Name: tunable hydrogels from pulmonary extracellular matrix for 3d cell culture
Uploaded: 2017-01-17T13:00:00
Description: Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

We would like to thank Smithfield farms for donating the intact porcine lung tissue. We would also like to thank Dr. Hu Yang, Dr. Christina Tang and the VCU Plastic Surgery Department for allowing us to use their equipment. Hydrogel and tissue samples were prepared for SEM at the VCU Department of Anatomy and Neurobiology Microscopy Facility supported, in part, by funding from NIH-NINDS Center Core Grant 5 P30 NS047463 and, in part, by funding form NIH-NCI Cancer Center Support Grant P30 CA016059. SEM imaging of samples at the VCU Nanotechnology Core Characterization Facility (NCC). This work was funded by the National Science Foundation, CMMI 1351162.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Sterile Filter</td>
			<td>Directions</td>
		</tr>
		<tr>
			<td>DiH2O</td>
			<td>Yes</td>
			<td>DiH2O; Sterile filtered</td>
		</tr>
		<tr>
			<td>0.1% Triton X-100 Solution</td>
			<td>Yes</td>
			<td>Under fume hood add 100 &#181;l Triton-X 100 Solution to 100 ml DiH2O and agitate until dissolved; 
			sterile filter.</t...

<ol>
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R., Panoskaltsis-Mortari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+decellularized+lung+bioreactor+system+for+bioengineering+the+lung:+the+matrix+reloaded.">Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.</a> Tissue Eng. Part A. 16 (8), 2581-2591 (2010).</li><li>Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+rheological+characterization+of+a+gel+form+of+the+porcine+urinary+bladder+matrix.">Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.</a> Biomaterials. 29 (11), 1630-1637 (2008).</li><li>Tilghman, R. W., 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=Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype.">Matrix rigidity regulates cancer cell growth and cellular phenotype.</a> PloS One. 5 (9), e12905 (2010).</li><li>Ratner, B. D., Bryant, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials:+where+we+have+been+and+where+we+are+going.">Biomaterials: where we have been and where we are going.</a> Annu. Rev. Biomed. Eng. 6, 41-75 (2004).</li><li>Fridman, R., Benton, G., Aranoutova, I., Kleinman, H. K., Bonfil, R. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acellular+Normal+and+Fibrotic+Human+Lung+Matrices+as+a+Culture+System+for+In+Vitro+Investigation.">Acellular Normal and Fibrotic Human Lung Matrices as a Culture System for In Vitro Investigation.</a> Am. J. Respir. Crit. Care Med. 186 (9), 866-876 (2016).</li><li>Matsuda, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+human+lung+stem+cells.">Evidence for human lung stem cells.</a> N. Engl. J. Med. 364 (19), 1795-1806 (2011).</li><li>Zuo, W., 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=p63+Krt5++distal+airway+stem+cells+are+essential+for+lung+regeneration.">p63+Krt5+ distal airway stem cells are essential for lung regeneration.</a> Nature. 517 (7536), 616-620 (2015).</li><li>Keane, T. J., Londono, R., Turner, N. J., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+ineffective+decellularization+of+biologic+scaffolds+on+the+host+response.">Consequences of ineffective decellularization of biologic scaffolds on the host response.</a> Biomaterials. 33 (6), 1771-1781 (2012).</li><li>Neill, J. D., 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=Decellularization+of+human+and+porcine+lung+tissues+for+pulmonary+tissue+engineering.">Decellularization of human and porcine lung tissues for pulmonary tissue engineering.</a> Ann Thorac Surg. 96 (3), 1046-1055 (2013).</li></ol>

One of the integral aspects of biology is the self-organization of molecules into hierarchal structures that perform a specific task.13 In the lab, self-assembly depends on numerous factors such as salt concentration, pH, and digestion duration. As shown, a self-organizing hydrogel forms when solubilized proteins return to a physiological temperature. The hydrogel formed is capable of promoting cellular attachment and proliferation in vitro.
Cellular response to biophysical...

