Paper Method

Method

• CT26 cell lines (from a mouse) were used to derive a colon carcinoma cell spheroid
• Well plate sides were covered in gel
• Cell suspensions seeded on gels
• Cells self assemble into spheroids (in less than 24hrs)
• Cells were cultured
• Confocal microscopy was used to check the spheroid shape
• Constant stress applied to tissue over long timescales using neutral polymer (which is not metabolized by mammalian cells)
• Also it was confirmed that it is neither a growth nor death factor by plating cells for 3 days with dextran and measuring cell concentration and viability

• First, indirect stress measurements were performed - this is done by positioning a growing spheroid inside a dialysis bag which is then placed in an external medium with added dextran. Oxmotic stress induces a force on the dialysis membrane, which is transmitted in the quasistatic equilibrium to the spheroid and calibrated 
• Stress exerted on this system can be seen as network stress that tends to reduce the volume occupied by the spheroid, and acts directly on the cells and not the interstitial fluid.
• Volume of spheroid is measured
• In absence of stress, spheroid reaches steady state of typical diameter 900
• When dextran is added to the medium, growth rate decreases as does the steady state volume
• Interestingly, after a release of stress, the growth resumes until it reaches the same steady state volume of ~900

• Also, direct experiments were conducted where the osmotic stress (the minimum pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane) is applied to the spheroid in the absence of the dialysis membrane (a type of semi-permeable membrane tubing used in separation techniques, that facilitates the removal or exchange of small molecules from macromolecules in solution based on differential diffusion).
• It was also verified that the dextran cannot diffuse inside the spheroid
• It was observed that the dependence of growth rate and the steady state size on stress is very similar to that observes in the indirect experiment, validating the approach.

• Finally, we investigate the spatial dependence of cell division and apoptosis using cyrosections and immunofluorescence. 
• Spheroids of comparable diameters are embedded in a freezing medium, frozen, cut into slices at the level of their equatorial line
• Recently divided cells were labelled, as were apoptotic cells