Name: two methods for decellularization of plant tissues for tissue engineering applications
Uploaded: 2018-05-31T14:00:00.000+00:00
Duration: 5 min 20 s
Description: Watch this Scientific Journal Video about Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications at JoVE.com

We would like to thank John Wirth of the Olbrich Gardens for graciously supplying the specimens used in this project. This work is supported in part by the National Heart, Lung, and Blood Institute (R01HL115282 to G.R.G.) National Science Foundation (DGE1144804 to J.R.G and G.R.G.), and the University of Wisconsin Department of Surgery and Alumni Fund (H.D.L.). This work was also supported in part by the Environmental Protection Agency (STAR grant no. 83573701), the National Institutes of Health (R01HL093282-01A1 and UH3TR000506), and the National Science Foundation (IGERT DGE1144804).

1. Decellularization of Plant Tissue Using the Detergent-based Approach
<ol>
	<li>Use fresh or frozen F. hispida, leaf samples. Freeze unused fresh samples in a -20 &#176;C freezer and store for future use (up to a year). 
	NOTE: Use stem or leaf tissue of nearly any desired plant. Extended storage times can cause damage to the tissues.
	<ol>
		<li>Determine the size and shape of samples to be processed on the basis of the sample&#8217;s intended use (i.e. samples cut into strips are well suited for mechanical testing applications, meanwhile 8 mm disc samples are useful in multi-well culturing applications). Cut the leaf into 8 mm discs with a sharp, clean biopsy punch while submerged under room temperature (20-25 &#176;C) deionized H2O. 
		Note: This protocol can be used on whole leaves and stems. However, smaller samples will decellularize faster.</li>
		<li>Incubate the samples for 5-10 min at room temperature (20-25 &#176;C) deionized H2O on a shake plate set to a low speed setting to wash and/or thaw them. Use enough deionized H2O to ensure all samples are thoroughly wetted.</li>
	</ol>
	</li>
	<li>Prepare a solution of 10% (w/v) sodium dodecyl sulfate (SDS) in deionized H2O. Place the samples in a suitable container (a glass or plastic dish is ideal) and add SDS solution to completely cover the samples. Incubate samples for 5 days at room temperature (20-25 &#176;C) on a shake plate set to a low speed to prevent damage to the samples. 
	NOTE: Do not overcrowd the container as this can slow down the decellularization process and lead to uneven treatment by the SDS. During this step, samples should acquire a brown hue.
	<ol>
		<li>After 5 days, replace the SDS solution with deionized H2O. Incubate the samples for an additional 10-15 min on the shake plate to thoroughly rinse off any residual SDS solution.</li>
	</ol>
	</li>
	<li>Prepare a 1% (v/v) non-ionic surfactant in 10% (v/v) bleach solution. To make a 500 mL solution, mix 5 mL of the non-ionic surfactant with 50 mL of bleach then add 445 mL of deionized H2O. Submerge samples in the freshly prepared solution. 
	NOTE: The non-ionic surfactant/bleach solution does not have a long shelf life, therefore, should be used within 48 h of preparation.</li>
	<li>Replace the non-ionic surfactant/bleach solution every 24 h until the samples are completely cleared (refer to Figure 1A for visual comparison). Then incubate the sample in deionized water on a shake plate for 2 min to rinse off the excess non-ionic surfactant/bleach solution. Set the shake plate to a low setting to prevent damaging the samples. 
	NOTE: The time required to clear the samples used varies, depending on the type and species of the plant. The complete discoloring of the samples is indicative of full decellularization (perform DNA quantification to ensure thorough clearing).
	<ol>
		<li>Lyophilize the samples to store them up to a year (flash freezing using liquid nitrogen is preferred, freezing samples in -80 &#176;C is also acceptable) and store them at room temperature (20-25 &#176;C) in low humidity.</li>
		<li>Reconstitute samples in Tris-HCl (10 mM, pH 8.5) using enough to coat samples. Rinse samples 2-3 times in serum-free media gently using a micropipette before use (i.e. use 150-300 &#181;L per rinse, in a standard 24 well plate). 
		NOTE: Tris-HCl buffer and serum-free media (i.e. DMEM, but any basic cell culture media can be substituted) are more effective in lowering the impact to cell viability of SDS treated samples than those treated with deionized H2O alone.</li>
	</ol>
	</li>
</ol>
2. Preparation of Samples Using the Detergent-free Decellularization Approach
NOTE: The initial steps of this procedure coincide with steps 1.1-1.1.2 (see above).
<ol>
	<li>Prepare a 5% (v/v) bleach (NaClO) and 3% (w/v) sodium bicarbonate (NaHCO3) solution. Warm the solution in a fume hood to 60-70 &#176;C for F. hispida leaf samples cut into 8 mm discs while stirring on a hot plate. 
	NOTE: The sodium bicarbonate can be substituted with sodium carbonate (Na2CO3), or sodium hydroxide (NaOH). The desired temperature varies greatly (from room temperature to 90 &#176;C) and should be adjusted according to the properties of the samples used.</li>
	<li>Once the solution reaches the desired temperature range, submerge the samples and reduce the stirring speed to avoid damaging them. After the samples are visibly cleared (refer to Figure 1B for visual comparison), remove from the bath carefully. Incubate samples once in deionized H2O for 1-2 min to remove excess bleach solution. 
	NOTE: The time required to clear the samples can vary widely. For example, cut parsley can be cleared in 10-15 min, while thicker and/or larger samples such as whole leaves or stems can take hours in high-temperature baths to clear completely.</li>
	<li>Lyophilize the samples and store them at room temperature (20-25 &#176;C) in low humidity.</li>
</ol>

