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>

<ol>
	<li>Shapiro, A. 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. 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=Extracellular+matrix+scaffold+and+hydrogel+derived+from+decellularized+and+delipidized+human+pancreas.">Extracellular matrix scaffold and hydrogel derived from decellularized and delipidized human pancreas.</a> Scientific Reports. 8 (1), 10452 (2018).</li><li>Kim, 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=3D+cell+printing+of+islet-laden+pancreatic+tissue-derived+extracellular+matrix+bioink+constructs+for+enhancing+pancreatic+functions.">3D cell printing of islet-laden pancreatic tissue-derived extracellular matrix bioink constructs for enhancing pancreatic functions.</a> Journal of Materials Chemistry B. 7 (10), 1773-1781 (2019).</li><li>Yi, H. G., 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=A+bioprinted+human-glioblastoma-on-a-chip+for+the+identification+of+patient-specific+responses+to+chemoradiotherapy.">A bioprinted human-glioblastoma-on-a-chip for the identification of patient-specific responses to chemoradiotherapy.</a> Nature Biomedical Engineering. 1, (2019).</li><li>Das, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix+bioinks+and+the+external+stimuli+to+enhance+cardiac+tissue+development+in+vitro.">Decellularized extracellular matrix bioinks and the external stimuli to enhance cardiac tissue development in vitro.</a> Acta Biomaterialia. , (2019).</li><li>Kim, H., 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=Shear-induced+alignment+of+collagen+fibrils+using+3D+cell+printing+for+corneal+stroma+tissue+engineering.">Shear-induced alignment of collagen fibrils using 3D cell printing for corneal stroma tissue engineering.</a> Biofabrication. 11 (3), 035017 (2019).</li><li>Huang, H. 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix:+a+step+towards+the+next+generation+source+for+bioink+manufacturing.">Decellularized extracellular matrix: a step towards the next generation source for bioink manufacturing.</a> Biofabrication. 9 (3), 034104 (2017).</li><li>Gaetani, R., 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=Evaluation+of+different+decellularization+protocols+on+the+generation+of+pancreas-derived+hydrogels.">Evaluation of different decellularization protocols on the generation of pancreas-derived hydrogels.</a> Tissue Engineering Part C: Methods. 24 (12), 697-708 (2018).</li><li>Gao, G., 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=Tissue+engineered+bio-blood-vessels+constructed+using+a+tissue-specific+bioink+and+3D+coaxial+cell+printing+technique:+a+novel+therapy+for+ischemic+disease.">Tissue engineered bio-blood-vessels constructed using a tissue-specific bioink and 3D coaxial cell printing technique: a novel therapy for ischemic disease.</a> Advanced Functional Materials. 27 (33), 1700798 (2017).</li><li>La, W. G., 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=Systemically+replicated+organic+and+inorganic+bony+microenvironment+for+new+bone+formation+generated+by+a+3D+printing+technology.">Systemically replicated organic and inorganic bony microenvironment for new bone formation generated by a 3D printing technology.</a> RSC Advances. 6 (14), 11546-11553 (2016).</li><li>Lee, H., 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=Development+of+liver+decellularized+extracellular+matrix+bioink+for+three-dimensional+cell+printing-based+liver+tissue+engineering.">Development of liver decellularized extracellular matrix bioink for three-dimensional cell printing-based liver tissue engineering.</a> Biomacromolecules. 18 (4), 1229-1237 (2017).</li><li>Choudhury, D., Tun, H. W., Wang, T., Naing, M. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+requirement+in+branching+morphogenesis.">Fibronectin requirement in branching morphogenesis.</a> Nature. 423 (6942), 876 (2003).</li></ol>

