3D Bioprinting Substrate Stiffness – Often Overlooked, But Always at Work

In the rising and promising field of tissue engineering, 3D hydrogels and 3D bioprinting are the forefronts of most conversations. The goal is often simplified as “how can I bioprint these cells?” or “I just want my cells to be in a 3D hydrogel.”

These are important steps in the right direction, but the composition and mechanical signals of the substrate or matrix surrounding the cells is frequently overlooked.

Cells are able to sense and respond to mechanical stimuli in a process called mechanotransduction. Various areas throughout the body have significantly different degrees of matrix stiffness. For example, the brain tissue is very soft (~0.2 kPa), whereas cartilage and bone tissue are very firm (>64 kPa). Cells receive critical information simply from the stiffness of the surrounding matrix. 


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The rigidity (stiffness) of the matrix surrounding cells can have a profound effect on cell propagation, differentiation, lineage specification, gene expression, morphology, self-renewal, pluripotency, and migration.

As mentioned before, substrate stiffness is often overlooked, but some recent publications indicate that the research community is starting to take notice. Look at the titles of these recent publications: 

  • “The role of substrate stiffness in enhancing the internalization efficiency of plasmid DNA in stem cells”
  • “Substrate stiffness induces astrogliosis in primary rat astrocytes”
  • “Fibroblast polarization is a matrix-rigidity-dependent process”
  • “Rigidity of silicone substrates controls cell spreading and stem cell differentiation”

Simply bioprinting a shape does not equate to bioprinting the correct micro and macro environment for cells to behave in the proper way. Putting cells in 3D, just for them to be in 3D, does not solve all the problems. 

One of the challenges of 3D hydrogels and bioprinting is the increase in variables. Protein concentration, photocrosslinking time and intensity, and combinations or mixtures of proteins, can all cause cells to behave differently, and add variability. 

To simplify, eliminate variables, and identify how the substrate stiffness affects cells, I recommend using a silicon substrate that has been tuned to a specific stiffness. 

CytoSoft® plates have a thin layer of silicone that has been functionalized to allow for protein binding and subsequent cell seeding on a specific rigidity or stiffness. Cells attach down on the surface and are able to respond to the mechanical stimuli via mechanotransduction. 

The CytoSoft® plates come in 0.2, 0.5, 2, 8, 16, 32 and 64 kPa rigidities, and are carefully measured and certified. The only difference between different stiffness plates is just that… stiffness. No variables are introduced with photocrosslinking, concentration, etc.

You can evaluate how the substrate stiffness affects your cells, and only worry about that one parameter. 

Though these plates are a “2D” application, they provide straightforward and purified data regarding substrate stiffness and cells. Once a certain “desired stiffness range” is discovered, you can start trying to target that stiffness in 3D applications. Having a target stiffness in mind, backed by data, will eliminate countless hours of blind experiments. 

The transition to 3D can be tricky, but it can be simplified by CytoSoft® and other tools on the market. For example, if you discover that your cells grow best in a 2 kPa plate, I recommend a 6-10 mg/ml atelocollagen for 3D hydrogels. If your cells grow best in a firmer environment such as 16-32 kPa, I recommend an 8 mg/ml telocollagen, or photo-crosslinkable ECM such as methacrylated collagen or gelatin.

Each application is different, so feel free to reach out if you want to brainstorm. Let’s all work together to move tissue engineering and 3D bioprinting in the right direction by talking about substrate stiffness.

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References:

Wilson, C. L., Hayward, S. L., & Kidambi, S. (2016). Astrogliosis in a dish: Substrate stiffness induces astrogliosis in primary rat astrocytes. RSC Advances, 6(41), 34447-34457. doi:10.1039/c5ra25916a

Modaresi, Saman, et al. “Deciphering the Role of Substrate Stiffness in Enhancing the Internalization Efficiency of Plasmid DNA in Stem Cells Using Lipid-Based Nanocarriers.” Nanoscale, vol. 10, no. 19, 2018, pp. 8947–8952., doi:10.1039/c8nr01516c.

Prager-Khoutorsky M, Lichtenstein A, Krishnan R, Rajendran K, Mayo A, Kam Z, Geiger B, Bershadsky AD. Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing. Nat. Cell Biol. 2011; 13:1457–1465.

Vertelov, G. et al. Rigidity of silicone substrates controls cell spreading and stem cell differentiation. Sci. Rep. 6, 33411; doi: 10.1038/srep33411 (2016).

About the Author:

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Bowman Bagley has a B.S. in Neuroscience from Brigham Young University and an MBA from the University of Utah. He currently is a co-owner of Advanced BioMatrix, where he has developed, manufactured and launched over 50 products related to tunable substrate stiffness. He has also helped write multiple white papers related to various collagen properties and characteristics, including viscosity, rheology, purity, osmolality, pH, concentration and electrophoresis. Away from work, he is a husband, father, and avid rock climber. 

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