Name: pancreatic tissue-derived extracellular matrix bioink for printing 3d cell-laden pancreatic tissue constructs
Uploaded: 2019-12-13T15:00:00
Description: Watch this Scientific Journal Video about Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs at JoVE.com

This research was supported by the Bio &#38; Medical Technology Development program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (2017M3A9C6032067) and &#34;ICT Consilience Creative Program&#34; (IITP-2019-2011-1-00783) supervised by the IITP (Institute for Information &#38; Communications Technology Planning &#38; Evaluation).

Porcine pancreatic tissues were collected from a local slaughterhouse. Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Asan Medical Center, Seoul, Korea.
1. Tissue decellularization
<ol>
	<li>Prepare the solutions for decellularization. 
	NOTE: 1x phosphate-buffered saline (PBS) used in all solution preparations is diluted by adding distilled water to 10x PBS.
	<ol>
		<li>For the 1% Triton-X 100 solution, dissolve 100 mL of 100% Triton-X 10...

<ol>
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J., Pokrywczynska, M., Ricordi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+pancreatic+islet+transplantation.">Clinical pancreatic islet transplantation.</a> Nature Reviews Endocrinology. 13 (5), 268 (2017).</li><li>Venturini, M., 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=Technique,+complications,+and+therapeutic+efficacy+of+percutaneous+transplantation+of+human+pancreatic+islet+cells+in+type+1+diabetes:+the+role+of+US.">Technique, complications, and therapeutic efficacy of percutaneous transplantation of human pancreatic islet cells in type 1 diabetes: the role of US.</a> Radiology. 234 (2), 617-624 (2005).</li><li>Xie, 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=Cytoprotection+of+PEG-modified+adult+porcine+pancreatic+islets+for+improved+xenotransplantation.">Cytoprotection of PEG-modified adult porcine pancreatic islets for improved xenotransplantation.</a> Biomaterials. 26 (4), 403-412 (2005).</li><li>Sackett, S. 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H., Ramachandran, K., Stehno-Bittel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+replacement+for+islet+equivalents+with+improved+reliability+and+validity.">A replacement for islet equivalents with improved reliability and validity.</a> Acta Diabetologica. 50 (5), 687-696 (2013).</li><li>Pati, F., 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=Printing+three-dimensional+tissue+analogues+with+decellularized+extracellular+matrix+bioink.">Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink.</a> Nature Communications. 5, 3935 (2014).</li><li>Hussey, G. S., Dziki, J. L., 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=Extracellular+matrix-based+materials+for+regenerative+medicine.">Extracellular matrix-based materials for regenerative medicine.</a> Nature Reviews Materials. 1, (2018).</li><li>Kim, B. S., Kim, H., Gao, G., Jang, J., Cho, D. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-derived+decellularized+extracellular+matrix:+a+game+changer+for+bioink+manufacturing?.">Organ-derived decellularized extracellular matrix: a game changer for bioink manufacturing?.</a> Trends in Biotechnology. 36 (8), 787-805 (2018).</li><li>Kurpios, N. 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=The+direction+of+gut+looping+is+established+by+changes+in+the+extracellular+matrix+and+in+cell:+cell+adhesion.">The direction of gut looping is established by changes in the extracellular matrix and in cell: cell adhesion.</a> Proceedings of the National Academy of Sciences. 105 (25), 8499-8506 (2008).</li><li>Sakai, T., Larsen, M., Yamada, K. 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This protocol described the development of pdECM bioinks and the fabrication of 3D pancreatic tissue constructs by using 3D cell printing techniques. To recapitulate the microenvironment of the target tissue in the 3D engineered tissue construct, the choice of bioink is critical. In a previous study, we validated that tissue-specific dECM bioinks are beneficial to promote stem cell differentiation and proliferation10. Compared to synthetic polymers, dECM can serve as a cell-favorable environment b...

