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 100 solution in 900 mL of 1x PBS using a magnetic stir bar with the stirring at 150 rpm for 6 h. Make 400 mL of 1% Triton-X 100 solution with 40 mL of 10% Triton-X 100 solution and 360 mL of 1x PBS prepared just before use. 
		NOTE: The 10% Triton-X 100 solution can be stored at room temperature until needed.</li>
		<li>For the 0.1% peracetic acid solution, dilute 8.5 mL of 4.7% peracetic acid into 22.8 mL of 70% ethanol with 368.7 mL of distilled water just before use.</li>
	</ol>
	</li>
	<li>Remove the peripheral tissues of the pancreas and slice the tissue before decellularization.
	<ol>
		<li>Wash the resected porcine pancreas with running tap water and remove the peripheral tissues using sterilized scissors.</li>
		<li>Transfer the pancreas into a plastic bag with forceps and freeze at -80 &#176;C for 1 h to help cut the pancreas effectively for the next step.</li>
		<li>Slice the frozen pancreas into 1 mm thick pieces using a grater.</li>
		<li>Transfer 50 g of the sliced tissue into a 500 mL plastic container. 
		NOTE: A plastic container with a lid is recommended here to protect tissues from contamination and prevent solution evaporation.</li>
	</ol>
	</li>
	<li>Treatment with reagents. 
	NOTE: The entire decellularization process should be carried out at 4 &#176;C on a digital orbital shaker at 150 rpm. In all decellularization steps, physical detachment using forceps is required to prevent the slices of pancreas from sticking together. Washing the container with distilled water is necessary to remove the residual reagents completely in the container.
	<ol>
		<li>Before any reagent treatment, wash 50 g of sliced pancreas with 300 mL of distilled water using a shaker.</li>
		<li>Stir the tissue continuously at 150 rpm until the cloudy water disappears (after approximately 12 h). Replace the distilled water every 2 h. 
		NOTE: Changing the distilled water every hour is recommended for efficiency to remove the cloudy water more quickly.</li>
		<li>Discard the water and treat the 50 g of tissues with 400 mL of 1% Triton-X 100 in 1x PBS solution for 84 h. Refresh the solution every 12 h. 
		NOTE: At this point, the amount of tissue will decrease because the cellular components start being removed.</li>
		<li>Treat with 400 mL of isopropanol (IPA) for 2 h to remove the remaining fat from the pancreas. 
		NOTE: It is normal for the tissue to become tough due to the removal of fat in this process.</li>
		<li>After 2 h, remove the IPA and wash the tissue with 400 mL of 1x PBS for 24 h. Refresh the 1x PBS every 12 h.</li>
		<li>To sterilize the decellularized tissue, discard the previous solution and treat with 400 mL of 0.1% peracetic acid in 4% ethanol for 2 h.</li>
		<li>To remove residual detergent, wash the tissue with 400 mL of 1x PBS for 6 h. Refresh the solution every 2 h.</li>
		<li>Collect the decellularized tissues in a 50 mL conical tube with forceps.</li>
		<li>Freeze the sample at -80 &#176;C for 1 h. Cover the conical tube with a lint-free wipe instead of the lid and fix with a rubber band for efficient lyophilization.</li>
		<li>Lyophilize the decellularized tissue at -50 &#176;C for 4 d. 
		NOTE: For step 1.3, 1 g of non-decellularized tissue should also be freeze-dried under the same conditions.</li>
	</ol>
	</li>
</ol>
2. Assessment of decellularized tissues
NOTE: To evaluate the residual amount of dsDNA, glycosaminoglycans (GAGs), and collagen in the decellularized tissue compared to native tissue, at least 1 g of each of the non-decellularized tissue (native tissue) and decellularized tissue are required for one batch of assessment. The amount of dsDNA, GAGs, and collagen can be calculated based on the dry weight of the tissue.
