunology, 20% of transplant recipients may experience acute rejection within 5 years, and within 10 years after transplantation, 40% of recipients may lose their transplanted graft or die1.
Advances in tissue engineering technologies have yielded in a new paradigm for transplantation of new organs without immune rejection using differentiated stem cells. After stem cell differentiation, a scaffold, called a synthetic extracellular matrix, is needed to facilitate the generation of three-dimensional organs and enable the new tissue to thrive within the recipient. Scaffolds from decellularized native organs have advantages, including a more effective environment for establishment of cells and enhancement of stem cell proliferation, although these mechanisms have not been fully elucidated2. In particular, the kidney is a suitable organ for scaffold generation because it has abundant circulation and a niche for stem cell establishment. Additionally, because of the complex structure of the kidney, it is difficult to artificially regenerate kidneys for organ transplantation.
In this report, we introduce a method of developing vascularized scaffolds using decellularized organs in a rat model to facilitate future animal studies for tissue engineering purposes.

<table><tbody><tr><td>1 cc syringe (inject probes and vehicle solutions</td><td>Becton Dickinson</td><td>305217</td><td></td></tr><tr><td>10-0 ethilon for vessel anastomosis</td><td>Ethicon</td><td>9032G</td><td></td></tr><tr><td>25 gauge inch guide needle(for vascular catheters)</td><td>Becton Dickinson</td><td>305145</td><td></td></tr><tr><td>3-0 PDS incision closure rat</td><td>Ethicon</td><td>Z316H</td><td></td></tr><tr><td>3-0 Prolene incision closure rat</td><td>Ethicon</td><td>8832H</td><td></td></tr><tr><td>3-0 silk spool vascular access/ligation in rat</td><td>Braintree Scientific</td><td>SUT-S 110</td><td></td></tr><tr><td>4-0 PDS incision closure mouse</td><td>Ethicon</td><td>Z773D</td><td></td></tr><tr><td>4-0 Prolene incision closure mouse</td><td>Ethicon</td><td>8831H</td><td></td></tr><tr><td>5-0 silk spool vascular access/ligation in mouse</td><td>Braintree Scientific</td><td>SUT-S 106</td><td></td></tr><tr><td>Fine Scissors to cut fascia/connective tissue</td><td>Fine Science Tools</td><td>14058-09</td><td></td></tr><tr><td>Halsey needle holder</td><td>Fine Science Tools</td><td>12001-13</td><td></td></tr><tr><td>Kelly Hemostat for rats: muscle clamp to minimize bleeding when cut</td><td>Fine Science Tools</td><td>13018-14</td><td></td></tr><tr><td>Polyethelyne 50 tubing, catheter tubing 100 ft</td><td>Braintree Scientific</td><td>.023&quot; &amp;times; .038&amp;rdquo;</td><td></td></tr><tr><td>Schwartz microserrefine vascular clamps</td><td>Fine Science Tools</td><td>18052-01 (straight)</td><td></td></tr><tr><td></td><td></td><td>18052-03 (curved)</td><td></td></tr><tr><td>Surgical Scissors to cut skin</td><td>Fine Science Tools</td><td>14002-12</td><td></td></tr><tr><td>Vannas-Tubingen Spring scissors for arteriotomy</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr></tbody></table>

preparation of decellularized kidney scaffolds in rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

