Creating 3D Controls: Bioprint Study

Wikis > Creating 3D Controls: Bioprint Study

New to the world of 3D biology, or testing out a new matrix material with your BioBot? This protocol is part of a series to help you learn how to design experiments and easily build with life!

Before you begin bioprinting, you need to make sure you have proper controls. Typical 2D culture controls are often not helpful comparisons to 3D printed constructs. 3D thin film controls help provide better insight and minimize variables as opposed to typical 2D controls. Before this first Bioprint study, be sure to complete an encapsulation study to create a 3D pipetted thin film control for this experiment.  Once you have a pipetted thin film control, complete this next study to create a bioprinted thin film control.

What’s a 3D Thin Film, and how do I make it?

Great question! A 3D thin film is a small volume of your material, ideally no thicker than 200 μm, that can be used to analyze cell viability and compare with bioprinted samples.

Ctrl_02_10X1X_200microns3D

Figure 1: PEGDA Thin Film. This 3D rendering of a PEGDA thin film was created through confocal microscopy. The thin film is no more than 200 μm thick and was analyzed through Live/Dead imaging. See the PEGDA bioreport here.

We create many of our pipetted thin films by simply pipetting small volumes of the material (5 – 10 μl) with encapsulated cells. Sometimes, we use a variety of methods to flatten the material after pipetting to ensure the height of the thin film is no thicker than 200 μm. Why does this thickness matter? In the body, cells can’t survive farther than this distance from blood vessels, or their source of nutrients. Ensuring the thickness is no larger than this distance removes the potential of decreased viability due to the geometry of the structures.

When bioprinting, we create these thin films by extruding small volumes of the material (5 – 10 μl) with encapsulated cells for a set amount of time. This can be accomplished by utilizing a custom .gcode like the one below:

; Sample Code – I can label my code and create comments by writing text after a semicolon
T0 ; select left extruder for this print(change this to T1 for right extruder) 
G92 E0 ; set position of extruder
G1 X0 Y0 Z0.2 F600 ; Move extruder to  (0,0,0.2) at a rate of 600 mm/min without extruding material
G1 X5 Y0 Z0.2 E0.1 ; Begin extruding material
G4 S10 ; extrude for 10 seconds (change the number 10 to desired amount of extrusion time) 
G92 E0 ;

Time needed for extrusion to create a specific volume can be determined through the volume test, which is described in more detailed in our wiki post about characterizing print parameters for a new material. You can use these thin films analyze the effects of the printing process on construct viability. If you are not able to achieve cell viability in your bioprinted thin films, for example, you’ll know there is an issue with your bioprinting parameters (such as pressure or needle type). Learn more about specific variables that can affect your construct viability here.

Setting Up Your Experiment

Determine Your Groups

What do you want to test in this study? Consider potential variables you can test that might be affected by the printing process. What pressure do you plan to print at? What needle type are you going to use? How long will your print take? At a minimum, you should have three groups: a 2D control, a 3D pipetted thin film control, and a 3D printed thin film experimental group.

For example, you can run an initial bioprint to analyze effects of different pressures. See example groups with our material Sodium Alginate:

  • 2% (w/v) sodium alginate, 27 gauge, 0.25″ needle, 5 psi bioprint thin film
  • 2% (w/v) sodium alginate, 27 gauge, 0.25″ needle, 10 psi bioprint thin film
  • 2% (w/v) sodium alginate, pipetted thin film control
  • 2D control

Alternatively, you may want to test different needle types. That study may contain groups such as:

  • 2% (w/v) sodium alginate, 27 gauge, 0.25″ needle, 10 psi bioprint thin film
  • 2% (w/v) sodium alginate, 30 gauge, 0.25″ needle, 10 psi bioprint thin film
  • 2% (w/v) sodium alginate, pipetted thin film control
  • 2D control

Determine Analytic Tests and Timepoints

How will you analyze your results? Your material and what variables you are testing will largely determine what assays are best to use. When testing for viability, Live/Dead staining is always a good qualitative control. We have successfully used Live/Dead staining (Calcein-AM, Ethidium Homodimer) with all of our matrix reagents.

Some other common assays used to analyze 3D samples include the AlamarBlue Assay and ATP Cell Titer Glo 3D Assay. However, these assays sometimes require you to break apart your samples, especially if the assay solution does not effectively permeate your specific material.gelma viability and atp with scale

Figure 2: GelMA Thin Film results. Before completing a bioprint study with GelMA, we first analyzed thin films of the data, utilizing a Cell Titer Glo assay and Live/Dead imaging. Here, results are shown from a bioprint study, where thin film results were compared with bioprinted samples. Timepoints of Day 1, 3 and 7 are used in this viability study.

If you plan to eventually test function, other assays or testing methods may be necessary. Finally, depending again on what variables you plan to analyze, you will have to decide what timepoints to use (When analyzing viability, we often complete a 7 day study with timepoints at days 1,3 and 7).

Determine Necessary Cell Number and Material Needed for Study

Now you just need to plan how much material and how many cells you will need for your experiment! You can use this cell encapsulation study calculator to help.

Once you complete this study, you can then use your 3D thin films as controls in future bioprinting experiments!

Examples

Check out some of these bioreports for results and methods with 3D thin films.