<ol>
	<li>Prepare solutions for biochemical assays.
	<ol>
		<li>Prepare papain solution for sample digestion. 
		NOTE: The amount of buffer to be made can be adjusted according to the number of samples.
		<ol>
			<li>Dissolve 119 mg of 0.1 M sodium phosphate (monobasic), 18.6 mg of 0.5 mM Na2-EDTA, and 8.8 mg of 5 mM cysteine-HCl in 10 mL of autoclaved water.</li>
			<li>Adjust the pH of the solution to 6.5 by adding 10 M NaOH solution.</li>
			<li>Add 125 &#181;L of 10 mg/mL papain stock solution to the above solution and vortex, allowing each element to mix evenly.</li>
		</ol>
		</li>
		<li>Prepare solutions for dimethyl-methylene blue (DMMB) assay.
		<ol>
			<li>To make DMMB dye, dissolve 8 mg of 1,9-dimethyl-methylene blue zinc chloride double salt, 1.52 g of glycine, and 1.185 g of NaCl in 500 mL of autoclaved water. Adjust the pH to 3 by adding 0.5 M HCl solution while measuring the change of pH using a bench-top pH meter. Then, filter this through a 500 mL bottle-top vacuum filter.</li>
			<li>Make 15 &#181;L of 10 mg/mL chondroitin sulfate A solution for standard.</li>
		</ol>
		</li>
		<li>Prepare solutions for hydroxyproline assay.
		<ol>
			<li>For the chloramine working solution, dissolve 2.4 g of sodium acetate, 1 g of citric acid, and 0.68 g of sodium hydroxide in 24 mL of distilled water and add 240 &#181;L of glacial acetic acid, 10 &#181;L of toluene, and 6 mL of IPA. 
			NOTE: Dissolve all powders in solution using a vortex mixer. Chloramine working solution can be stored at 4 &#176;C for up to three months.</li>
			<li>For chloramine T solution, dissolve 0.35 g of chloramine T in 20 mL of chloramine working solution and add 2.5 mL of IPA. Vortex to mix all components. 
			NOTE: Prepare immediately before use.</li>
			<li>For P-DAB solution, put 3.75 g of P-DAB into 6.5 mL of perchloric acid and 15 mL of IPA; wrap it in aluminum foil. 
			NOTE: Prepare immediately before use.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Add 1 mL of papain solution to 10 mg of lyophilized tissue in a 1.5 mL microcentrifuge tube and vortex the tube to better digest the sample.</li>
	<li>Place the 1.5 mL microcentrifuge tube in a rubber rack and digest the samples in a 500 mL beaker that contains 300 mL of water at 60 &#176;C for 16 h.</li>
	<li>Centrifuge at 9,500 x g for 20 min, collect the supernatant, and transfer it into a new tube.</li>
	<li>Quantify the residual DNA and major proteins in the decellularized tissue.
	<ol>
		<li>Load 1 &#181;L of digested sample into the spectrometer and measure the amount of dsDNA according to the manufacturer&#39;s instructions. 
		NOTE: The experimenter should tie their hair back and wear a mask to avoid contaminating the sample.</li>
	</ol>
	</li>
	<li>Perform a DMMB assay to quantify the amount of GAGs that remains in the decellularized tissue.
	<ol>
		<li>Mix 1 &#181;L of chondroitin sulfate A solution and 499 &#181;L of 1x PBS to make standards. Dilute the chondroitin sulfate A solution with distilled water at concentrations of 0, 4, 8, 12, 16, and 20 &#181;g/mL.</li>
		<li>Load triplicates of 50 &#181;L of each concentration of the standard and digested samples into a 96-well plate.</li>
		<li>Add 200 &#181;L of the DMMB dye to each well using a multi-channel pipette.</li>
		<li>Immediately read the absorbance at 525 nm on a microplate reader.</li>
	</ol>
	</li>
	<li>Perform a hydroxyproline assay to quantify the amount of collagen.
	<ol>
		<li>To conduct a hydroxyproline assay, incubate 250 &#181;L of digested sample with equal volumes of HCl at 120 &#176;C for 16 h.</li>
		<li>Dry the residues at room temperature for 3 h to cool the samples and then re-dissolve the samples in 1 mL of 1x PBS.</li>
