Bioengineering
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Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs
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Summary December 13th, 2019
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.
Transcript
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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