Recently, pancreatic islet transplantation has been considered a promising treatment for patients with type 1 diabetes. The relative safety and minimal invasiveness of the procedure are great advantages of this treatment1. However, it has several limitations such as the low success rate of isolating islets and the side effects of immunosuppressive drugs. Furthermore, the number of engrafted islets decreases steadily after transplantation due to the hostile environment2. Various biocompatible materials such as alginate, collagen, poly(lactic-co-glycolic acid) (PLGA) or polyethylene glycol (PEG) have been applied to pancreatic islet transplantation to overcome these difficulties.
3D cell printing technology is emerging in tissue engineering due to its great potential and high performance. Needless to say, bioinks are known as important components for providing a suitable microenvironment and enabling the improvement of cellular processes in printed tissue constructs. A substantial number of shear-thinning hydrogels such as fibrin, alginate, and collagen are widely used as bioinks. However, these materials show a lack of structural, chemical, biological, and mechanical complexity compared to the extracellular matrix (ECM) in native tissue3. Microenvironmental cues such as the interactions between islets and ECM are important signals for enhancing the function of islets. Decellularized ECM (dECM) can recreate the tissue-specific composition of various ECM components including collagen, glycosaminoglycans (GAGs), and glycoproteins. For example, primary islets that retain their peripheral ECMs (e.g., type I, III, IV, V, and VI collagen, laminin, and fibronectin) exhibit low apoptosis and better insulin sensitivity, thus indicating that tissue-specific cell-matrix interactions are important for enhancing their ability to function similarly to original tissue4.
In this paper, we elucidate protocols for preparing pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide beneficial microenvironmental cues for boosting the activity and functions of pancreatic islets, followed by the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Biological Safety Cabinets</td>
			<td>CRYSTE</td>
			<td>PURICUBE 1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Deep Freezer</td>
			<td>Thermo Scientific Forma</td>
			<td>957</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital orbital shaker</td>
			<td>DAIHAN Scientific</td>
			<td>DH.WSO04010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry oven</td>
			<td>DAIHAN Scientific</td>
			<td>WON-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze dryer</td>
			<td>LABCONCO</td>
			<td>7670540</td>
			<td></td>
		</tr>
		<tr>
			<td>Fridge</td>
			<td>SANSUNG</td>
			<td>CRFD-1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Grater</td>
			<td>ABM</td>
			<td>1415605793</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscopes</td>
			<td>Leica</td>
			<td>DMi1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>CRYSTE</td>
			<td>PURISPIN 17R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Thermo Fisher Scientific</td>
			<td>Multiskan GO</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>DAIHAN Scientific</td>
			<td>CF-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Hotplate Stirrers</td>
			<td>DAIHAN Scientific</td>
			<td>SMHS-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-LITE-PR</td>
			<td></td>
		</tr>
		<tr>
			<td>pH benchtop meter</td>
			<td>Thermo Fisher Scientific</td>
			<td>STARA2110</td>
			<td></td>
		</tr>
		<tr>
			<td>Rheometer</td>
			<td>TA Instrument</td>
			<td>Discovery HR-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>DAIHAN Scientific</td>
			<td>VM-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Cirurgical Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors</td>
			<td>Hirose</td>