		<li>Centrifuge at 2,400 x g for 10 min at 4 &#176;C.</li>
		<li>Prepare 100 &#181;g/mL hydroxyproline solution as standard.</li>
		<li>Dilute hydroxyproline solution with distilled water at a concentration of 0, 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, 30 &#181;g/mL for a standard solution.</li>
		<li>Load triplicates of 50 &#181;L of samples and standard solution into a 96-well plate.</li>
		<li>Add 50 &#181;L of Chloramine T solution then incubate for 20 min at room temperature.</li>
		<li>Add 50 &#181;L of p-DAB solution and incubate for 30 min at 60 &#176;C. 
		NOTE: Aliquot in a dark room. After the addition, wrap the plate with aluminum foil.</li>
		<li>Incubate at room temperature for 30 min.</li>
		<li>After cooling, measure the absorbance at 540 nm on a microplate reader.</li>
	</ol>
	</li>
</ol>
3. Bioink preparation
NOTE: pdECM powder can be stored stably at -80 &#176;C for at least one year. Before pH adjustment, the digested pdECM solution can be stored at -20 &#176;C for one month. Prior to use, thaw the sample of frozen pdECM solution at 4 &#176;C overnight. The pH-adjusted pdECM solution can be stored at 4 &#176;C for up to one week. The digested pdECM solution can be stored at 4 &#176;C for at least a few days but should not exceed 1 week.
<ol>
	<li>Digest the freeze-dried pdECM with pepsin.
	<ol>
		<li>For effective digestion of bioink, pulverize the lyophilized pdECM with liquid nitrogen using a mortar and pestle.</li>
		<li>Collect 200 mg of the pdECM powder in the 50 mL conical tube and add 20 mg of the pepsin and 8.4 mL of the 0.5 M acetic acid (final concentration is 2 w/v%).</li>
		<li>Place the magnetic stir bar in the 50 mL conical tube and stir at 300 rpm for 96 h.</li>
	</ol>
	</li>
	<li>Adjust pH of digested pdECM solution. 
	NOTE: In order to avoid gelation before pH adjustment, this process should be conducted on ice.
	<ol>
		<li>Filter out the undigested particles in the pdECM solution using a 40 &#181;m cell strainer using a positive displacement pipette on ice to obtain the optimal digestion of parts.</li>
		<li>Add 1 mL of the 10x PBS and vortex before using NaOH.</li>
		<li>Adjust the pH to 7 with 10 M NaOH checking the pH with pH indicator strips. 
		NOTE: Vortex each time NaOH is added so that the bioink is thoroughly mixed with the other reagents.</li>
	</ol>
	</li>
</ol>
4. Rheological analysis
<ol>
	<li>Experimental setup
	<ol>
		<li>Prepare the 1.5% (w/v) of pdECM bioink to assess the rheological properties.</li>
		<li>Establish a 20 mm cone plate geometry (cone diameter of 20 mm with a 2&#176; angle) in rate-controlled mode of a rheometer.</li>
		<li>Create experimental sequences in the installed software (TRIOS) to measure the viscosity, gelation kinetics, and dynamic modulus of the pdECM bioink.
		<ol>
			<li>Viscosity: Place the pdECM bioink on the plate. Measure complex viscosity (Pa&#183;s) of pdECM bioink under an increasing shear rate from 1 to 1,000 s-1 at a constant temperature of 15 &#176;C.</li>
			<li>Gelation kinetics: Place the pdECM bioink on the plate. Calculate the complex modulus (G*) by measuring the storage and loss modulus of pdECM bioink at 4-37 &#176;C with an incremental increase rate of 5 &#176;C/min (time-sweep mode).</li>
			<li>Dynamic modulus: Place the pdECM bioink on the plate at 37 &#176;C for 60 min prior to measurement. Measure the frequency-dependent storage modulus (G&#39;) and loss modulus (G&#39;&#39;) of the pdECM bioink in the range of 0.1-100 rad/s at 2% strain.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. 3D cell printing of pancreatic tissue constructs using islet
<ol>
	<li>Preparation of isolated islets
	<ol>
