The overall goal of the following experiment is to measure the effects of different microenvironments on cellular functions of adherent cell types by exposing them in parallel to a diverse collection of Combinator microenvironments. This is achieved by first fabricating printing substrates to support absorption of mixtures of microenvironmental proteins. Next master plates of microenvironmental proteins are prepared, which are used to print ME arrays using either a quill or capillary pin microarray printing robot.
Next master plates of microenvironmental proteins are prepared. Then adherent cells are cultured on the ME array with the option of limiting cell number. The results obtained show the impact of the different microenvironments on cellular functions such as differentiation or proliferation based on immunofluorescence analysis.
We first had the idea for this method when we wanted to quantify the effects of tissue microenvironments on human memory stem cell fate decisions, but we realized that working in vivo is impossible. Now that it's developed, it has many applications in cell biology, research and drug discovery. PDMS is used as a substrate that mimics stiffer tissues as it is inexpensive, easy to prepare and easy to print.
However, PDMS hydrophobicity can make it incompatible with some cell lines when working with PDMS done. Non latex gloves as latex can inhibit PDMS polymerization. To begin in a disposable plastic cup, combine cigar 184 silicone elastomer base with the curing agent at a 10 to one ratio and mix it vigorously with a wooden or plastic tongue depressor.
Next, degas the mixture in a room temperature vacuum bell for 30 minutes. Now center a standard microscope slide on the actuated vacuum chuck of a spin Coer then drizzle 0.5 milliliters of the mixed elastomer polymer onto the center of the slide surface and spin coat at 6, 000 RPM for 60 seconds. Cure the PDMS coated slides at 70 degrees Celsius for four hours to overnight.
The slides can be stored air tight at room temperature for several months. PA is a hydrogel that can mimic softer tissues in the range on PA cells will only attach to spots where there are proteins that support cell adhesion. Begin by etching slides with sodium hydroxide.
Place the slides on a heat block at 80 degrees Celsius. Drop one milliliter of 0.1 normal sodium hydroxide onto each slide. Be certain to cover the entire slide surface or the PA will fail.
Allow the sodium hydroxide to evaporate leaving a white film. Repeat this process if the surface is not completely covered. The slide can be stored at room temperature for several days if needed.
Next, coat the slides with a PES in a fume hood. Place slides in a 15 milliliter dish. Add 300 microliters of a PES on each sodium hydroxide etched slide.
Let the A PES react with the sodium hydroxide Slides for precisely five minutes. Not longer. Wash out the A PES thoroughly with deionized water two to three times on both sides of the slides.
Otherwise, it will oxidize to form a brown deposit on the slides in the next step. Now aspirate all the solutions from the slide surfaces to each dish. Add 25 milliliters of 0.5%glutaraldehyde in PBS.
Place the slides in the dark and wait 30 minutes. After 30 minutes, aspirate all the glutaraldehyde solution from the slide surface. Then dry the slides carefully using absorbent lint-free paper.
The slides can now be stored at room temperature overnight if needed. After preparing and degassing the PA mixtures, place them on ice to slow down their polymerization. Just before preparing the slides to each mixture on ice, add ammonium per sulfate and temid and tritrate them using a pipette for five to 10 seconds.
Now pipette the PA mixture onto the slide surfaces. The amount of PA varies according to the required gel hardness. Then place glass cover slips on top of the PA without applying any pressure to avoid any bubble formation.
Let the PA gel polymerize for two hours and then remove the cover slips under deionized water. Wash the PA gel slides with water in large coplan jars overnight. To remove the unreactive acrylamides the next day, dry the slides until the PA gel completely hardens.
The slides can then be stored at four degrees Celsius for one month in a sealed slide box. To begin, a plastic chamber is fitted to surround the printed array from a two chamber slide. Remove the chambers and cut them in half with a razor blade.
Use a one milliliter pipette tip to apply a thin bead of aquarium silicone to the edge of a chamber and press it to the surface of an Emmy array. Avoid touching the silicone to any part of the array features. Ensuring good attachment of the cultural whales to the Emmy array is crucial because peria at attach whales allow media to leak from the chambers, which can result in widespread cell death.
For PDMS coated slides, block the slides by incubating them in aqueous 1%onic F 108 for 15 minutes under vacuum for either gel type, rinse the arrays with cell culture media with antibiotics three times for five minutes per wash. This removes unreactive monomers, which can be toxic to the cells in the case of PA gels. After the third wash, allow an additional 30 minutes incubation in the media to rehydrate the gel.
Now place four to five arrays into a 15 centimeter sterile Petri dish. To the dish, add half of the final media volume with the cells of interest at the required concentration for their application. ME arrays should be placed in an incubator appropriate for maintenance of the cell type being used.
Check for uniform attachment of the cells to the printed features under a microscope every 15 to 20 minutes while observing. Gently shake the ME arrays to distinguish attached and floating cells. Attachment time will vary, but is typically complete within two hours on a PA coded ME array.
After attachment saturation or a time limit has been reached, aspirate the unbound cells and fill the dish to the final volume on PDMS coated ME arrays. Removing all of the media will kill the cells. So for all media changes on these slides successively replace half of the media volume and then fill the dish with the final volume.
Culturing can proceed for many days with normal media changes. Common fixatives such as paraform, aldehyde, and methanol acetone are compatible with the Emmy array systems. Do not forget that even when staining PDMS coated, ME arrays must remain wet.
So continue with the half volume replacement technique to prepare slides of the stained ME arrays. Use a razor blade to remove the chambers and mount cover slips using flora. Mount G protein was deposited on a printed PDMS coded ME array using square tipped silicon pins on a quill pin.
Microarray printing robot deposition of various proteins was verified by immunofluorescence using antibodies. Dilutions of the protein solutions in the master plate were reflective of the amount that was deposited on the printing substrata surface cells attached to the printed features in an obvious patterned manner. Likewise, the same obvious pattern of cell attachment occurs when an Emmy array is printed on a 40, 000 pass scale.
PA gel two microenvironment proteins elicited concentration dependent keratin expression profiles in a human multipotent mammary epithelial progenitor cell line. The log ratio of keratin eight signals to keratin 14 signals illustrates the variation and reproducibility of the signal. After 24 hours of culture, unbound cells are washed away In an analysis of 12 me arrays.
Standard statistical analysis were used to determine whether the different dilutions of type one collagen and recombinant human p cadherin cost changes in keratin expression. Initially, there was no variation from the mean among cells on the features just after attachment. However, after 24 hours high type one collagen concentrations elicited significantly higher keratin eight expression.
Whereas high PCA herein concentrations elicited a strong keratin 14 signal. This finding is consistent with previous reports on bi potent memory progenitor cells. Due to the dynamic and reciprocal nature of these cell microenvironment interactions, these cells themselves can alter the composition of the printed microenvironments.
Therefore, to understand the acute impacts of the microenvironments you printed, the time of exposure should not exceed 48 hours Following this procedure. Any theory process that can be measured, fluorescently or chally can be asset as a function of microenvironment in order to answer additional questions like, how do different microenvironments alter drug responses in cancer lines? It's major by changes in the ptosis and proliferation.
Or how do microenvironments alter progenitor differentiation.
