This work was supported by the National Natural Science Foundation of China (82000624), Natural Science Basic Research Program of Shaanxi (2022JQ-899 &#38; 2021JM-268), Shaanxi Province Innovation Capability Support Program (2023KJXX-030), Shaanxi Province Key R&#38;D Plan University Joint Project-Key Project (2021GXLH-Z-047), Institutional Foundation of The First Affiliated Hospital of Xi&#39;an Jiaotong University (2021HL-42 &#38; 2021HL-21).

This study was approved by the Committee on the Ethics of Animal Experiments of Xi&#39;an Jiaotong University and carried out in accordance with the guidelines for the Care and Use of Laboratory Animals.
1. Spleen harvesting
<ol>
	<li>Use male Sprague Dawley rats weighing 250-280 g. House the rats in rooms with controlled temperature and humidity, and provide them with food and water ad libitum, except for fasting before surgery.</li>
	<li>Subcutaneously inject buprenorphine (0.05 mL/kg) as an analgesic 1 h before operation. Anesthetize the rat by isoflurane inhalation. Use a flow rate of 1.5 L/min of 5% isoflurane for induction anesthesia in a plexiglass box and maintain anesthesia with a flow rate of 0.6-0.8 L/min of 2% isoflurane through a mask. Confirm the depth of anesthesia by pinching the toes.</li>
	<li>Use an electric shaver to shave the skin over the entire abdomen. Secure the rat in a supine position on the surgical table. Inject 2 mL of heparinized saline (1,000 U of heparin) via the penile dorsal vein to achieve systemic anticoagulation. Disinfect the shaved skin with a povidone-iodine solution and cover with a sterile draping cloth.</li>
	<li>Make a cruciform incision using surgical scissors in the abdomen, expose the abdominal cavity by stretching with the hemostatic forceps, and flip the liver towards the diaphragm. Exteriorize the gastrointestinal tract to the right side of the abdomen and cover it with moist gauze.</li>
	<li>Carefully separate and cut the splenogastric ligament to expose the splenic hilum. 
	NOTE: The spleen, which appears as a reddish elongated structure, approximately 3.0 cm x 0.6 cm x 0.6 cm in size, can be identified in the left abdomen.</li>
	<li>Gradually separate and expose the common hepatic artery, gastroduodenal artery, and splenic artery by dissecting along the splenic hilum. Ligate and cut the gastroduodenal artery and common hepatic artery while progressively dissociating the surrounding tissue.</li>
	<li>Flip the spleen towards the right side to expose the abdominal aorta. Gently perform blunt dissection and expose the abdominal aorta and celiac trunk using cotton swabs. Place a 3-0 silk suture, approximately 3 cm in length, above and below the branches of the celiac trunk, and place a 6-0 silk suture, approximately 10 cm in length, at the branch of the celiac trunk.</li>
	<li>Ligature the abdominal aorta below and above the branches of the celiac trunk. Make a small incision at the arterial branch. Gently lift the 6-0 silk suture, insert a 24 G venous catheter into the splenic artery along the celiac trunk, and ligate and secure it.</li>