			<td>HC.13-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Korea Ace Scientific</td>
			<td>HC.203-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml glass vial</td>
			<td>Scilab</td>
			<td>SL.VI1243</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>5 L beaker</td>
			<td>Dong Sung Science</td>
			<td>SDS 2400</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL cornical tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL beaker</td>
			<td>Korea Ace Scientific</td>
			<td>KA.23-08</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL bottle-top vacuum filter</td>
			<td>Corning</td>
			<td>431118</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL plastic container</td>
			<td>LOCK&#38;LOCK</td>
			<td>INL301</td>
			<td></td>
		</tr>
		<tr>
			<td>96well plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>DAEKYO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Kimtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic bar</td>
			<td>Korea Ace Scientific</td>
			<td>BA.37110-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar and pestle</td>
			<td>DAIHAN Scientific</td>
			<td>SC.MG100</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipettor</td>
			<td>Eppendorf</td>
			<td>4982000314</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>SPL</td>
			<td>10100</td>
			<td></td>
		</tr>
		<tr>
			<td>pH indicator strips</td>
			<td>Sigma-Aldrich</td>
			<td>1095350001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sieve filter mesh</td>
			<td>DAIHAN Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Decellularization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x pbs</td>
			<td>Hyclone</td>
			<td>SH30258.01</td>
			<td></td>
		</tr>
		<tr>
			<td>4.7% Peracetic acid</td>
			<td>Omegafarm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>SAMCHUN CHEMICALS</td>
			<td>E0220 SAM</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IPA</td>
			<td>SAMCHUN CHEMICALS</td>
			<td>samchun I0348</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Biosesang</td>
			<td>T1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Biochemical assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,9-Dimethyl-Methylene Blue zinc chloride double salt</td>
			<td>Sigma-Aldrich</td>
			<td>341088</td>
			<td></td>
		</tr>
		<tr>
			<td>10 N NaOH</td>
			<td>Biosesang</td>
			<td>S2018</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramine T</td>
			<td>Sigma-Aldrich</td>
			<td>857319</td>
			<td></td>
		</tr>
		<tr>
			<td>Chondroitin sulfate A</td>
			<td>Sigma-Aldrich</td>
			<td>C4384</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Supelco</td>
			<td>46933</td>
			<td></td>
		</tr>
		<tr>
			<td>Cysteine-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>C1276</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Merok</td>
			<td>100063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>410225</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>SAMCHUN CHEMICALS</td>
			<td>S2097</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma-Aldrich</td>
			<td>p4762</td>
			<td></td>
		</tr>
		<tr>
			<td>P-DAB</td>
			<td>Sigma-Aldrich</td>
			<td>D2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>311421</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S5636</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Supelco</td>
			<td>SX0607N</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate(monobasic)</td>
			<td>Sigma-Aldrich</td>
			<td>RDD007</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Sigma-Aldrich</td>
			<td>244511</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Charicterized FBS</td>
			<td>Hyclone</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma-Aldrich</td>
			<td>P7215</td>
			<td></td>
		</tr>
		<tr>
			<td>Rose bengal</td>
			<td>Sigma-Aldrich</td>
			<td>198250</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
	</tbody>
</table>