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The authors have nothing to disclose.

Herein, two methods to decellularize plant tissues are described. The results presented here, coupled with the results of prior studies25, suggest that the protocols put forth are likely applicable to a wide spectrum of plant species and can be performed on both stems and leaves. These procedures are simple and do not require specialized equipment, so plant decellularization can be carried out in most laboratories. It is noteworthy that after decellularization, the scaffolds must be functionalized...

Tissue engineering emerged in the 1980s to create living tissue substitutes, and potentially address significant organ and tissue shortages1. One strategy has used scaffolds to stimulate and guide the body to regenerate missing tissues or organs. Although advanced manufacturing approaches such as 3-D printing have produced scaffolds with unique physical properties, the ability to manufacture scaffolds with a diverse range of achievable physical and biological properties remains a challenge2,3. Moreover, due to a lack of a functional vascular network, these techniques have been limited in regenerating 3-dimensional tissues. The use of decellularized animal and human tissues as scaffolds has aided in circumventing this problem4,5,6,7. However, high cost, batch-to-batch variability, and limited availability may limit widespread use of decellularized animal scaffolds8. There are also concerns about potential disease transmission to patients and immunologic reaction to some decellularized mammalian tissues9.
Cellulose, derived from plant and bacterial sources, has been extensively used to generate biomaterials for a wide range of applications in regenerative medicine. Some examples include: bone10,11, cartilage12,13,14 and wound healing15. Scaffolds that are comprised of cellulose have an added benefit in that they are durable and resistant to being broken down by mammalian cells. This is due to the fact that mammalian cells do not produce the enzymes necessary to break down cellulose molecules. In comparison, scaffolds produced using macromolecules from the extracellular matrix, such as collagen, are readily broken down16 and may not be well suited to long-term applications. Collagen scaffolds can be stabilized by chemical cross-linking. However, there is a trade-off due to the inherent toxicity of the cross-linkers that affect the biocompatibility of the scaffolds17. Conversely, cellulose has the potential to remain present at the site of implantation for prolonged periods of time because it is impervious to enzymatic degradation by mammalian cells18,19,20. This can be altered by tuning the rate of degradation through hydrolysis pretreatment and co-delivery of the scaffolds with cellulases21. The biocompatibility of decellularized plant-derived cellulose scaffolds in vivo has also been demonstrated in a study done on mice22.
Through hundreds of millions of years of evolution, plants have refined their structure and composition to increase the efficiency of fluid transport and retention. Plant vascular vessels minimize hydraulic resistance by branching into smaller vessels, similar to the mammalian vasculature according to Murray&#39;s law23. After decellularization, the plant&#39;s complex network of vessels and interconnected pores is maintained. Considering the vast number of distinct plant species readily available, plant-derived scaffolds have the potential to overcome design limitations currently affecting scaffolds in tissue engineering24,25. For instance, Modulevsky et al. demonstrated that angiogenesis and cell migration occurred when decellularized apple tissue was implanted subcutaneously on the back of a mouse22. Similarly, Gershlak et al. showed that endothelial cells could be grown within the vasculature of decellularized leaves24. In a separate experiment, Gershlak et al. were also able to show that cardiomyocytes could be grown on the surface of leaves and were able to contract24.
Plants also include complex organization from the cellular to the macroscopic scale, which is difficult to achieve even with the most advanced manufacturing techniques developed to date. The complex hierarchical design of plant tissues makes them stronger than the sum of their constituents26. Plants possess a plethora of different mechanical properties ranging from rigid and tough components such as stems, to much more flexible and pliable ones such as leaves27. Leaves vary depending on species in terms of size, shape, break strength, the degree of vascularization, and can carry different degrees of hydrophilicity. Overall, these plant properties suggest that decellularized plants can serve as unique and highly functional medical devices, including as tissue engineering scaffolds.
This protocol focuses on two methods to decellularize plant tissues, such as leaves and stems, for use as scaffolds in tissue engineering. The first method is a detergent-based technique that uses a series of baths to remove DNA and cellular matter, which has been adapted from a widely used technique to decellularize mammalian and plant tissues6,22,25,28,29,30. The second method is detergent-free and is adapted from a &#34;skeletonization&#34; protocol generally used to remove the soft tissues of leaves31. Prior work showed that simmering leaves in a bleach and sodium bicarbonate solution facilitated separation of the vasculature from the surrounding soft tissue31. This technique can be cited back to experiments carried out in the 17th and 18th centuries, such as the work of Albertus Seba32 and Edward Parrish33. These experiments centered around leaving plant matter, such as leaves and fruit, submerged in water for extended periods of time (weeks to months) and allowing the softer tissues to decay away naturally. Here the &#34;skeletonization&#34; approach is adapted to use milder conditions, such as longer incubation times at lower temperatures, to remove cellular residues and to avoid significantly disrupting the soft tissue structure. For the experiments detailed herein, three plant types were used: Ficus hispida, Pachira aquatica and a species of Garcinia. Results of DNA quantification, mechanical tests, and impact on cellular metabolic activity from both methods are described.

<table><tbody><tr><td>Sodium dodecyl sulfate</td><td>Sigma Life Science</td><td>75746-1KG</td><td></td></tr><tr><td>Triton X-100</td><td>MP Biomedicals, LLC</td><td>807426</td><td>Non-ionic surfactant referenced in paper. Very viscous reagent, can help to cut end of pipette tip when drawing it up.</td></tr><tr><td>Concentrated bleach (8.25% sodium hypochlorite)</td><td>Clorox</td><td>Item #: 31009</td><td>Standard concentrated bleach.</td></tr><tr><td>Sodium bicarbonate</td><td>Acros Organics</td><td>217120010</td><td>Can be substituted with sodium hydroxide or sodium carbonate.</td></tr><tr><td>8 mm Biopunch</td><td>HealthLink</td><td>15111-80</td><td>Cuts samples that fit well in 24 well plate</td></tr><tr><td>Belly Dancer-Shake table</td><td>Stovall Life Sciences</td><td>BDRAA115S</td><td>Use low speeds to not damage tissues. Can use any model/brand of shake table.</td></tr><tr><td>Isotemp hot/stir plate</td><td>Fisher Scientific</td><td></td><td>Can use any style/brand of hot/stir plate.</td></tr><tr><td>Beaker</td><td>Any</td><td></td><td>Can use any size beaker as long as it will fit your samples and not overcrowd them.</td></tr><tr><td>Tris Hydrochloride</td><td>Fisher Scientific</td><td>BP153-500</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>MT50003PC</td><td></td></tr><tr><td>Quant-iT Picogreen dsDNA assay</td><td>Life Technologies</td><td>P11496</td><td>Can use any dsDNA quantification mehtod on hand.</td></tr></tbody></table>

two methods for decellularization of plant tissues for tissue engineering applications

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

Both methods yielded scaffolds that were suitable for cell culture and tissue engineering applications. Figure 1 shows the general workflow of the decellularization process using an intact leaf for the detergent-based method and cut samples (8 mm diameter) for the detergent-free method. Successful decellularization of Ficus hispida tissues following both methods yielded clear and intact samples (Figure 1A and 1B<...

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Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

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

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

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Research

JoVE Journal

Bioengineering

14.5K Views.  University of Wisconsin-Madison. Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Video: Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

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, ...

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 ...

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 ...

Developmental Biology

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

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 ...

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

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

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

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

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

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

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

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

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