Cancer invasion and progression involves a modal cell phenotype, which is under complex regulation by growth factors, cytokines and extracellular matrix components within the tumor microenvironment. Hyaluronic acid or HA is one stromal extracellular matrix component that is known to facilitate tumor progression by enhancing invasion growth and angiogenesis interaction of HA with its cell surface receptor. CD 44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread spread to study cancer cell adhesion, migration and growth on exogenous ha.
Standard photolithography is used to fabricate a micro pattern elastomeric stamp, which is then used to create a patterned array of amino seline molecules on glass substrates. Next carbo dite chemistry is performed to covalently attach HA to micro pattern amino seline. Thus creating micro patterns of HA cultured cancer cells can then be plated on the HA micro patterns and interactions of cancer cells with exogenous hyaluronic acid can be visualized using light microscopy, high resolution imaging techniques such as scanning electron microscopy or immunofluorescent microscopy.
The method for creating micro pattern HA surfaces presented in this video article can help answer key questions in the cancer research field, such as further elucidating the role of exogenous hyaluronic acid in cancer progression. To fabricate a micropattern Polymethyl Zane or PDMS stamp, which will be used for imprinting glass slides with ha. Begin by picking up a new silicon wafer with tweezers.
Then over a lab sink. Use a wash bottle to rinse the wafer with ethanol dry with a stream of air. Turn off the room lights.
Then using forceps, transfer the wafer to a spin coder using a transfer pipette cover at least 80%of the wafer surface with SU 2025 negative photo resist spin coat for 10 seconds at 600 RPM. Then for 30 seconds at 3000 RPM, transfer the photo resist coated wafer to a hot plate and soft bake at 95 degrees Celsius for three minutes. Then remove it from the hot plate and allow it to cool for one minute.
Place a prefabricated mask with the desired pattern on top of the photoresist covered wafer and uv. Irradiate it for 20 seconds. Next, transfer the wafer to a hot plate and hard bake at 110 degrees Celsius for six minutes.
Turn on the lights and remove the wafer from the hot plate. Distinct patterns should be visible at this point with a transfer pipette. Carefully rinse the wafer with SU eight photoresist developer after three rinses with developer solution, rinse with ethanol.
If any white residue is present, repeat the rinse with photoresist developer solution or simply submerge the wafer in developer solution for two minutes and rinse again with ethanol, wash with water and dry with a stream of air in a large whey boat. Mixed PDMS elastomer solution and curing agent in a 10 to one weight ratio for the wafer shown here, 22 grams is sufficient. Pour into a centrifuge tube and spin at 900 RPM for five minutes.
To remove any air bubbles, place the patterned wafer in an appropriately sized Petri dish. Pour enough PDMS solution on it to completely cover the wafer with a thickness of about a quarter inch. If there are any bubbles present on the surface of the wafer, place it in a vacuum desiccate until they disappear.
Cure overnight at room temperature to form a complimentary elastomeric stamp. If the PDMS is not completely cured after 12 hours, place the wafer in an oven at 60 degrees Celsius for 30 minutes. Carefully remove the PDMS from the wafer.
Then using a razor, cut the stamp to the desired size. Place the stamp in a glass speaker with ethanol, preferably within a plastic holder and sonicate for 15 minutes. Wafers can be used multiple times to create new elastomeric stamps to covalently immobilize ha onto glass surfaces in defined patches.
Begin by cleaning all of the slides to be used in a plasma cleaner according to the instructions in the accompanying text. After the glass slides are cleaned, use tweezers to remove them from the plasma cleaner. Place the slides in a plastic petri dish and set them aside for later use.
Next, prepare the amino cline solution to be stamped on the glass slide to bind HA in a 15 milliliter polystyrene centrifuge tube. Prepare a fresh 3%volume per volume solution of three amino propyl trimeth oxy cline or A-P-T-M-S in 95%Volume per volume. Ethanol for a total volume of five milliliters, allow the solution to react for five minutes at room temperature.
During this period, A-P-T-M-S is converted by hydrolysis to a ciol. Next, touching only the sides place the PDMS stamp pattern up on a spin coder chuck. Use a transfer pipette to cover the pattern surface with A-P-T-M-S.