	<li>Using a syringe pump at a rate of 4 mL/min, infuse heparinized normal saline (25 U/mL) at a volume of 50 mL. At the same time, sever the portal vein as an outflow channel to allow the infused fluid to flow out of the spleen. The animal is euthanized by exsanguination.</li>
	<li>Carefully dissect the surrounding tissue of the spleen, avoiding damage to the pancreas, while preserving the major accessory vessels.</li>
	<li>Check for any leakage around the spleen, then remove the spleen and pancreas and rinse them in normal saline. 
	NOTE: The spleen and pancreas of rats are closely connected, with the pancreas wrapping around the splenic artery. If the spleen is removed separately, it can be challenging to ligate numerous small blood vessels. In this procedure, the spleen is removed together with the pancreas. After decellularization, the spleen and pancreas become transparent and the microvasculature is visible, which facilitates the preservation of the spleen with intact blood vessels.</li>
	<li>Transfer the spleen into a 50 mL centrifuge tube filled with normal saline and store it in a -80 &#176;C freezer. 
	NOTE: The spleen and the venous catheter inserted into the splenic artery will be cryopreserved together for convenient connection during the perfusion experiments.</li>
</ol>
2. Spleen decellularization
<ol>
	<li>Repeat the freeze-thaw cycle 3x in a sterile container to lyse the spleen cells. 
	NOTE: The freeze-thaw cycle is a physical method used for scaffold decellularization. The spleen tissue is placed in a -80 &#176;C freezer overnight for freezing, then removed from the low-temperature environment and placed in a room temperature or 37 &#176;C water bath for thawing. This freezing and thawing process is repeated 3-6 times, which disrupts cell membranes and leads to cell lysis.</li>
	<li>Set up a perfusion system within a clean bench comprising a peristaltic pump, a 2 L reservoir, a silicone tube with an inner diameter of 2.4 mm, and a bubble trap (Figure 1).</li>
	<li>Fill the perfusion system with deionized water (ddH2O) and keep it running for 10 min.</li>
	<li>Carefully transfer the harvested spleen to the ddH2O-filled container.</li>
	<li>Connect the silicone tube to the venous catheter that has been inserted into the splenic artery.</li>
	<li>Start perfusion with ddH2O at a rate of 2 mL/min for 30 min.</li>
	<li>Continue perfusion with ddH2O at a rate of 4 mL/min for 30 min.</li>
	<li>Perfuse with 0.1% (w/v) SDS solution for 4 h.</li>
	<li>Perfuse with 1% (v/v) Triton X-100 solution for 2 h.</li>
	<li>Perfuse with PBS at a rate of 4 mL/min for 4 h to wash the DSM. 
	NOTE: Using the peristaltic pump for unidirectional perfusion of all liquids.</li>
	<li>After the perfusion is completed, ligate and transect the vascular branches between the pancreas and the spleen, and then remove the pancreas. Store the DSM in a clean and sealed 50 mL centrifuge tube soaked in PBS containing 10% penicillin-streptomycin at -20 &#176;C until ready to use for future experiments.</li>
</ol>

