The authors thank the Wake Forest Institute of Regenerative Medicine for their help with development of this protocol. We acknowledge support by grants from the Great Ormond Street Hospital charity, the Foundation Eugenio Litta (Geneva, Switzerland), the Medical Research Council, the Royal College of Surgeons of England, the Sparks Children's Medical Charity, the British Foreign Office for the UK/USA Stem Cell Collaboration Award and the Mittal Research Fund. We would also like to thank the Royal Society/ Wolfson Foundation for the tissue engineering laboratory refurbishment grant obtained for the Pediatric Surgery Department in the Institute of Child Health. PDC and SE are supported by the Great Ormond Street Hospital Children's Charity.

1. Isolation of Rat Intestine
<ol>
	<li>Prepare a 1 L solution of Phosphate Buffered Saline (Sigma, UK) with 1% antibiotic/antimycotic (Sigma, UK) (PBS/AA). Fit the peristaltic pump (Masterflex L/S variable speed) with two tubes. Run 70% ethanol (EtOH) through the tubes of the peristaltic pump for 15 min at maximum speed, followed by PBS/AA for another 15 min.</li>
	<li>Euthanize adult Sprague-Dawley rats weighing approximately 250-350 g by CO2 inhalation and cervical dislocation in accordance with local animal guidelines and approvals (in the UK, Home Office guidelines under the Animals (Scientific Procedures) Act 1986).</li>
	<li>Autoclave the surgical instruments before use. Spray and wipe the abdomen of the animal using 70% EtOH.</li>
	<li>Perform a xifo-pubic laparotomic incision on the abdomen to ensure a clear surgical field.</li>
	<li>Eviscerate the small intestine on the right side of the animal onto a paper towel sprayed with 70% EtOH. Take care to continuously wet the intestine with PBS/AA so as to avoid dehydration of the tissue and denaturation of the extracellular matrix (ECM).</li>
	<li>Locate the superior mesenteric artery (SMA) arising in a 90&#176; angle from the aorta towards the intestine. Use a 27 G cannula to enter the SMA from the aorta and quickly withdraw the needle to avoid tearing the wall of the vessel. Advance the plastic tube of the cannula into the SMA and secure using sutures (Vicryl 5/0).</li>
	<li>Confirm cannulation by injecting 1 ml of PBS/AA. Use spring scissors to incise the aorta proximal and distal to the SMA so as to release the cannula.</li>
	<li>To avoid leakage of feces when dissecting out the large intestine, place one (Silk 5/0) suture knot on the proximal end of the ileocaecal junction and one at the distal end of the sigmoid colon. Then cut the terminal ileum and colon between the two knots and remove the large intestine.</li>
	<li>Cut the intestine at the jejuno-duodenal junction and free the intestine from any connective tissue connections it might have with the abdomen.</li>
	<li>Transfer the small intestine in a Petri dish with PBS/AA. Cannulate the proximal section of the intestine with a plastic Pasteur pipette (LSL Laboratory Consumables) and secure it in place with sutures. Cut the pipette so it will fit the Luer-Lok of a 60 ml syringe (BD Plastipak). Flush the intestinal lumen 2x with 60 ml of PBS/AA to remove feces and debris.</li>
</ol>
2. Decellularization Methodology
<ol>
	<li>Connect the vascular cannula to a three-way stopcock. This will ease handling, minimize tension on the vascular tree and allow removal of bubbles from the tubing. </li>
	<li>Start running deionized water (resistivity 18.2 M&#937;/cm) through the Masterflex L/S variable speed roller pump at 0.6 ml/hr. Ensure no bubbles are present within tubing prior to connecting with the intestinal lumen stopcock. Some bubbles may be present within the lumen from step 1.9, which may impair the decellularization process. Vary the speed of the pump and handle the tissue with forceps carefully to ensure the bubbles exit from the distal end. Do not connect the vascular cannula until this process is finished since high speeds may damage the vascular tree.</li>
	<li>Transfer apparatus to the cold room, since the following step of decellularization is performed at 4 &#176;C. The low temperature allows removal of cells whilst minimizing tissue decomposition. Place the Petri dish in a container that will collect the overflowing solution. Perfuse both the intestinal lumen and the vascular tree with deionized water (resistivity 18.2 M&#8486;/cm) for 24 hr at 0.6 ml/hr. For the first hour check the apparatus regularly to ensure all connections are secure, leakage is minimal and no bubbles are present within the intestine.</li>
	<li>After 24 hr of treatment with deionized water, move decellularization apparatus outside the cold room as the rest of the steps are carried out in room temperature (RT).</li>
	<li>Weigh sodium deoxycholate (Sigma, UK) to prepare 300 ml of a 4% solution in deionized water. Weigh sodium deoxycholate under a suction hood, as it is an oral and eye irritant. Mix using a vortex until the solution is clear. The solution may be maintained at 4 &#176;C for up to 1 month.</li>
	<li>Change the deionized solution to sodium deoxycholate, check the connections, remove any bubbles and perfuse at 0.6 ml/hr for 4 hr.</li>
	<li>Following the sodium deoxycholate step wash with PBS/AA for 30 min to avoid interaction between the sodium deoxycholate and DNase-I.</li>
	<li>Weigh out deoxyribonuclease-I (DNase-I, Sigma) and prepare 250 ml of a 2,000 kU solution in 1 M sodium chloride, pH 7.4. Mix using a vortex until the solution is clear. This solution must be prepared immediately before use and can&#39;t be stored.</li>
	<li>Perfuse with DNase-I for 3 hr at RT at a speed of 0.6 ml/hr.</li>
	<li>Following the DNase-I step, transfer the scaffold into a tissue culture hood and change plates and instruments. It is suggested to use aseptic technique.</li>
	<li>Wash with PBS/AA for 30 min to ensure all remnants of the decellularization solutions are removed. Place the scaffold in a new Petri dish and transfer to a UV Crosslinker (Spectroline, US). Run two sterilization cycles. Store in 5% PBS/AA and change the solution every 3 days.</li>
</ol>

