Product Description

This CytoSoft® 6-well plate product has a defined elastic modulus (see certificate of analysis) in a standard flat-bottom 6-well plate.  The thickness of the silicone gel is uniform with a ~0.5 mm thick layer of silicone in each well that is fully compatible with mammalian cell cultures. The silicone gels are activated and ready to bind to a purified ECM, such as PureCol® type I collagen (#5005) prior to cell addition. The plates are packaged individually and sterilized, and provided with 5 plates per package.

The rigidity of the substrate to which cells adhere can have a profound effect on cell morphology and gene expression. CytoSoft® products provide a tool to culture cells on substrates with various rigidities covering a broad physiological range.  On the bottom of each well, there is a thin layer of specially formulated biocompatible silicone, whose elastic modulus (rigidity) is carefully measured and certified. The surfaces of the gels in CytoSoft® products are functionalized to form covalent bonds with amines on proteins.  This chemical functionalization is stable and the reaction does not require a catalyst, facilitating the coating of the gel surfaces with matrix proteins and plating cells.

The silicone substrates of CytoSoft® products are optically clear and have a low auto-florescence. The layer of silicone in each well is firmly bonded to the bottom of the well. Unlike hydrogels (such as polyacrylamide gels), silicone gels are not susceptible to hydrolysis, do not dry nor swell, are resilient and resistant to tearing or cracking, and their elastic moduli (rigidities) remain nearly unchanged during extended storage periods.

CytoSoft® products accommodate the harvesting of cells using enzymes such as trypsin and collagenase. There is no biochemical breakdown of the substrate during or after enzyme treatment, and there are no residuals of the substrate in the sample retrieved from a CytoSoft® plate.

 

Parameter, Testing, and Method CytoSoft® 6-Well Plates 
Sterilization Method Ozone
Plate Size 6-Well Plates
Quantity per Package 5 Plates
Rigidty (Elastic Modulus) 0.2, 0.5, 2, 8, 16, 32, 64 kPa (exact values provided on the CofA)
Storage Temperature Room Temperature
Shelf Life Minimum of 6 months from date of receipt
Plate Surface Material Functionalized Silicone

Growth Area per Well

9.5 cm2

Typical Working Volume per Well

2.0 to 3.5 mL

Cell Attachment Assay

Pass



Directions for Use

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Coating Procedure

Note: Use these recommendations as guidelines to determine the optimal coating conditions for your culture system.

Remove the CytoSoft® product from the protective sleeve in a sterile hood.

  1. Prepare extracellular matrix material by neutralizing in amine-free buffer pH 7.4 to 7.9 (such as 1X DPBS). We do not recommend using gelatin as your ECM protein.

Note:  Pre-warm the coating solution to approximately room temperature before use.

  1. Dilute as needed, and dispense 3 ml of solution into each well to coat the surface.

Note: Recommended dilution for PureCol® Type I collagen is 1:30 (~100 µg/ml).

Note: The hydrophobic surface requires larger volumes to cover the surface than do conventional plastic dishes

  1. Incubate ECM coated CytoSoft® at room temperature, covered for 0.5-1 hour.
  1. After incubation, aspirate any remaining material and rinse coated surfaces immediately two times with culture medium or PBS. Leave about 2.5 ml of medium per well to keep surface covered.

Note:  Do not allow the CytoSoft® surface to become dry once the surface has been wetted.

5.   Coated surfaces are ready for use.

Standard harvesting procedures used for removing cells from cultureware can be employed for harvesting cells from the CytoSoft® product including use of trypsin, Accutase® and non-enzymatic cell detachment solutions.

Product Q & A

We recommend a 60X objective for the imaging plates.

The elastic modulus is measured by tracking beads on the gel surface under a wide-field fluorescence microscope without any other specialized equipment. The measurements have small and simple to estimate errors and their results are confirmed by conventional tensile tests. A master curve is obtained relating the mixing ratios of the two components of Sylgard 184 with the resulting elastic moduli of the gels.

Using prewarmed media will decrease gas solubility and help prevent bubbles on the surface of the silicone. 

The silicone gel can crack if the surface becomes dry.

No. Cell matrix proteins are attached to the surface of the gel via covalent bonding. It is difficult to form a new ECM layer after cell detachment. There are no longer any reactive groups on the surface of the gel after the initial cell culture.

The change in the refractive index of the silicone distorts things a little, so DIC will be possible but not perfect. Also, DIC is only possible on the glass bottom imaging plates, not the 6-well plastic plates.

1.41

The two main issues are:

1. Ineffecient coating of the matrix proteins due to low pH of the coating solution.

2. Insufficient wash of leftover matrix after the coating procedure. 

3. After coating, plates needed to be allowed to incubate longer for improved ECM attachment.

We have mostly tested CytoSoft with ECM proteins such as collagen or fibronectin, but a few publications have recently come out using Poly-D-lysine and Poly-L-Lysine instead (while following the rest of the protocol as is).