Decellularized Rat Spleen Matrix Preparation for Liver Tissue Engineering

<ol>
	<li>Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+of+the+art+and+perspectives+in+liver+transplantation.">State of the art and perspectives in liver transplantation.</a> Hepatobiliary Pancreat Dis Int. 22 (1), 1-3 (2023).</li><li>Hautz, 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=Immune+cell+dynamics+deconvoluted+by+single-cell+RNA+sequencing+in+normothermic+machine+perfusion+of+the+liver.">Immune cell dynamics deconvoluted by single-cell RNA sequencing in normothermic machine perfusion of the liver.</a> Nat Commun. 14 (1), 2285 (2023).</li><li>Cardini, B., 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=Live+confocal+imaging+as+a+novel+tool+to+assess+liver+quality:+insights+from+a+murine+model.">Live confocal imaging as a novel tool to assess liver quality: insights from a murine model.</a> Transplantation. 104 (12), 2528-2537 (2020).</li><li>Ding, Y., 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=Mesenchymal+stem+cell-derived+exosomes:+a+promising+therapeutic+agent+for+the+treatment+of+liver+diseases.">Mesenchymal stem cell-derived exosomes: a promising therapeutic agent for the treatment of liver diseases.</a> Int J Mol Sci. 23 (18), 10972 (2022).</li><li>Yaghoubi, 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=Prednisolone+and+mesenchymal+stem+cell+preloading+protect+liver+cell+migration+and+mitigate+extracellular+matrix+modification+in+transplanted+decellularized+rat+liver.">Prednisolone and mesenchymal stem cell preloading protect liver cell migration and mitigate extracellular matrix modification in transplanted decellularized rat liver.</a> Stem Cell Res Ther. 13 (1), 36 (2022).</li><li>Uygun, B. 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=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. 16 (7), 814-820 (2010).</li><li>Xiang, 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=The+effect+of+riboflavin/UVA+cross-linking+on+anti-degeneration+and+promoting+angiogenic+capability+of+decellularized+liver+matrix.">The effect of riboflavin/UVA cross-linking on anti-degeneration and promoting angiogenic capability of decellularized liver matrix.</a> J Biomed Mater Res A. 105 (10), 2662-2669 (2017).</li><li>Liu, 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=Implantation+strategy+of+tissue-engineered+liver+based+on+decellularized+spleen+matrix+in+rats.">Implantation strategy of tissue-engineered liver based on decellularized spleen matrix in rats.</a> J South Med Univ. 38 (6), 698-703 (2018).</li><li>Xiang, 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=Decellularized+spleen+matrix+for+reengineering+functional+hepatic-like+tissue+based+on+bone+marrow+mesenchymal+stem+cells.">Decellularized spleen matrix for reengineering functional hepatic-like tissue based on bone marrow mesenchymal stem cells.</a> Organogenesis. 12 (3), 128-142 (2016).</li><li>Gao, 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=Hepatocyte+culture+in+autologous+decellularized+spleen+matrix.">Hepatocyte culture in autologous decellularized spleen matrix.</a> Organogenesis. 11 (1), 16-29 (2015).</li><li>Liu, 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=Hemocompatibility+improvement+of+decellularized+spleen+matrix+for+constructing+transplantable+bioartificial+liver.">Hemocompatibility improvement of decellularized spleen matrix for constructing transplantable bioartificial liver.</a> Biomed Mater. 14 (2), 25003 (2019).</li><li>Somuncu, &. #. 2. 1. 4. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+concept+in+regenerative+medicine.">Decellularization concept in regenerative medicine.</a> Adv Exp Med Biol. 1212, 71-85 (2020).</li><li>Neishabouri, A., Soltani, K. A., Daghigh, F., Kajbafzadeh, A. M., Majidi, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+in+tissue+engineering+and+regenerative+medicine:+evaluation,+modification,+and+application+methods.">Decellularization in tissue engineering and regenerative medicine: evaluation, modification, and application methods.</a> Front Bioeng Biotech. 10, 805299 (2022).</li><li>Brown, M., Li, J., Moraes, C., Tabrizian, M., Li-Jessen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix:+New+promising+and+challenging+biomaterials+for+regenerative+medicine.">Decellularized extracellular matrix: New promising and challenging biomaterials for regenerative medicine.</a> Biomaterials. 289, 121786 (2022).</li><li>Gui, L., Muto, A., Chan, S. A., Breuer, C. K., 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=Development+of+decellularized+human+umbilical+arteries+as+small-diameter+vascular+grafts.">Development of decellularized human umbilical arteries as small-diameter vascular grafts.</a> Tissue Eng Pt A. 15 (9), 2665-2676 (2009).</li><li>Li, T., Javed, R., Ao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenogeneic+decellularized+extracellular+matrix-based+biomaterials+For+peripheral+nerve+repair+and+regeneration.">Xenogeneic decellularized extracellular matrix-based biomaterials For peripheral nerve repair and regeneration.</a> Curr Neuropharmacol. 19 (12), 2152-2163 (2021).</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></ol>

The authors have declared no conflicts of interest.

The BALs represent an effective approach for the treatment of end-stage liver disease, particularly in cases where liver transplantation is hindered by the current shortage of donor organs6. A promising option for creating BALs is the utilization of DLM, which preserves the native liver&#39;s natural ECM and vascular structure. However, the scarcity of human DLM and the potential risks of infection and immunogenicity associated with animal DLM pose significant limitations. To address this challeng...

