Bioprinting Protocol

This section is currently under development.

In this workflow protocol, the construction of geometries for Bioprinting, path generation and the actual printing process will be described. Some of the steps are flexible and should be adapted to your needs (i.e. cell source, approach of experiment, materials used, designed construct, etc.)

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 better support 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.

Create a .stl file

You should create a geometry to be printed either on your own or just use a available .STL file (from sites as thingiverse or 3D-NIH.com for example). You can find tools for Computer aided Design (CAD) in our knowledge base

Generate a custom gcode

Use a Slicer Programm and the appropriate settings to generate custom gcode. A "Slicer" is a program that generates a layer path for the printer out of the geometry information of the construct data file, we focus here on the Prusa Slic3r, other software like CURA etc. probably needs different settings! ATTENTION: Be aware you have to change the bed size in the slicer according to your "container"! This is necessary for proper path generation in the gcode! Note: The settings may also change or be adapted depending on the used material involved. The settings from Nils for simple, rather geometric (cubus, spheres, etc.) structures work for the most part, more delicate and complex structures (hollow, many overhanging parts, fine small details) may require some tweaking and printing with different settings. You can also export the same construct with different settings (remember to name them aptly!) and generate complete G-Code with the composer for differently sliced constructs or different constructs all together for one print run (this happens to be way more convenient than to print everything in a single run, trust us)!

Use of the "Composer"

What the Composer basically does is to connect the pathplanning data from the construct gcode and the geometry of the container to be printed in. This is very important to let the machine knows where it has to "go" for proper printing!

Start your composer (via command in Linux or Mac or navigate into the folder where your files are on Windows and launch the launch-windows.bat). NOTE: You have to have electron installed. Choose the fitting container structure (i.e. 6-well) for your bio-structure via the respective Open/Load buttons. set your parameters (needlelength, intitial infill, etc.). NOTE: These parameters heavily depend on the object you want to print. click on compose .gcode and then after generation in the left upper coroner you can save the .gcode via clicking on the button. You can now either use the generated .gcode file to print via a connected program like Pronterface or transfer the file on a SD-Card and print from that.

Microgel/Gelatin Bath Preparation

The gelatin is prepared directly in a mixer jar. 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 centrifuged 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 good starting point: 1 mio. cells/ml in a 1:1 cells/bioink ratio. Ink/Cell solutions are filled into standard 10ml syringes. Different needle lengths can be used for printing, we use 0.5 " (approx 12,7mm).

Print Process

Using sterile equipment is highly recommended. The microgel should be filled in the construct (petri dish or 6-well plate, etc.) in which the model has to be printed in, preferably without any bubbles, then left for around 15 minutes at room temperature to let the hydrogel adjust. Before attaching the syringe holder with the bioink , you have to run the calibrating routine on your printer. This is done automatically after choosing your structure file and clicking print. After the calibration is done, the ink and the dish can be placed and the print started. When the print is finished, the dish should be placed in the incubator at 37°C for around 20- 30 minutes until the slurry bath is melted, so that the bath can be changed to (prewarmed) mHEBS medium for washing. Remove first the liquid gelatine gently via pipette (do not use the vacuum dispenser uner the flow hood since a) hardened gelatine might clog it b) you may accidentally destroy/flush away your construct. After submerging in mHEBS medium, it can be cultured like all standard cell culture, which also includes frequent medium change (use again just pipettes and be careful not to destroy the construct) or staining procedures.