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No conflicts of interest declared.

The most difficult steps in setting up this experiment involve the cannulation of the SMA and the establishment and maintenance of sterility. Cannulating the SMA in rodents without rupturing the wall can be quite difficult due to the size and position of the vessel. Alternatively, a suture may be placed around the proximal aorta prior to the SMA origin, followed by cannulation of the aorta itself distally, directing the plastic cannula into the SMA. During decellularization a combination of poor cannula placement and high flow rates may result in losing the vascular access. Sterility is a major issue due to the amount of bacterial flora present in the small intestine. Washing with PBS/AA following harvest is very important, and any sign of fecal matter or debris must be removed from the lumen. Placing a portion of the scaffold in a falcon tube with DMEM in the incubator following UV sterilization should be an indicator if sterility has been achieved. In the case of bacterial colonization, the media will change the pH with its color turning from red to yellow. To deal with this, further UV cycles are advised as well as washing with PBS containing a high concentration of antibiotic/antimycotic.
A possible modification to obtain a scaffold with both vascular and venous access would be to cannulate the inferior vena cava (IVC) as well as the SMA. Decellularization from the venous and vascular sides should be attempted intermittently for all three solutions. Continuous decellularization may burst the capillaries by having positive pressure on both sides.
Over the years there have been a number of efforts in intestinal TE using different cell-scaffold combinations in vitro and in vivo6,9,12. The majority of the work has been performed using tubular nonwoven 95% PGA-5% PLGA scaffolds coated with collagen type I6,7,13,22. Following seeding with intestinal epithelial organoid units (OUs) and a period of implantation into the omentum of mice, they form cysts with muscle on the outside and epithelium on the inside which can then be tubularized. However, the porosity and simplicity in the design of these scaffolds does not permit for the generation of large pieces of artificial intestine in an in vitro environment such as that of a bioreactor. Additionally, the lack of an innate vascular network that can be connected to the recipient further limits the use of this scaffold for clinical translation. Besides the experiments with the PGA-PLGA scaffolds, other groups have used collagen or SIS scaffolds, both of which do not replicate the intricacy of the intestinal tract. SIS in particular is the only previous published methodology of intestinal tissue engineering and has been used in more than 150 medical settings, demonstrating the ability of decellularized scaffolds to provide mechanical stability and promote cell growth whilst leading to no immunogenic response. However, the lack of appropriate macro- and micro-architecture, has led to poor results in its&#39; use for intestinal TE purposes9-11,23.
The benefits of the decellularization methodology we have described include the preservation of microscopic characteristics such as the luminal crypt-villus architecture that represent an appropriate environment for the repopulation by the intestinal stem cell niche. In the macroscopic aspect, the presence of a hierarchical vascular network will enable attachment to the recipient, allowing provision of nutrients and oxygen to all layer of the TE-intestine21. What is more, the maintenance of ECM components such as collagen, elastin and glyocosaminoglycans has an important role not only for mechanical characteristics but also directing cellular proliferation and differentiation. Most importantly, the ability of the decellularization methodology to be scaled up into larger tissues whilst maintaining the same characteristics is an important feature for clinical translation of TE.
The development of a natural intestinal matrix with a vascular network allows the creation of larger segments of artificial intestine that can be connected to the host.