Then spin coat the surface of the stamp with A-P-T-M-S at 3, 500 RPM for 30 seconds. After removing the PDMS stamp from the spin COER inverted so that the pattern side is facing downward, place one end of the PDMS stamp in the center of the glass slide and gently press so the entire stamp is in contact with the glass Slide for one minute. Next, gently remove the PDMS stamp and allow the A-P-T-M-S patterned glass slide to sit at room temperature.
After 30 minutes, rinse the pattern surface with ethanol and dry with a stream of air sonicate. The PDMS stamp for 15 minutes to remove any excess A-P-T-M-S. Then place the slides on a hot plate for one hour at 115 degrees Celsius to heat the surfaces and remove excess water.
Rinse with ethanol and dry with air using tweezers. Transfer the pattern slide to a glass petri dish. Use a transfer pipette to completely cover the glass slide with a freshly prepared polyethylene glycol or peg cline solution, which will serve as a non-adhesive region surrounding the patterns.
Following a 45 minute incubation at 75 degrees Celsius, pour off the excess peg saline solution. Then while holding the glass slide with tweezers, rinse the surface of patterned glass slides with toluene and then again with water dry with a stream of air. Place the slides in a humidified slide box.
Then place the box in a biological safety cabinet and turn on the UV lamp for one hour to perform germicidal irradiation. Proceed in the biological safety cabinet from this point forward. To maintain sterility outside the hood, prepare one milliliter aqueous sterile HA solution consisting of 10 millimolar.
One ethyl three three dimethyl amino propyl carbimide or EDC five millimolar and hydroxy CIN aide and 50 micrograms per milliliter. Sterile fluorescein labeled HA EDC is a zero length cross-linking agent that reacts with HA carboxyl groups to form amine reactive intermediates. As this intermediate is susceptible to hydrolysis, NHS is added to increase carbo dite reaction efficiency.
Next, apply 200 microliter aliquots of sterile HA solution to the patterned glass until the patterned area is covered. And close the slide box to protect the slides from light. The pattern substrates should be left under the hood undisturbed for 16 to 24 hours as the HA solution interacts with micro patterned A-P-T-M-S-A stable MI bond forms between the HA intermediate and the primary amine of A-P-T-M-S.
The next day use APA tear pipette to aspirate off the HA solution. Wash the slides with PBS, then cover the surface with 1%BSA in PBS for one hour. The surface is now ready for cell culture.
Place ha micropattern surfaces in a sterile petri dish to culture on HA micropattern surfaces. Seed single cell suspensions of cancer cells with densities between 0.4 and one times 10 to the sixth cancer cells per 18.75 centimeter squared of glass surface in one milliliter of cell media to cover the surface of the glass slide carefully place the cells in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide. Taking care that the medium does not spill over cell adhesion should occur within 24 hours and can be observed using an inverted light microscope.
Cell culture surfaces can be maintained for up to five days. In standard incubator conditions change the media every other day. Cell morphology and growth can be observed using an inverted light microscope.
Additional cellular assays can then be applied to analyze responses to the HA surface in the example shown here. HA pattern surface visualized using a fluorescent microscope and image. J fluorescence intensity analysis demonstrate the pattern demobilization of ha.
The HA micro pattern surfaces enable the study of cancer cell interactions with exogenous HA.Cancer cell adhesion on HA micropattern surfaces is shown here. Both colon and breast carcinoma cells preferentially adhered onto HA patches with 24 hours of culture. High resolution imaging using scanning electron microscopy can be used to analyze interactions of cultured cells with HA immobilized surfaces.
In this scanning electron micrograph colon cancer cell interaction with HA can be seen higher magnification is shown on the right. Breast cancer cells cultured on HA surfaces are shown here again, higher magnification is shown on the right. This analysis clearly demonstrates the presence of adhesive protrusions that enhance the spreading of cancer cells on HA micro patterns.
After watching this video, you should have a good understanding of how to prepare HA micro pattern services and apply them to study cancer cell response.