The gross morphology of rat kidneys was dark red (Figure 1A). After decellularization, the kidney became pale and translucent (Figure 1D). Residual genomic DNA was assessed with a commercial kit according to the manufacturer&#8217;s instructions, in decellularized kidney scaffolds and compared with that in native kidneys (control). Quantitative analysis confirmed that tissue genomic DNA was almost eliminated after decellularization. From 14 cases, the average DNA contents were 115.05 ng/&#181;L for the control and 1.96 ng/&#181;L for the decellularized scaffold. In total, 98.3% of DNA was removed (Figure 2), although the three-dimensional structure was maintained, and acellular gromeruli were preserved in the cortical parenchyma (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61856/61856fig01.jpg" /> 
Figure 1: Rat kidneys subjected to renal arterial perfusion decellularization.&#160;(A) Immediately after the start of decellularization. (B) After Triton X-100 treatment. (C) After SDS buffer treatment. (D) After overnight scaffold washing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61856/61856fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61856/61856fig02.jpg" /> 
Figure 2: DNA concentrations in control and decellularized rat kidneys, showing reduced DNA contents after decellularization. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61856/61856fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61856/61856fig03.jpg" /> 
Figure 3: Hematoxylin and eosin staining of control and decellularized kidney samples.&#160;(A) control cortex (A`) decellularized cortex (B) control medulla (B`) decellularized medulla (C) control vein (C`) decellularized vein. Scale bar, 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61856/61856fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation of Decellularized Kidney Scaffolds in Rats at JoVE.com

Preparation of Decellularized Kidney Scaffolds in Rats

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

preparation-of-decellularized-kidney-scaffolds-in-rats

Nanyang Technological University

Research

JoVE Journal

Medicine

4.1K Views.  Pusan National University Hospital. Dit protocol maakt het mogelijk om gevasculariseerde steigers te ontwikkelen met behulp van nieren in een rattenmodel. De nier is het meest geschikte orgaan voor steigers van gedifferentieerde stamcellen. Dit is een zeer succesvol protocol voor decellularisatie van de nier.Het verwijdert effectief de kernen met minimale vernietiging van de ECM-ultrastructuur. Deze techniek kan worden toegepast op transplantatiegeneeskunde en weefseltechniek. Kim Joo Hyoung, een MD/PhD universitair hoof...

Video: Preparation of Decellularized Kidney Scaffolds in Rats

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 ...

Bioengineering

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 ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. 
One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. 
When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.

In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various ...

Developmental Biology

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

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

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

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

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

Decellularized Rat Spleen Matrix Preparation for Liver Tissue Engineering

Fabrication of Decellularized Spleen Matrix Derived from Rats

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the development of alternative therapies. Bioartificial livers (BALs) utilizing decellularized liver matrix (DLM) have emerged as promising solutions. However, sourcing suitable DLMs remains challenging. The use of a decellularized spleen matrix (DSM) has been explored as a foundation for BALs, offering a readily available alternative. In this study, rat spleens were harvested and decellularized using a combination of freeze-thaw cycles and perfusion with decellularization reagents. The protocol preserved the microstructures and components of the extracellular matrix (ECM) within the DSM. The complete decellularization process took approximately 11 h, resulting in an intact ECM within the DSM. Histological analysis confirmed the removal of cellular components while retaining the ECM's structure and composition. The presented protocol provides a comprehensive method for obtaining DSM, offering potential applications in liver tissue engineering and cell therapy. These findings contribute to the development of alternative approaches for the treatment of end-stage liver disease.

Author Spotlight: Utilization of Decellularized Spleen Matrix for Bioartificial Livers

The decellularized spleen matrix (DSM) holds promising applications in the field of liver tissue engineering. This protocol outlines the procedure for preparing rat DSM, which includes harvesting rat spleens, decellularizing them through perfusion, and evaluating the resulting DSM to confirm its characteristics.

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the ...

Analyzing Cell-Seeded Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering

Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering In Vitro and In Vivo

Plant-derived&#160;cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as&#160;its ability to effectively support osteogenic differentiation. The present study further&#160;examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then&#160;assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and&#160;energy-dispersive spectroscopy (EDS)&#160;confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young&#39;s modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived&#160;scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.

Author Spotlight: Insights into the Use of Apple-Derived Cellulose Scaffolds for Bone Tissue Engineering 

In this study, we detail methods of decellularization, physical characterization, imaging, and in vivo implantation of plant-based biomaterials, as well as methods for cell seeding and differentiation in the scaffolds. The described methods allow the evaluation of plant-based biomaterials for bone tissue engineering applications.

Preparation of Decellularized Kidney Scaffolds in Rats

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