Embedded Bioprinting of Tissue-like Structures Using κ-Carrageenan Sub-Microgel Medium

3D bioprinting in a microgel-based suspension bath has provided a versatile platform for fabricating complex 3D structures using soft hydrogels, but it is still limited by the low print resolution and accuracy, hindering the practical applications of the printed products in tissue engineering. The present microgel baths often exhibit large sizes and poor dispersions, resulting in a big diameter and irregular morphology of the printed filaments. Therefore, it poses a significant challenge in preparing a microgel bath with a small particle size and uniform morphology.

This protocol offers the fabrication of a homogenous and biocompatible cationic cross-linked kappa-carrageenan sub-microgel bath for high-fidelity bioprinting through an easy-to-operate mechanical grinding strategy. Since kappa-carrageenan sub-microgels exhibit a uniform methodology with a small particle size of 565 nanometer, and a low germ state of 0.35%enabling precise tissue and organ construction with remarkable fidelity and high resolution. Begin by adding 1.75 grams of kappa-carrageenan powder to 500 milliliters of PBS in a 1000 milliliter glass bottle.

to prepare a kappa-carrageenan suspension bath, then introduce a 70 millimeter magnetic stirrer bar into the glass bottle. Tighten the glass bottle cap and then loosen it by half a turn. Heat the glass bottle in a 70 degree Celsius water bath.

Then turn on the magnetic stirrer at 300 RPM. Place the bottle and stir until the polymer is completely dissolved. Using a 0.22 micrometer filter, filter the completely dissolved kappa-carrageenan solution into the sterilized glass bottle.

Store the kappa-carrageenan solution at four degrees Celsius to induce a cation cross-linked gelation for 12 hours. Then using a 60 millimeter magnetic stirrer, mechanically grind the kappa-carrageenan hydrogels until they successfully transform into a liquid state. To begin, switch on the micro extrusion pump controller.

Select the corresponding volume syringe and set the extrusion rate at 0.08 milliliters per minute. Then divide the hydrogel volume by the extrusion rate and coordinate it with the desired printing time to calculate the duration of extrusion. Next, fit the syringe with a needle featuring an inner diameter of 210 micrometers into the syringe slot of the bio-printer.

Adjust the syringe controller to ensure that the plunger of the syringe is in close contact with the screw. Then place three milliliters of the prepared kappa-carrageenan suspension bath into a 35 millimeter cell culture dish. Based on the code settings, position the dish on the platform and confirm that the printing head is one millimeter above the bottom of the dish.

After that, insert the SD card containing the code into the 3D bio-printer. Activate the code file, start the bio-printer, and click the start button on the controller. Then expose the printed construct to 405 nanometers of blue light for one minute to initiate photo cross-linking.

Using a one milliliter pipette, remove the kappa-carrageenan gel, followed by the addition of three milliliters of PBS for washing and subsequent removal. To begin, take the cultured esophageal smooth muscle cells once they attain 80%confluency. Aspirate the medium and wash the endothelial smooth muscle cells with five milliliters of PBS.

Then add two milliliters of 0.2%trypsin EDTA to the flask and incubate for two minutes to digest the cells. Add two milliliters of DMEM to terminate the digestion process, and transfer the cell suspension to a 15 milliliter centrifuge tube. Centrifuge the tube at 675G for three minutes.

Carefully aspirate the supernatant and resuspend the cell pellet in the composite gelatin methacrylate and silk fibroin methacryloyl bio inks. After bioprinting and washing the 3D esophageal muscle layer with PBS, carefully aspirate the PBS and replace it with three milliliters of DMEM supplemented with 10%FBS and 1%penicillin or streptomycin.