The surface is decorated with anhydride functional groups.

Plasma use is not recommended. Plasma will induce formation of a hard crust on the surface and will change the mechanical properties of the CytoSoft® products.

Do not freeze the CytoSoft® products. 

When frozen, there is a good chance that the silicone surface gets hydrolyzed and absorbs moisture, which would inactivate the binding sites and make the product not-usable.

If you froze the product and it is still frozen, warm the CytoSoft® up at 60C with the bag vented. That will minimize the chance of them absorbing moisture – but there is still a chance that they will no longer be functional.

Using gelatin for coating CytoSoft is often problematic because gelatin often has low molecular weight impurities that block binding sites on the activated surface of the silicone. We recommend using highly purified ECM's instead.

Fibroblast cells secrete their own ECM. The Silicone surface is not efficient for passive absorption of ECM and this is why we use covalent bonding to attach the ECM molecules to its surface for the initial coating.

Fibroblasts use the attached ECM molecules (from the initial coating) to attach and spread on the plate. Fibroblasts are very good at laying down new ECM and after prolonged culture they build a thick layer of ECM on the top of silicone surface.

The problem is that the cell-secreted ECM is not sticking very well to the silicone gel surface and is easy to be pulled by cellular forces.  One way to prevent this from happening is to put some sort of 3D hydrogel on top of the cells after they attach so that the hydrogel absorbs or incorporates some of that secreted ECM. A possible downside would be that now the cells are attached to one stiffness (the silicone) but then surrounded by another (the hydrogel).

 

Product Cell Assay

CytoSoft® plates can also be used to show how fibroblasts are able to discriminate between the underlying stiffness. This is manifested in both adhesion size and stress fibers, as seen below. It appears that the cells on the 8 kPa stiffness have reduced intracellular tension and increased adhesion.

Product References

References for CytoSoft® Products:

1. 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.

2. Wilson, Christina L. In Vitro Models Of Brain For Study Of Molecular Mechanisms In Brain Disorder. The University of Nebraska-Lincoln, 2016.

3. 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

4. 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.

5. Gutierrez E, Tkachenko E, Besser A, Sundd P, Ley K, Danuser G, Ginsberg MH, Groisman A. High Refractive Index Silicone Gels for Simultaneous Total Internal Reflection Fluorescence and Traction Force Microscopy of Adherent Cells. PLoS One. 2011; 6:e23807.

6. Merkel R, Kirchgessner N, Cesa CM, Hoffmann B. Cell force microscopy on elastic layers of finite thickness. Biophys. J. 2007; 93:3314–23.

7. Schellenberg, A. et al. Matrix elasticity, replicative senescence and DNA methylation patterns of mesenchymal stem cells. Biomaterials 35, 6351–8 (2014).

8. Cesa, C. M. et al. Micropatterned silicone elastomer substrates for high resolution analysis of cellular force patterns. Rev. Sci. Instrum. 78, 034301 (2007).

9. Gutierrez, E. & Groisman, A.  Measurements of Elastic Moduli of Silicone Gel Substrate with a Micro fluidic Device. Plos One 6 (2011).

10. Mori, H., Takahashi, A., Horimoto, A., and Hara, M. Migration of glial cells differentiated from neurosphere-forming neural stem/progenitor cells depends on the stiffness of the chemically cross-linked collagen gel substrate. Neuroscience Letters, Vol. 555, October (2013)

11. Banerjee, I., Carrion, K., Serrano, R., Dyo, J., Sasik, R., Lund, S. et al. Cyclic stretch of embryonic cardiomyocytes increases proliferation, growth, and expression while repressing Tgf-β signaling. J Mol Cell Cardiol. 2015; 79: 133–144

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

13. Tkachenko E, Rawson R, La E, et al. Rigid Substrate Induces Esophageal Smooth Muscle Hypertrophy and EoE Fibrotic Gene Expression. The Journal of allergy and clinical immunology. 2016;137(4):1270-1272.e1. doi:10.1016/j.jaci.2015.09.020.

14. Sao, K. et al. Myosin II governs intracellular pressure and traction by distinct tropomyosin-dependent mechanisms. Molecular Biology of the Cell30,1170–1181 (2019).

15. Cooper, J. G. et al. Spinal Cord Injury Results in Chronic Mechanical Stiffening. Journal of Neurotrauma (2019). doi:10.1089/neu.2019.6540

Product Certificate of Analysis

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Safety and Documentation

Safety Data Sheet

Certificate of Origin

Product Disclaimer

This product is for R&D use only and is not intended for human or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.