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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;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&#160; Co., Ltd</td>
			<td>J31020</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Heparinized Saline</td>
			<td>SPH No.1 Biochemical &#38; Pharmaceutical Co., LTD&#160;</td>
			<td></td>
			<td>prevent the formation of thrombosis&#160;</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&#160;</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&#215;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

589 Views.  The First Affiliated Hospital of Xi'an Jiaotong University. 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.

Video: Author Spotlight: Utilization of Decellularized Spleen Matrix for Bioartificial Livers

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.  ...

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 ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

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 ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed from JBNTs and FN through self-assembly in an aqueous solution. JBNTs measure 200-300 &#181;m in length with inner hydrophobic hollow channels and outer hydrophilic surfaces. JBNTs are positively charged and FNs are negatively charged. Therefore, when injected into a neutral aqueous solution, they are bonded together via noncovalent bonding to form the NM bundles. The self-assembly process is completed within a few seconds without any chemical initiators, heat source, or UV light. When the pH of the NM solution is lower than the isoelectric point of FNs (pI 5.5-6.0), the NM bundles will self-release due to the presence of positively charged FN.
NM is known to mimic the extracellular matrix (ECM) morphologically and hence, can be used as an injectable scaffold, which provides an excellent platform to enhance hMSC adhesion. Cell density analysis and fluorescence imaging experiments indicated that the NMs significantly increased the anchorage of hMSCs compared to the negative control.

The goal of this protocol is to show the assembly of a biomimetic nanomatrix (NM) with Janus base nanotubes (JBNTs) and fibronectin (FN). When co-cultured with human mesenchymal stem cells (hMSCs), the NMs exhibit excellent bioactivity in encouraging hMSCs adhesion.

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

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 ...