pancreatic tissue-derived extracellular matrix bioink for printing 3d cell-laden pancreatic tissue constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularization of pancreatic tissues 
We developed the process for preparing pdECM bioink to provide pancreatic tissue-specific microenvironments for enhancing functionality of islets in a 3D bioprinted tissue construct (Figure 2A). After the decellularization process, 97.3% of dsDNA was removed and representative ECM components such as collagen and GAGs remained at 1278.1% and 96.9% compared to that of the native pan...

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Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

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

pdECM bioink can provide a beneficial microenvironment for pancreatic islets using tissue-specific components and architecture and has optimal neurological properties that reduce cell deaths and increase their printability. The main advantage of this technique is that the tissue-specific composition can be preserved within the pdECM bioink promoting a constructive crosstalk between 3D pancreatic tissue constructs and encapsulated islets. The development of islet encapsulation in pdECM bioink can be applied to the fabrication of transplantable pancreatic tissue constructs for the treatment of type one diabetes.This bioink can be used to broaden the application of transplantable constructs as well as in vitro tissue models for diabetes, diabetes-related complications and pancreatic cancer. For the decellularization of a frozen porcine pancreas tissue sample, use a grater to slice the frozen pancreas into one millimeter thick pieces and transfer 50 grams of the sliced tissue into a 500 milliliter plastic container. Wash the tissue with 300 milliliters of distilled water at four degrees Celsius on a digital orbital shaker at 150 rotations per minute for approximately 12 hours.When the cloudy water disappears, replace the water with 400 milliliters of 1%Triton-X 100 in PBS for 84 hours. At the end of the detergent treatment, incubate the tissue with 400 milliliters of isopropanol for two hours to remove the remaining fat from the pancreas, followed by a 24-hour wash in 400 milliliters of PBS. At the end of the wash, sterilize the decellularized tissue with 400 milliliters of 0.1%peracetic acid in 4%ethanol for two hours before washing the sample with 400 milliliters of fresh PBS for six hours to remove any residual detergent.At the end of the wash, use forceps to transfer the tissue pieces into a 50 milliliter conical tube and refreeze the sample at minus 80 degrees Celsius for one hour. Then cover the conical tube with a lint-free wipe fixed with a rubber band and lyophilize the decellularized tissue at minus 50 degrees Celsius for four days. To prepare the bioink, transfer 200 milligrams of freeze dried pdECM into a new 50 milliliter conical tube and add 20 milligrams of pepsin and 8.4 milliliters of 0.5 molar acetic acid to the tissue sample.Then place a magnetic stir bar into the tube for a 96-hour stirring incubation at 300 rotations per minute. At the end of the incubation, use a 40 micrometer cell strainer and a positive displacement pipette to filter the solution into a new tube on ice to filter out any undigested particles and add one milliliter of 10X PBS to the tube. After vortexing, use sodium hydroxide to adjust the pH of the solution to seven.To prepare the bioink for the 3D cell printing of a pancreatic construct with a patterned structure, stain one aliquot of pdECM bioink with 0.4%Trypan Blue and one aliquot with Rose Bengal Solution at a one to 20 ratio. Then use a positive displacement pipette to gently mix each bioink solution with a medium suspended islet solution at a three to one ratio to a final 1.5%bioink concentration and a cell density of three times 10 to the three islet equivalent per milliliter. For 3D cell printing of multimaterial-based pancreatic tissue constructs, load each bioink islet mixture into individual sterilized syringes equipped with 25 gauge nozzles and 3D print each bioink under optimized printing conditions at 18 degrees Celsius in the shape of a lattice with alternating lines of blue and red.To cross-link the bioink, place the printed construct in a 37 degree Celsius and 5%carbon dioxide cell culture incubator for 30 minutes. Then immerse the printed construct into RPMI 1640 medium supplemented with 10%fetal bovine serum and 100 units per milliliter of penicillin and streptomycin. After the decellularization process, 97.3%of the double-stranded DNA is removed and the representative extracellular matrix components such as collagen and glycosaminoglycans remain at 1278.1%and 96.9%compared to that of the native pancreatic tissue respectively.In this representative analysis, the pdECM bioink exhibited a shear thinning behavior with a value of approximately 10 pascals per second at the shear rate of one per second indicating that the bioink demonstrated the appropriate rheological characteristics for extrusion through a nozzle. Further, the complex modulus of the bioink began to increase when the temperature reached 15 degrees Celsius and rapidly increased when the temperature was maintained at 37 degrees Celsius indicating the sol-gel transition of the solution. The dynamic G prime and G double prime of the pdECM bioink were investigated at physiologically relevant temperatures to ensure its stability after the printing process resulting in achieving a stable modulus under the frequency sweep condition.Using a multihead printing system, various types of 3D constructs could then be fabricated as demonstrated using the developed pdECM demonstrating the versatility of pdECM for the purpose of 3D bioprinting to harmonize two or more types of living tissues in a tissue-like arrangement. Be gentle when mixing the bioink with the cells to avoid cell death and bubble formation as the presence of bubbles lowers the printing resolution and reduces the cell viability. The functionality of the encapsulated pancreatic islets within the 3D pancreatic tissue constructs can be assessed by immunofluorescence staining or a glucose tolerance test.This pdECM bioink and 3D bioprinting technique can potentially be used for the development of functional pancreatic tissue constructs in the field of tissue engineering. Researchers should wear the appropriate personal protective equipment to prevent injuries when handling the sharp grater and reagents.

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Research

JoVE Journal

Bioengineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

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.

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 ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

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 ...

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 cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

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