Tissue engineering (TE) provides a therapeutic alternative to organ transplantation, bypassing issues of immunosuppression and organ shortage. TE has recently had successful applications in the clinic, with replacement of organs such as the bladder1, urethra2 and trachea, both in adults3,4 and children5.
Building a tissue-engineered organ necessitates the combination of a scaffold with the appropriate cells. A scaffold can be prepared using naturally-derived (e.g. collagen) and synthetic (e.g. poly-L-glycolic acid; PLGA) materials, or be obtained by the decellularization of native organs and tissues. Scaffolds that have been used thus far for intestinal TE have mainly been either decellularized (small intestinal submucosa) or synthetic (poly-L-glycolic acid and poly-lactic acid)6-13. These biomaterials are very simple in both macro- and micro-architecture, which may not be ideal if tissue-engineered intestine is to be clinically translated. An optimal biomaterial for the intestine should have an innate vascular tree that can be connected to the host&#39;s blood supply, a tiered tubular wall with different properties to reflect the layers of the intestinal wall and a villus-crypt axis on the luminal side to aid with repopulation by epithelial stem cells.
Decellularization is a novel methodology that produces scaffolds by removing cells from whole organs whilst maintaining their original architecture14. This is preferable to already existing scaffolds since they do not only replicate the structure of the organ but also contain chemical cues embedded within the extracellular matrix (ECM) that aid cellular proliferation and differentiation. In 2008 a cadaveric trachea was decellularized14, seeded with the patient&#39;s own cells, and transplanted to replace the main left bronchus in a young man3. Since then, a number of groups have reported the production of decellularized scaffolds for the heart15, liver16,17, and lung18-20 in small and large animals.
We have also adapted the same methodology to produce a small intestinal decellularized scaffold21. The goal of the method described herein is to produce decellularized intestinal matrices that maintain the macroscopic characteristics of the original tissue such as the blood supply, as well as the microscopic architecture of the villus-crypt axis in the intestinal lumen. We believe this methodology could ultimately be adopted for other organs to improve the efficiency of decellularization.

<table><tbody><tr><td>Ethanol solution, 70% in H&lt;sub&gt;2&lt;/sub&gt;0</td><td>Sigma</td><td>02877</td><td></td></tr><tr><td>Phosphate buffered saline tablets</td><td>Sigma</td><td>79382</td><td></td></tr><tr><td>Antibiotic Antimycotic Solution (100x)</td><td>Sigma</td><td>A5955</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>D6750</td><td>Oral and eye irritant; use protection</td></tr><tr><td>Sodium chloride</td><td></td><td></td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma</td><td>D5025</td><td></td></tr></tbody></table>

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.

