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

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

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

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

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

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

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

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

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

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

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

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

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

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