Creating 3D Controls: Encapsulation Study
New to the world of 3D biology, or testing out a new matrix material with your BioBot? This protocol is the first 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. Complete this study first to create 3D pipetted thin film controls for easier troubleshooting and improved controls.
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.
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 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.
You can use these thin films to quickly and easily test a variety of variables before adding even more in bioprinting. If you are not able to achieve cell viability in your thin films, for example, you’ll know there is an issue with your material (such as cell density or toxicity) as opposed to your bioprinting parameters (such as pressure or shear stress).
Setting Up Your Experiment
Determine Your Groups
What do you want to test in this study? Consider potential variables you can test prior to bioprinting that might be affected by your material. Does the concentration of your material affect encapsulated cell viability? What about crosslinking parameters, or encapsulated cell density? At minimum, you should have two groups: a 2D control and a 3D encapsulated thin film.
For example, you can run an encapsulation study to analyze material concentration. See example groups with our material Sodium Alginate:
- 2% (w/v) sodium alginate
- 5% (w/v) sodium alginate
- 10% (w/v) sodium alginate
- 2D control
Alternatively, you may want to test cell concentration. That study may contain groups such as:
- 2% (w/v) sodium alginate, 1 million/ml cell concentration
- 2% (w/v) sodium alginate, 5 million/ml cell concentration
- 2% (w/v) sodium alginate, 10 million/ml cell concentration
- 2D controls at each cell concentration
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 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.
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!
Check out some of these bioreports for results and methods with 3D thin films.