Liver transplantation remains the only definitive treatment for end-stage liver disease1,2,3. However, the critical shortage and declining quality of donor organs have heightened the need for alternative treatments4. In the realm of regenerative medicine, bioartificial livers (BALs) utilizing decellularized liver matrix (DLM) have emerged as promising solutions5,6,7. The DLM preserves the original liver structure, including its intricate microvascular network and components of the ECM, offering a scaffold for creating transplantable BALs that could potentially alleviate liver diseases.
Despite the promise, the adoption of this technology faces challenges, particularly in sourcing suitable DLMs. Human-derived DLMs are in short supply, while those from animal sources carry the risks of disease transmission and immune rejection. In an innovative approach, our research has explored the use of a decellularized spleen matrix (DSM) as a foundation for BALs8,9,10,11. Spleens are more readily available in various medical situations, such as portal hypertension, traumatic rupture, idiopathic thrombocytopenic purpura, and donation after cardiac death. Therefore, spleens are more widely available than livers for research purposes. Patients who have undergone splenectomies do not suffer from severe conditions, further confirming the dispensability of the spleen. The microenvironment of the spleen, particularly the extracellular matrix and sinusoids, is similar to that of the liver. This makes the spleen a suitable organ for cell adhesion and proliferation in hepatocyte transplantation research. Based on these findings, our previous investigations have demonstrated that DSMs share comparable microstructures and components with DLMs and can support the survival and function of hepatocytes, including albumin and urea production. Furthermore, DSMs have been shown to enhance the hepatic differentiation of bone marrow mesenchymal stem cells, leading to improved and consistent functionality.
By employing DSMs treated with heparin, we have engineered functional BALs capable of demonstrating effective short-term anticoagulation and partial liver function compensation11. Consequently, this three-dimensional DSM holds significant promise for the advancement of liver tissue engineering and cell therapy. In this work, we present the detailed methods of harvesting rat spleens and preparing DSM that preserve the microstructures and components of the ECM.

<table><tbody><tr><td>Anesthesia Machine</td><td>Harvard Apparatus</td><td>tabletop</td><td>animal anesthesia</td></tr><tr><td>bubble trap</td><td>Shandong Weigao Group Medical Polymer Co., Ltd.</td><td>pore diameter: 5 &amp;mu;m</td><td>prevent air bubbles</td></tr><tr><td>Buprenorphine</td><td>TIPR Pharmaceutical Responsible Co.,Ltd</td><td></td><td>an analgesic</td></tr><tr><td>Hemostatic Forceps</td><td>Shanghai Medical Instruments&amp;nbsp; Co., Ltd</td><td>J31020</td><td>surgical tool</td></tr><tr><td>Heparinized Saline</td><td>SPH No.1 Biochemical &amp;amp; Pharmaceutical Co., LTD&amp;nbsp;</td><td></td><td>prevent the formation of thrombosis&amp;nbsp;</td></tr><tr><td>Isoflurane</td><td>RWD life Science Co.</td><td></td><td>anesthetic:for the induction and maintenanceof anesthesia</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Beyotime Biotechnology Co., Ltd.</td><td>C0222</td><td>antibiotics in vitro to prevent microbial contamination</td></tr><tr><td>Peristaltic Pump</td><td>Baoding Longer Precision Pump Co., Ltd.</td><td>BT100-1L</td><td></td></tr><tr><td>Phosphate-Buffered Saline</td><td>Shanghai Titan Scientific Co., Ltd.</td><td>4481228</td><td>phosphoric acid buffer salt solution</td></tr><tr><td>Silicone Tube</td><td>Baoding Longer Precision Pump Co., Ltd.</td><td>2.4&amp;times;0.8mm</td><td></td></tr><tr><td>Silk Suture</td><td>Yangzhou Jinhuan Medical Instrument Factory</td><td>6-0 and 3-0</td><td>ligate blood vessels</td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Shanghai Titan Scientific Co., Ltd.</td><td>151-21-3</td><td>ionic detergent, dissolves both cell and nuclear membranes</td></tr><tr><td>Syringe Pump</td><td>Shenzhen Mindray Bio-Medical Electronics Co., Ltd</td><td>BeneFusion SP5</td><td>intravenous infusion</td></tr><tr><td>Triton X-100</td><td>Shanghai Titan Scientific Co., Ltd.</td><td>9002-93-1</td><td>non-ionic detergent, disrupts lipid-lipid, lipid-protein, and DNA-protein interactions</td></tr><tr><td>Venous Catheter</td><td>B. Braun Company</td><td>24G</td><td>inserting the spleen artery</td></tr></tbody></table>

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.

