Bioprinting Protocol

Preparations

Before you can start your print project, first you have to prepare the bioink and the slurry bath. The bioink consist of a hydrogel as well as cells (if necessary). While printing the bioink in the prepared slurry bath, you can higher the stability of your construct and avoid blur of the printed slices. Besides the "wet" steps, you have to create a .stl file with a CAD software and generate a custom .gcode beforehand.

microgel/gelatin bath preparation

The gelatin is prepared directly in a mixer bin. A 4,5 % w/v gelatin solution in 0,1 M CaCl_2 solution is prepared (in this case: 6,7g gelatin and 150 mL 0,1 CaCl_2 solution) and heated in the microwave until the liquid starts to boil. The solution is stored in the fridge overnight to solidify. The next day a small amount of the 0,1 M CaCl_2 solution is poured on the gel and the gelatin is loosened carefully with a spatula. Afterwards the bin is filled up with 0,1 M CaCl_2 solution until spilled to prevent air bubbles and closed with the lid. The gelatin is stored for 30 min in a -20°C freezer to prevent heat production during blending and blended for 2 min afterwards. To remove residues of dissolved gelatin, the mixture is washed. For this purpose, the blended gel is evenly distributed over falcon tubes and centrifuges at 3.000 rcf and 4°C for 2 min. The supernatant is discarded, and the tube is filled up with cold 0,1 M CaCl_2 solution to the 40 mL mark. The gel is resuspended by shaking and vortexing. This step is repeated three times. After the third step, the supernatant is discarded, and the tubes filled up with 0,1 M CaCl_2 solution to the 25 mL mark each. The gel is resuspended via shaking and vortexing and the contents of two tubes united, followed by an additional centrifugation step. These steps are repeated once. Finally, the supernatant is discarded again, the tubes filed up to the 25 mL mark, followed by resuspension. The Gel is sterilized by exposure to UV light (1000 J/cm^2) twice with the tubes turned by 180° before the second exposure. The microgel is stored at 4°C and can be used up to two weeks after preparation.

Bioink Preparation

Preparation of a 4% alginate hydrogel solution (dissolve 1g of alginate in 25 ml ddH_2O). To prevent contamination, the filter has to be filtered with a 0,45 µm mesh. The temperature of the hydrogel should be 37°C before using and should be stored at 8°C if necessary. The concentration of the cells and the total amount of the bioink should be choosen depending on the individual experiment, as well as the ratio of cells and bioink. The following protocol should be a fine for many interrogations: 1 mio. cells/ml in a 1:1 cells/bioink ratio.

Print Process

Using sterile equipment is highly recommended. The microgel should be filled in the construct (petri dish or 6-well plate) in which the model has to be printed in, preferably without any bubbles, then left for around 15 minutes at room temp. to let the hydrogel to adjust in the dish. Before attach the bioink and the hydrogel dish to the printer, you have to run the calibrating routine on your printer. After the calibration is done, the ink and the dish can be placed at their places and run the print. When the print is finished, the dish should be placed in the incubator at 37°C for aroudn 20 minutes until the slurry bath is melted, so that the bath can be changed to (prewarmed) mHEBS medium.