		<li>Isolate primary islets from a rat according to the protocols described in a previous work5.</li>
		<li>To separate debris and dead cells from the isolated islet, pass the cell suspension through a 70 &#181;m cell strainer. Islets with a diameter smaller than 70 &#181;m are considered dead or abnormal.</li>
		<li>Suspend the isolated islets in RPMI-1640 medium and place them on the petri dish. Remove islets larger than 300 &#181;m in diameter by using a P200 volume pipette under the microscope (4x objective lens) in the biosafety cabinet.</li>
	</ol>
	</li>
	<li>Islet encapsulation into pdECM bioink
	<ol>
		<li>Prepare pH adjusted pdECM bioink and isolated islet. 
		NOTE: To avoid gelation before pH adjustment, this process should be performed on ice.</li>
		<li>Gently mix the the pdECM bioink and the media suspended with islets (ratio 3:1) using a positive displacement pipette until uniformly mixed. 
		NOTE: The final concentration of the pdECM bioink is 1.5% and the cell density in the pdECM bioink is 3,000 IEQ/mL.</li>
	</ol>
	</li>
	<li>3D cell printing of pancreatic tissue constructs
	<ol>
		<li>Prepare a sterilized syringe and 22 G nozzle. 
		NOTE: This gauge was selected for printing islets with a diameter of 100-250 &#181;m.</li>
		<li>Load islet-laden pdECM bioink into the syringe.</li>
		<li>Print the bioink with the optimized printing condition (feed rate: 150 mm/min; pneumatic pressure: 15 kPa) at 18 &#176;C in the shape of a lattice.</li>
		<li>To crosslink the bioink, place the printed construct in the incubator for 30 min.</li>
		<li>Immerse the printed construct into the islet culture media which is RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin.</li>
	</ol>
	</li>
</ol>
6. 3D cell printing of pancreatic construct with patterned structure
<ol>
	<li>Preparation of two types of the bioink
	<ol>
		<li>To validate the printing versatility using multiple bioinks, prepare two sets of pdECM bioinks and stain them by adding 0.4% Trypan Blue and Rose Bengal solution into each pdECM bioink at a ratio of 1:20, respectively. 
		NOTE: To avoid gelation before pH adjustment, this process should be conducted on ice.</li>
		<li>Gently mix the the pdECM bioink and the media suspended with islets (ratio 3:1) using a positive displacement pipette until uniformly mixed. 
		NOTE: The final concentration of the pdECM bioink is 1.5% and the cell density in the pdECM bioink is 3,000 IEQ/mL.</li>
	</ol>
	</li>
	<li>3D cell printing of multimaterial-based pancreatic tissue constructs
	<ol>
		<li>Prepare sterilized syringes and a 25 G nozzle.</li>
		<li>Load each bioink (blue and red) into two different syringes, respectively.</li>
		<li>Print the bioink with optimized printing condition (feed rate: 150 mm/min; pneumatic pressure: 15 kPa) at 18 &#176;C in a shape of a lattice with alternating lines of blue and red.</li>
		<li>To crosslink the bioink, place the printed construct in the incubator for 30 min.</li>
		<li>Immerse the printed construct into islet culture media which is RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin.</li>
	</ol>
	</li>
</ol>

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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...

Watch this Scientific Journal Video about Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs at JoVE.com

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 ...

pancreatic-tissue-derived-extracellular-matrix-bioink-for-printing-3d

Research

JoVE Journal

Bioengineering

10.8K Views.  Pohang University of Science and Technology. 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.

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

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 ...