3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Differences in simplified 2D in vitro cultures versus 3D tissue-like environments have increased the interest in 3D systems to represent the spatial and chemical complexity of living tissues. The fabrication process does not require facility or photolithography techniques. However, the 3D PDMS device includes the necessary vectors for 3D physiological environment applications.

For mesofluidic device preparation, use an appropriate three-dimensional computer-aided design software program to design the mask of the mold for the polydimethylsiloxane or PDMS device and print the mold using stereo lithography equipment with a thermoresistant resin. When the mold is ready, roughly mix three milliliters of PDMS monomer solution per mold with a curing agent at a 10 to one ratio and use a vacuum to degas the mixture in a vacuum desiccator for one hour. At the end of the desiccation, use a piece of adhesive tape to remove any dust from the surface of the mold and carefully fill the mold with the degassed PDMS solution.

Next, cure the PDMS at 80 degrees Celsius for two hours before allowing the mold to cool at room temperature. When the mold is cooled to the touch, use a blade to cut the boundary between the mold and PDMS and carefully peel the PDMS from the mold. Trim the PDMS to fit a 22 by 22 millimeter coverslip and punch a hole at the inlet.

Using low lint tissues and 70%ethanol, wipe the device and coverslip and dab the device with a new piece of adhesive tape to remove any large dust particles. Then autoclave the PDMS device and coverslip at 121 degrees Celsius for eight minutes wet and 15 minutes dry and treat the bottom surface of the PDMS part and coverslip with a corona discharge gun for five minutes in a tissue culture hood before bonding the pieces together. To load the device, first resuspend the cells of interest in an appropriate volume of growth medium supplemented with 1%penicillin and streptomycin and 1%insulin-transferrin-selenium-x.

Next mix 50 microliters of the mammary gland cell suspension with 425 microliters of freshly prepared neutralized collagen solution and fill a 200 microliter pipette with the collagen cell suspension. Inject the suspension through the inlet into the PDMS device until the entire chamber is filled and place the device in the incubator at 37 degrees Celsius with 5%carbon dioxide for one hour to induce gelation before filling the reservoirs on both sides with growth medium and return the device to the incubator until confocal imaging. For 3D imaging and quantification, install a live cell imaging culture chamber onto a confocal microscope and preset the temperature and carbon dioxide to 37 degrees Celsius and 5%respectively.

Add water to the imaging chamber reservoir to humidify the chamber, placing wet wipes into the chamber as necessary and place the PDMS device into the chamber. Add epidermal growth factor to one of the reservoirs as a source of the factor in the gradient formation leaving the other reservoir to serve as a sink for the development of the spatially graded epidermal growth factor distribution. Then set the range of the Z stack covering the volume of organoids of interest and start the imaging.

At the end of the experiment, use an appropriate image analysis program to measure the length and angle of the branches extending from the organoids or the migration of individual cells. Both in silico and in vitro tests revealed the formation of a stable linear epidermal growth factor gradient across the cell culture area that last for approximately two days without replenishment of the source or sink reservoirs suggesting that the epidermal growth factor, a 6.4 kilodalton protein, can form a stable diffusion-based gradient within a collagen gel prepared as demonstrated over a relatively short period of time. Mammary organoids form multiple branches in the presence of spatially uniform 2.5 nanomolar epidermal growth factor with no directional bias over three days if epidermal growth factor is added in the linear gradient to have 0.5 nanomolar per milliliter.

However, the branch formation displays a significant directional bias. Of note, the addition of gap junction inhibitors suppresses the ability of the organoids to respond to the gradient. The pH of collagen solution is critical for the gelation and cell viability.

If the collagen is not neutralized before mixing, the cell viability will be low. This method can be extended to accommodate more realistic tissue modeling and could accommodate vascular network formation from individual cells within the gel.