Upon successful decellularization (Figure 1), the scaffold should have a macroscopic appearance similar to the one of the native intestine, but with translucent walls (Figure 2A). The preservation of macro-architecture should also be evident by the patency of the vascular tree. When dye is injected through the SMA cannula, it should distribute evenly throughout the scaffold and reach the capillary tree (Figure 2B). The scaffold should be free of nuclear and cytoplasmic material, something that can be assessed using a DNA quantification kit and simple histological analysis. Hematoxylin and eosin staining (H&#38;E) should demonstrate an absence of nuclear and cytoplasmic material whilst preserving the small intestinal layers (Figure 3A). In particular, the maintenance of the villi and crypts on the luminal side should be evident following scanning electron microscopy (Figure 4). A conservation of extracellular matrix components such as collagen, elastin and glycosaminoglycans should also be expected. For example, Masson&#39;s Trichrome staining displays intact connective tissue in the scaffold (Figure 3B).
<img alt="Figure 1" fo:content-width="6.5in" src="/files/ftp_upload/50658/50658fig1.jpg" width="600px" /> 
Figure 1. Experimental plan. Timeline of decellularization procedure; PBS/AA: phosphate buffered saline with antibiotic-antimycotic, NaDeoxy: Sodium deoxycholate, RT: room temperature, DNAse-I: deoxyribonuclease-I. <a href="https://www.jove.com/files/ftp_upload/50658/50658fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6.5in" src="/files/ftp_upload/50658/50658fig2.jpg" width="600px" /> 
Figure 2. Scaffold macroscopic characteristics. Macroscopic appearance of the rat small intestine following successful decellularization (A). Injection of Rosso Ponceau dye through the SMA demonstrates an intact vascular network (B) ; SMA: superior mesenteric artery <a href="https://www.jove.com/files/ftp_upload/50658/50658fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6.5in" src="/files/ftp_upload/50658/50658fig3.jpg" width="600px" /> 
Figure 3. Histology. H&#38;E (A) and MT (B) staining indicate an absence of cellular material with preservation of the connective tissue architecture; H&#38;E: hematoxylin and eosin, MT: Masson&#39;s trichrome. Scale bar: 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/50658/50658fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="6.5in" src="/files/ftp_upload/50658/50658fig4.jpg" width="600px" /> 
Figure 4. Electron Microscopy. Scanning electron microscopy indicates the preserved villus-crypt architecture in the scaffold lumen; Scale bar: 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/50658/50658fig4highres.jpg" target="_blank">Click here to view larger image</a>.

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

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, ...

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29.1K Views.  University College London. 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.

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

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.  ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Developmental Biology

Decellularization of the Murine Cardiopulmonary Complex

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and composition. It is based on a microsurgical procedure designed to catheterize the trachea and aorta of a euthanized mouse to perfuse the heart and lungs with decellularizing agents. The decellularized cardiopulmonary complex can subsequently be immunostained to reveal the location of structural ECM proteins. The whole procedure can be completed in 4 days.
The ECM scaffolds resulting from this protocol are free of dimensional distortions. The absence of cells enables structural examination of ECM structures down to submicron resolution in 3D. This protocol can be applied to healthy and diseased tissue from mice as young as 4-weeks old, including mouse models of fibrosis and cancer, opening the way to determine ECM remodeling associated with cardiopulmonary disease.

This protocol aims to decellularize the heart and lungs of mice. The resulting extracellular matrix (ECM) scaffolds can be immunostained and imaged to map the location and topology of their components.

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and ...

Cancer Research

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

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

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

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

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 ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix ECM Production

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets&#8217; microenvironment in vitro and, potentially, in vivo settings.

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those ...

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of surrounding cells due to its content of diverse bioactive molecules. Due to these characteristics, the ECM has been recently considered a promising candidate for the creation of biological scaffolds to boost tissue regeneration. Emerging studies have demonstrated that decellularized human tissues could resemble the native ECM in their structural and biochemical profiles, preserving the three-dimensional (3D) architecture and the content of fundamental biological molecules. Hence, decellularized ECM can be employed to promote tissue remodeling, repair, and functional reconstruction of many organs. Selecting the appropriate decellularization procedure is crucial to obtain acellular tissues that retain the characteristics of the ideal microenvironment for cells. 
The protocol described here provides a detailed step-by-step description of the decellularization method to obtain a reproducible and effective cell-free biological ECM. Skin fragments from patients undergoing plastic surgery were scaled down and decellularized using a combination of sodium dodecylsulfate (SDS), Triton X-100, and antibiotics. To promote the regular and homogeneous transport of the solution through the samples, they were enclosed in embedding cassettes to ensure protection from mechanical insults. After the decellularization procedure, the snow-white color of skin fragments indicated complete and successful decellularization. Additionally, decellularized samples showed an intact and well-preserved architecture. The results suggest that the proposed decellularization method was effective, fast, and reproducible and protected samples from architectural damages.

Decellularized human skin is suitable for tissue regeneration. A major issue of decellularization is the preservation of the native architecture, along with the appropriate content of structural proteins, glycosaminoglycans (GAGs), and growth factors. The method proposed allows fast and effective decellularization, producing decellularized skin with well-preserved native features.

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of ...

Matrix Comparison for Efficient Human Intestinal Organoid Generation from iPSCs

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

Author Spotlight: Advancing Organoid Generation for Drug Development Using iPSCs

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

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

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Chapters in this video

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Decellularization of the Murine Cardiopulmonary Complex

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

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

Novel Process for 3D Printing Decellularized Matrices

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation