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...

<ol>
	<li>Vacanti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+and+regenerative+medicine:+from+first+principles+to+state+of+the+art.">Tissue engineering and regenerative medicine: from first principles to state of the art.</a> Journal of Pediatric Surgery. 45 (2), 291-294 (2010).</li><li>Kim, S., 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=Survival+and+function+of+hepatocytes+on+a+novel+three-dimensional+synthetic+biodegradable+polymer+scaffold+with+an+intrinsic+network+of+channels.">Survival and function of hepatocytes on a novel three-dimensional synthetic biodegradable polymer scaffold with an intrinsic network of channels.</a> Annals of Surgery. 228 (1), 8-13 (1998).</li><li>Park, A., Wu, B., Griffith, L. <a target="_blank" 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S.</a> Philosophical Transactions. 36 (407), 441-444 (1775).</li><li>Parrish, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Phantom+Boutique:+A+Popular+Treatise+on+the+Art+of+Skeletonizing+Leaves+and+Seed-Vessels+and+Adapting+Them+to+Embellish+the+Home+of+Taste.">The Phantom Boutique: A Popular Treatise on the Art of Skeletonizing Leaves and Seed-Vessels and Adapting Them to Embellish the Home of Taste.</a> The Phantom Boutique: A Popular Treatise on the Art of Skeletonizing Leaves and Seed-Vessels and Adapting Them to Embellish the Home of Taste. , (1865).</li><li>Coffin, S., Gaudette, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aprotinin+extends+mechanical+integrity+time+of+cell-seeded+fibrin+sutures.">Aprotinin extends mechanical integrity time of cell-seeded fibrin sutures.</a> Journal of Biomedical Materials Research Part A. 104 (9), 2271-2279 (2016).</li><li>Zangala, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Genomic+DNA+from+Mouse+Tails.">Isolation of Genomic DNA from Mouse Tails.</a> Journal of Visualized Experiments: JoVE. 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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 border="0" cellpadding="0" cellspacing="0" style="width:771px;" width="771">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Sodium dodecyl sulfate</td>
			<td style="width:111px;">Sigma Life Science</td>
			<td style="width:136px;">75746-1KG</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">Triton X-100</td>
			<td style="width:111px;">MP Biomedicals, LLC</td>
			<td style="width:136px;">807426</td>
			<td style="width:277px;">Non-ionic surfactant referenced in paper. Very viscous reagent, can help to cut end of pipette tip when drawing it up.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Concentrated bleach (8.25% sodium hypochlorite)</td>
			<td style="width:111px;">Clorox</td>
			<td style="width:136px;">Item #: 31009</td>
			<td style="width:277px;">Standard concentrated bleach.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Sodium bicarbonate</td>
			<td style="width:111px;">Acros Organics</td>
			<td style="width:136px;">217120010</td>
			<td style="width:277px;">Can be substituted with sodium hydroxide or sodium carbonate.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">8 mm Biopunch</td>
			<td style="width:111px;">HealthLink</td>
			<td style="width:136px;">15111-80</td>
			<td style="width:277px;">Cuts samples that fit well in 24 well plate</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Belly Dancer-Shake table</td>
			<td style="width:111px;">Stovall Life Sciences</td>
			<td style="width:136px;">BDRAA115S</td>
			<td style="width:277px;">Use low speeds to not damage tissues. Can use any model/brand of shake table.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Isotemp hot/stir plate</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;"></td>
			<td style="width:277px;">Can use any style/brand of hot/stir plate.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Beaker</td>
			<td style="width:111px;">Any</td>
			<td style="width:136px;"></td>
			<td style="width:277px;">Can use any size beaker as long as it will fit your samples and not overcrowd them.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Tris Hydrochloride</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;">BP153-500</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DMEM</td>
			<td style="width:111px;">Corning</td>
			<td style="width:136px;">MT50003PC</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Quant-iT Picogreen dsDNA assay</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:136px;">P11496</td>
			<td style="width:277px;">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<...

Watch this Scientific Journal Video about Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications at JoVE.com

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 ...