This protocol utilized a combination of repeated freeze-thaw cycles and perfusion with decellularization reagents for the decellularization of rat spleen. The complete decellularization of the spleen was achieved in approximately 11 h (Figure 2A). Throughout the decellularization process, the spleen&#39;s color gradually transitioned from deep red to a mottled, light red, and eventually, a white translucent appearance (Figure 2B). The overall morphology remained...

Watch this Scientific Journal Video about Utilization of Decellularized Spleen Matrix for Bioartificial Livers at JoVE.com

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.

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 ...

fabrication-of-decellularized-spleen-matrix-derived-from-rats

Research

JoVE Journal

Bioengineering

599 Views.  The First Affiliated Hospital of Xi'an Jiaotong University. We ontwikkelen een nieuwe strategie waarbij een gedecellulariseerde miltmatrix wordt gebruikt als een alternatieve steiger voor biokunstmatige levers. We toonden de gedetailleerde procedure van het oogsten van de milt van ratten en het voorbereiden van een gedecellulariseerde miltmatrix die de macrostructuren en de componenten van de extracellulaire matrix behoudt. Ons protocol biedt een direct beschikbare miltmatrix als alternatief voor schaarse donorlevers.Ons protocol presenteert ee...

Video: Auteur in de schijnwerpers: gebruik van gedecellulariseerde miltmatrix voor biokunstmatige levers

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

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, ...

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 ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

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

Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases susceptibility to severe blood infections and sepsis. Auto-transplantation of spleen slices has been used clinically to replace lost tissue and restore immune function. However, the mechanism driving robust and immunologically functional spleen tissue regeneration has not been fully elucidated. Here, we aim to develop a method for aggregating and encapsulating spleen cells within a semi-solid matrix in order to investigate the cellular requirements for spleen tissue formation. Basement membrane matrix encapsulated cell constructs are amenable to both in vitro tissue culture of three-dimensional organoids as well as transplantation under the kidney capsule to directly assess in vivo tissue formation. By manipulating the input cells for aggregation and encapsulation, we demonstrate that graft-derived PDGFR&#946;+MAdCAM-1- neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.

This article describes a protocol for aggregating and encapsulating spleen cells within a semi-solid basement membrane matrix. Basement membrane matrix constructs can be used in three-dimensional culture for studying organoid development, or for in vivo transplantation and tissue regeneration studies.

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases ...

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 ...

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.

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 ...

Medicine

Preparation of Decellularized Rat Kidney: A Procedure to Generate Acellular Vascularized Whole-organ Scaffold from a Harvested Rat Kidney

Harvest the left kidney with the abdominal aorta and inferior vena cava. Ligate the ureter, thoracic aorta, superior vena cava, and branches of the abdominal aorta. Keep the organ hydrated in DPBS in a 10-centimeter Petri dish. 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 the organ with 500 milliliters of DPBS and 16 units per milliliter heparin for 90 minutes at 5 rpm and 37 degrees Celsius. To decellularize the kidney, perfuse it with 1% Triton X-100 for 3 hours and then with 0.75% SDS solution for 6 hours at a constant pressure of 40 milliliters of mercury.
To remove residual SDS, perfuse the sample with 1% penicillin in distilled water for 18 hours and then with sterile DPBS and 16 units per milliliter heparin for 90 minutes.

Fabrication of Decellularized Spleen Matrix Derived from Rats

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video