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 because of the tissue-specific composition and architecture11. Therefore, the decellularization process should be seriously considered for the high retention of major components in the dECM.
The selection of different detergents for decellularization of pancreatic tissue varies the residual ECM constituent12. In the process of decellularization, we noticed that the use of sodium dodecyl sulfate (SDS) can affect loss of the ECM proteins13. Thus, we modified our previous protocol by eliminating the step for the treatment of SDS solution, which is an ionic surfactant used in many cleaning and decellularization processes featuring relatively harsh characteristics compared to the others such as Trion-X 100, or 3-[(3-cholamidopropyl) dimethyl-lammonio]-1-propanesulfonate (CHAPS). In this protocol, we used 1% Triton-X 100 solution for 84 h instead of SDS solution, which was able to remove the cellular components effectively while preserving GAGs and collagenous proteins. In addition, we noted that removal of residual lipids by treating with IPA is also a very crucial process for inducing the crosslinking of pdECM bioink and it can be understood in the same context as a previously published article4. Treatment with peracetic acid solution was also applied for the sterilization of decellularized tissue. In addition, removal of the remaining detergents and chemicals in the decellularized tissue is a crucial step to prevent the inflammatory host response. However, we did not discuss that issue in this protocol. Protocols that include a sanitization process at the end of decellularization will improve the biocompatibility of the decellularized material. Furthermore, standards for evaluation criteria should be considered to ensure that detergents and chemicals are completely removed.
The digestion of pdECM bioink with pepsin was performed to achieve the homogenous mixing of pdECM powder in the acidic solution by cleavage of the telopeptide region in the collagenous protein. In the pH adjustment process, keeping the pdECM bioink on ice is critical for the preservation of gelation. Afterward, we can produce physically crosslinkable pdECM pre-gel bioinks that can enter a gel state by incubating at 37 &#176;C, which is one of the main advantages of dECM-based bioinks. Selection of the proper concentration of the pdECM bioink is also important10. An ideal bioink should protect cells from external damage that occurs during the printing process such as pneumatic pressure and temperature change. It is known that the applied shear force may cause damage to the cells and reduce the cell viability in the printed constructs10. Also, enhancing concentrations of bioink could induce cell death5. In contrast, low concentrations of bioink induces low viscosity which means poor printability and shape-fidelity during printing. It is necessary to check the viscosity of bioink and optimize its concentration.
Currently, researchers are actively studying the development of various types of tissue-derived bioinks for printing 3D tissue constructs14,15,16. The results of these studies indicate that the bioink could provide tissue-specific microenvironments for cells. These unique conditions can promote the differentiation or maturation of stem cells and the proliferation of cells. Moreover, utilizing the multi-head equipped 3D cell-printing system makes it possible to print multiple types of bioinks with high precision simultaneously. Using this technique, a structure with a specific pattern can be produced, thus showing design versatility. In addition, it is feasible to encapsulate different types of cells into each bioink to mimic native cell arrangement17. These patterned structures can be utilized in the induction of vascularization or co-culture effect by improving cell-to-cell interactions, which can be key factors in the long-term survival of specific cells18,19.

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><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>&lt;strong&gt;Cirurgical Instruments&lt;/strong&gt;</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>&lt;strong&gt;Materials&lt;/strong&gt;</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 &amp;micro;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&amp;amp;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>&lt;strong&gt;Decellularization&lt;/strong&gt;</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>&lt;strong&gt;Biochemical assay&lt;/strong&gt;</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>&lt;strong&gt;Bioink&lt;/strong&gt;</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 pancreatic tissue, respectively (Figure 2B).
Bioink preparation 
To apply the pdECM in the printing process, the pdECM powder was solubilized in weak acid with pepsin and neutralized using 10 M NaOH solution. The digested pdECM solution could then be diluted through mixing with a cell culture medium or 1x PBS. In this study, we prepared pdECM bioink at a final concentration of 1.5% for further study. The pdECM bioink maintained a solution phase when it was placed under room temperature and instantly converted into a gel phase after incubation at 37 &#176;C for 30 min. To investigate the effect of the pdECM bioink on islets, isolated islets were encapsulated in the pdECM, alginate and collagen bioinks at a concentration of 1.5%. The result of the glucose-stimulated insulin secretion test showed islets in the pdECM bioink represented the highest index (approximately 3.174) among the experimental groups, indicating higher functionality over the widely applied hydrogels for islet encapsulation5.
Rheological analysis 
Viscosity is one of the critical characteristics when considering a printable biomaterial. We measured viscosity of the pdECM bioink at a frequency ranging from 1 to 1,000 Hz at 15 &#176;C for printing various dECM bioinks6,7,8. The pdECM bioink showed shear-thinning behavior and the value was approximately 10 Pa&#183;s at the shear rate of 1/s, indicating the pdECM bioink had appropriate rheological characteristics for extrusion through a nozzle (Figure 3A). The gelation kinetics at a temperature ranging from 4 to 37 &#176;C indicated the gelation behavior of the pdECM bioink at physiologically relevant temperatures. The complex modulus started to increase when the temperature reached 15 &#176;C, and it increased rapidly when the temperature was maintained at 37 &#176;C, indicating the sol-gel transition of the pdECM bioink (Figure 3B). The dynamic G&#39; and G&#34; of pdECM bioink were investigated at physiologically relevant temperatures to ensure its stability after the printing process, which resulted in having a stable modulus under the frequency sweep condition (Figure 3C).
3D cell printing 
3D cell-laden pancreatic tissue constructs were fabricated by using a microextrusion-based printing process. To build a construct containing at least 3,000 Islet equivalents (IEQ), that corresponds to the tissue volume of a perfectly spherical islet with a diameter of 150 &#181;m9, we designed the construct with a dimension of 10 mm x 10 mm x 3 mm (Figure 4A). The process parameters and conditions for printing pancreatic islets were selected to encapsulate islets, which are large cellular clusters in sizes ranging 100-250 &#181;m in diameter (Figure 4B). Using a multi-head printing system, various types of 3D constructs-such as the shape of the lattice having alternate lines of blue and red-were fabricated by using the developed pdECM (Figure 4C), indicating the versatility of pdECM for the purpose of 3D bioprinting to harmonize two or more types of living cells in a tissue-like arrangement.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60434/60434fig1a.jpg" /> 
Figure 1: Schematic of the development of decellularized pancreatic tissue, evaluation of pdECM bioink and fabrication of 3D pancreatic tissue constructs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60434/60434fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60434/60434fig2a.jpg" /> 
Figure 2: Representative images of the decellularization process and biochemical characterization of pdECM. (A) Overview of the decellularization of porcine pancreatic tissue. (B) Results of biochemical assays of native tissue and pdECM. Error bars show standard deviation. Copyright (2019) The Royal Society of Chemistry5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60434/60434fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60434/60434fig3a.jpg" /> 
Figure 3: Rheological analysis of pdECM bioink. (A) Viscosity of pdECM and collagen bioinks that exhibited shear thinning behavior. (B) Gelation kinetics of pdECM and collagen bioinks during temperature change. (C) The complex modulus of crosslinked pdECM and collagen bioinks. Copyright (2019) of The Royal Society of Chemistry5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60434/60434fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60434/60434fig4a.jpg" /> 
Figure 4: 3D cell printing of cell-laden pdECM bioink for 3D pancreatic tissue constructs. (A) The dimensions of 3D pancreatic tissue constructs. (B) Pancreatic islet-laden and (C) multimaterial-based 3D pancreatic tissue constructs. Copyright (2019) of The Royal Society of Chemistry5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60434/60434fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.9K Views.  Pohang University of Science and Technology. pdECM bioink kan een gunstige micro-omgeving bieden voor alvleeskliereilandjes met behulp van weefselspecifieke componenten en architectuur en heeft optimale neurologische eigenschappen die celsterfte verminderen en hun afdrukbaarheid verhogen. Het belangrijkste voordeel van deze techniek is dat de weefselspecifieke samenstelling kan worden bewaard binnen de pdECM bioink ter bevordering van een constructieve crosstalk tussen 3D-alvleesklierweefsel constructies en ingekapselde eilandjes. De on...