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6. 
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research. 
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

<table border="0" cellpadding="0" cellspacing="0" width="841">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100&#160;</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td style="width:371px;">Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Deoxycholate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6750-100g</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1016-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>D5025-150KU</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>258148-500ML</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepsin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>P6887-5G</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Hydroxide</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP359-500</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Genipin</td>
			<td style="width:135px;">Wako Chemicals</td>
			<td style="width:119px;">078-03021</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 10x</td>
			<td style="width:135px;">Quality Biological</td>
			<td>119-069-151</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:135px;">VWR</td>
			<td>45000-448</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter Paper</td>
			<td>Whatman</td>
			<td align="right">8519</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hand pump</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>10-239-1</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graduate Beaker</td>
			<td style="width:135px;">VitLab</td>
			<td align="right" style="width:119px;">445941</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomill</td>
			<td>SPEX</td>
			<td>6700</td>
			<td>Use cryogloves and eye protection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lyophilizer</td>
			<td>FTS FlexiDry</td>
			<td></td>
			<td>Use gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rheometer</td>
			<td>Discovery</td>
			<td>HR-2</td>
			<td>Use gloves and eye protection.</td>
		</tr>
	</tbody>
</table>

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

Using this method, we have produced hydrogels from normal pig, rat, and mouse lungs (Figure 1). Processed lungs provide an estimated 5 mg, 40 mg, and 10 g of ECM powder respectively. An overview of the process is shown in Figure 2. Key visualizations during the process include: white appearance of the lungs after rinsing deoxycholate; after the pregel formation, the solution should be opaque and the solution should appear homogenous for months if stored a...

Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

The overall goal of this method to make customized hydrogels is to study cells and culture conditions that replicate the complexity of the lung extracellular matrix. This method can help answer key questions in the pulmenary field such as how do lung cells interact with ECM under healthy and pathalogical conditions. The main advantage of this technique is that the lung ECM hydrogels are tailorable to your application in 2D or 3D culture.Generally, individuals new to this method will struggle because of the complexity of the decellularization techniques, sometimes requiring multiple people and attention to detail to optimize the amount of viable tissue. For this protocol, have a porcine lung ready for decellularization. Details on its preparation can be found in the text protocol.Once the lung is prepared, use a hand pump that is cannulated to fit the pulmonary artery. First, perfuse around one liter of water into the vasculature, and then 1.5 liters into the trachea. Do this three times, increasing the volume of water perfused as cellular debris is removed.Next, perfuse the tissue with Triton X-100, also using the hand pump. Then, submerge the tissue in a bath of Triton X-100 solution at four degrees Celsius for 24 hours. After 24 hours, rinse the vasculature and trachea three times with distilled water.After the rinses, use the hand pump to perfuse the tissue with deoxycholate. And then submerge tissue into deoxycholate for 24 hours at four degrees Celsius. The next day, rinse the tissue three times with distilled water, as before.After the rinses, use the hand pump to perfuse the tissue with filtered sodium chloride. Then submerge the tissue in filtered sodium chloride for an hour at four degrees Celsius. After the bath, perfuse the vasculature and trachea three times with distilled water to rinse.After the rinses, perfuse the tissue with DNase solution, and then submerge it in DNase solution for one hour at four degrees Celsius. After an hour, perfuse the vasculature and trachea five times using PBS, not water. Now, only using opaque white tissue, dissect away all the tubules that are two millimeters in diameter or larger.They are primarily found around the hilum and medial portions of the lungs. Leave the respiratory zones intact, which are mostly in the periphery. After removing the tubules, dissect the tissue into one inch sections or smaller.The orientation of these blocks is not important. After draining the tissue blocks, transfer them to a 50 milliliter conical tube and freeze them at 80 degrees Celsius. A few blocks can be set aside for histology to confirm the removal of the cells and debris.To process the lung tissue into decellularized powder, replace the cover on the frozen tissue tube with a filter paper, and secure this with an elastic band. Then, lyophilize the tissue until all the excess liquid is gone using a freeze dryer according to manufacture's directions. Then, store the tissue at 80 degrees Celsius until it can be milled.To ready the mill, first load it with liquid nitrogen, leaving room for the mill tube. Next, from the mill tube, remove the magnetic mill bar and cover the bottom of the tube with tissue. Then, replace the mill bar and loosely pack in more tissue to fill the tube.Then, close the mill tube, place it in the freezer/mill, and fill the mill with liquid nitrogen up to its maximum capacity. Now, run the mill for approximately five minutes with an impaction rate of about 600 per minute. Then, store the decellularized powder at 80 degrees Celsius, until needed.To make the pre-gel material, add decellularized tissue powder and pepsin to a conical tube. Then add 0.01 molar hydrochloric acid to the tube, for a final concentration of 1%decellularized tissue powder, and 0.01%pepsin. Keep this solution under constant agitation at room temperature for 48 hours to digest the tissues.After 48 hours, put the solution on ice for five minutes. Now adjust the solution's pH to 7.4 using cold 0.1 molar sodium hydroxide, and then bring the solution's PBS concentration to 1X by adding 10X PBS. We have some experience with altering times and the pH of the pre-gel.There is some flexibility in the range that can be used, but the properties can change dramatically if the procedure is altered in minor ways. The solution is not considered pre-gel and it can be used to coat non-treated plates to form hydrogels to form cells on, to form hydrogels to form cells in, or it can be further modified before forming hydrogels to increase the hydrogels'rigidity. To culture cells on two-dimensional micro-porous gel coating, add 20 microliters of pre-gel solution to wells of a 96-well non-treated tissue culture plate.Then, refrigerate the plate overnight at four degrees Celsius so proteins are absorbed onto the plate. The next day, aspirate the pre-gel solution and rinse the wells with PBS. Then, add 3, 200 cells to each well and top up the media volume in each well to 100 microliters.The plates can then be incubated. For a three-dimensional cell culture with cells inside the micro-porous gel, resuspend the cells of interest at one million per milliliter in pre-gel solution. Then, quickly dispense 16 microliters of the cell suspension into each well of a 96-well plate to form a 3D cell culture that is about 500 microns thick.The gel will form in 30 minutes once incubated at 37 degrees Celsius. After the gels form, add media to the wells up to the 100 microliter marks. For a three-dimensional cell culture with cells on top of the micro-porous gel, add the 100 microliters of the pre-gel solution to the wells of an untreated 96-well plate.Then, incubate the plate at 37 degrees Celsius for 30 minutes for gelation. Once the gels form, add 3, 200 cells in 100 microliter media on top of the hydrogels and proceed to incubate the plate. Hydrogels were produced from normal pig, rat, and mouse lungs using the described method.Rheological testing confirmed that over 90%of the gelling occurs in the first minutes after reaching 37 degrees Celsius. The formed hydrogels were stable for long periods in PBS, but cell attachment and proliferation may change with time. The pre-gel solution was tested as a coding to improve cell attachment.Human umbilical vein endothelial cell attachment and proliferation was measured using an MTTSA. Improvements were observed by coating the wells with ECM. In making 3D gels, adding Genipin increased the hydrogel crosslinking.Using frequency sweeps on a rheometer, hydrogel stiffness was directly correlated to the Genipin concentration, with the median and most relevant concentration being 0.01%Testing the more rigid gels, cell proliferation was measured to be higher by an MTTSA on the gels with increased crosslinking. Once mastered, decellularization can be performed in eight hours over four days. The pre-gel solution can be made in one hour over two days, and the gel formation can be accomplished in two hours over two days.Following this procedure, other methods like AFM, western blots, staining, and immunohistochemistry can be performed in order to answer additional questions, like elastic modulus, protein composition, and cellular response to the material.

Research

JoVE Journal

Bioengineering

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The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

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Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...