These methods can help address key questions in the biomaterials field such as the development of a scaffold with excellent fluid transfer capabilities with sophisticated ultra-structures and scalable manufacturing. The main advantage of these techniques is that they can be applied to most plants for stem tissues without the need for specialized equipment. First, collect fresh or frozen samples of F.hispida leaves.Store any remaining fresh sample at minus 20 degrees Celsius for future use. Let the leaf remain emersed in deionized water at 20 to 25 degrees Celsius. Use a clean biopsy punch to cut the leaf into eight millimeter diameter discs.Next, to wash the leaf samples, incubate them on a shake plate at low speed for five to 10 minutes, emersed in deionized water. Then prepare 10%weight by volume of SDS in deionized water. After preparing the SDS solution, place the leaf samples on a plastic dish.Then add the SDS solution to completely cover the samples. Place the SDS emersed leaf samples, covered, on a shake plate to prevent evaporation. Then incubate the leaf sample at room temperature for five days.After five days of incubation, replace the SDS solution with deionized water. Incubate the samples soaked in deionized water for 10 to 15 minutes on a shake plate. Next, mix five milliliters of non-ionic surfactant with 50 milliliters of bleach.Add this mixture to 445 milliliters of deionized water. Then emerse the samples in the freshly prepared non-ionic surfactant bleach solution. Continue changing the non-ionic surfactant bleach solution every 24 hours till the samples are cleared.Then leave the cleared samples emersed in deionized water for two minutes on a shake plate. For detergent free decellularization, prepare five percent volume by volume bleach in 3%weight by volume sodium bicarbonate solution. Next, warm the solution to 60 to 70 degrees Celsius on a hot plate within a fume hood.As soon as the solution reaches the desired temperature, soak the leaf samples. Then reduce the speed of the stir plate to prevent damaging the samples and look for the visibly cleared samples and transfer them from the bath carefully. Finally, incubate the samples in deionized water for one to two minutes.As all the samples are intrinsically different, it's recommended to check on the progress of decellularization approximately every 15 minutes to ensure the samples are not being damaged. MTM, SAF, and UTS parameters are first studied to investigate the unique decellularized scaffold properties of both P.aquatica and F.hispida species. In fact, all three parameters display similar mechanical properties using both the detergent based and detergent free decellularization methods.However, the UTS mechanical property of F.hispida shows a significantly higher average value using SDS rather than bleach for decellularization. The genomic DNA content of the cleared plant species studied is also significantly reduced upon decellularization when compared to the non-decellularized samples. Next, the metabolic activity of human dermal thyroblasts is also measured in the presence of decellularized P.aquatica or Garcinia scaffolds.Interestingly, detergent free based scaffolds elicited a reduced impact on cellular metabolic activity, but washing with Trishydrochloricde buffer, followed by serum-free medium after SDS clearing decreases the impact on cellular metabolic activity When compared to washing with deionized water alone. Finally, growth of the mesenchymal stem cells stained with calcine on the surface of the F.hispida leaf structure is shown here. Once mastered, these techniques can be completed from around seven days to as little as 15 minutes when performed properly.When performing these procedures, it's important to remember to monitor the samples as they decellularize because the samples can vary from batch to batch which can result in different clearing times. Following this procedure, other methods such as dequantification or mechanical testing can be applied to answer additional questions such as to what extent are the samples cleared and to what other tissues the samples are mechanically similar to. After its development, this technique has helped pave the way for researchers in the field of tissue engineering to manufacture perfusable scaffolds.After watching this video you should have a good understanding of how to decellularize plant tissues for use as scaffolds. Don't forget that working with detergents and bleach fumes can be hazardous and precautions such as using proper protective equipment and using a fume hood should always be taken while performing these procedures.

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Research

JoVE Journal

Bioengineering

14.2K Görüntüleme.  University of Wisconsin-Madison. These methods can help address key questions in the biomaterials field such as the development of a scaffold with excellent fluid transfer capabilities with sophisticated ultra-structures and scalable manufacturing. The main advantage of these techniques is that they can be applied to most plants for stem tissues without the need for specialized equipment. First, collect fresh or frozen samples of F.hispida leaves.Store any remaining fresh sample at minus 20 degrees Celsius for future ...