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

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

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 ...

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 ...

Advancing 3D Bioprinting with &#954;-Carrageenan Sub-Microgel Medium

Embedded Bioprinting of Tissue-like Structures Using &#954;-Carrageenan Sub-Microgel Medium

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic scaffolds. However, engineering a suitable gel suspension medium that balances precise bioink deposition with cell viability and function presents multiple challenges, particularly in achieving the desired viscoelastic properties. Here, a novel &#954;-carrageenan gel supporting bath is fabricated through an easy-to-operate mechanical grinding process, producing homogeneous sub-microscale particles. These sub-microgels exhibit typical Bingham flow behavior with small yield stress and rapid shear-thinning properties, which facilitate the smooth deposition of bioinks. Moreover, the reversible gel-sol transition and self-healing capabilities of the &#954;-carrageenan microgel network ensure the structural integrity of printed constructs, enabling the creation of complex, multi-layered tissue structures with defined architectural features. Post-printing, the &#954;-carrageenan sub-microgels can be easily removed by a simple phosphate-buffered saline wash. Further bioprinting with cell-laden bioinks demonstrates that cells within the biomimetic constructs have a high viability of 92% and quickly extend pseudopodia, as well as maintain robust proliferation, indicating the potential of this bioprinting strategy for tissue and organ fabrication. In summary, this novel &#954;-carrageenan sub-microgel medium emerges as a promising avenue for embedded bioprinting of exceptional quality, bearing profound implications for the in vitro development of engineered tissues and organs.

Author Spotlight: Development of Homogeneous &#954;-Carrageenan Sub-Microgel Baths for High-Resolution 3D Bioprinting

This study introduces a novel &#954;-carrageenan sub-microgel suspension bath, displaying remarkable reversible jamming-unjamming transition properties. These attributes contribute to the construction of biomimetic tissues and organs in embedded 3D bioprinting. The successful printing of heart/esophageal-like tissues with high resolution and cell growth demonstrates high-quality bioprinting and tissue engineering applications.

Embedded three-dimensional (3D) bioprinting utilizing a granular hydrogel supporting bath has emerged as a critical technique for creating biomimetic ...

Engineering

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

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 ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix ECM Production

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets&#8217; microenvironment in vitro and, potentially, in vivo settings.

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those ...

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression.
After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (Fc&#949;RI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (Fc&#949;RI).

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto ...

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

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