The overall goal of this procedure is to visually inspect and characterize cancer cell migration through a compartmentalizing microfluidic device. This is accomplished by first dissociating, pre-existing neurospheres of brain tumor stem cells grown in serum free medium. The second step is to fabricate a Bilayered SU eight master and using soft lithography mold A-P-D-M-S stamp that has a compartmentalizing design.
Next, assemble the microfluidic device and load it with brain tumor stem cells. Finally, long-term time-lapse imaging of brain tumor stem cell migration is accomplished using an all-in-one microscopic system. Ultimately, results show that brain tumor stem cells exhibit a revolving sequence of morphological stages during their migration through ultra confined space.
The major advantage of this technique over existing masses like Transwell Boyton Chamber, is that micro device allow for visual inspection of migration process controlled placement of cells, and the precise delivery of factors. Therefore, micro foric technology has features of reliable, efficient, and cost effective single cell selection and navigation. The implications of this technique extend toward therapy of cancer metastasis and recurrence because single cell analysis of highly migratory cells may identify potential future chemotherapeutic targets.
Though this method can provide insight into migration behavior of cancer system, it can also be applied to other systems such as embryonic development, immune responses, wound healing, and organ regeneration. Beginning with the cell suspension of BTSC derived neuros spears. Pool the cells in a 15 milliliter conical tube and centrifuge them at 900 RPM for five minutes, but not any faster.
Aspirate the super natin. Then allow the neurospheres to loosen by incubating them in 0.5 milliliters of prewarm Accutane for five to 10 minutes at 37 degrees Celsius. After the incubation, mechanically disrupt the cells with 10 to 20 gentle strokes of a P 100 pipette.
Then add 1.5 milliliters of stem cell medium to neutralize the Accutane centrifuge, the cell suspension at 1300 RPM for five minutes and resuspend the cells in one milliliter of stem cell medium. Check the cell density with a hemo cytometer and adjust the density to 20, 000 cells per microliter. To begin the fabrication of the SU eight master, prepare two photo masks using Adobe Illustrator and laser printing transparency film with Image Setter incorporated.
The first layer photo mask consists of two fiducial markers and an array of micro channels, and the second layer of photo mask has the seating and receiving chambers. Clean both mask layers with isopropanol and allow them to air dry. Now spin coat a handle wafer with three micron thick SU eight photo resist, followed by soft baking.
Then with the first layer photo mask in close contact uv, expose the photo, resist and allow it to cure. Then post bake and develop the cured wafer. Cover the fiducial marker areas of the handle wafer with scotch tape.
Then spin coat the wafer with 250 micron thick photo resist. Afterwards, peel off the tape to reveal the markers for alignment purpose. Place the second layer of photo resist and align the fiducial markers.
Then UV expose and cure the second layer photo mask followed by post baking and developing. Now ize the SU eight master via vapor deposition to reduce surface tackiness. Place the wafers into a desiccate with several drops of Tri Chloro cline solution under vacuum for at least one hour.
The master will then be ready for use to mold PDMS with the master. Begin by thoroughly mixing the pre polymer base with cure at a 10 to one ratio. Then place the mix in a desiccate for vacuum degassing until no air bubble is seen.
Pour the mixture on the mask and degas again. If new air bubbles are introduced, then cure the mold on a level hot plate for two hours at 90 degrees Celsius. After it cools to room temperature, gently release the PDMS stamp.
Thus culturing channels are engraved in PDMS and ready for assembly of the microfluidic device. The master is reusable for molding until it is cracked or worn. When the master becomes sticky, the antis stick coating can be reapplied.
Begin by making inlets and outlets in the microfluidic device through the PDMS stamp. Using a biopsy hole puncture, clean the PDMS stamp by soaking it in 70%ethanol for 30 minutes, and then rinsing it with deionized water for 10 minutes. Sterilize and complete the cross-link reactions of the PDMS stamp through autoclaving.
Now lay the PDMS stamp on a glass cover slip coated with poly L lysine. The device is then coated with laminate by filling the chambers with laminate solution through reservoir inlets and incubated overnight at 37 degrees Celsius. The next day, aspirate the laminate from the device.
Then wash the device by flooding it twice with stem cell medium. Now load 11 microliters of dissociated cells into one of the seeding reservoirs If needed, use vacuum aspiration to force the cells into the chamber. Then load an additional seven microliters of cells into the adjoining seating reservoir.
After five minutes, the seating and receiving reservoirs will be flooded with media. Now place a 0.5 to one millimeter thick sterile PDMS sheet on the device. The natural surface adhesion of PDMS with itself will seal the device once sealed, it is ready for transport, incubation and microscopic imaging.
Pre equilibrate the biot, IM by running it for 45 minutes. To stabilize its temperature, moisture, and air supply, add several dishes of water to the sample chamber to obtain the proper humidity. Load the prepared device in the sample chamber of the microscope.
Center the device using micro tweezers. Now set the focus position points and time points using the Biot software and start the time lapse recordings if necessary. After five days in culture, replace the culture media.
The BTCs seeded in the device were continuously recorded by phase image. For five days in the seating chamber, spindle shaped cells stayed in the pre-migration stage. As they approached the microchannel entrance, a few cells expanded and generated adhesive protrusions.
Only one cell was able to occupy the entrance and explore the migration direction. Once the cell determined the migration direction, it proceeded to cruise through the entire microchannel at steady high speed. At the end of Microchannel, the cell proceeded to explore the open space of the receiving chamber before finally entering it.
By observing the cells with two second imaging intervals migrating power appeared to be generated mostly by blabbing activity similar to that of a OID cells. While attempting the procedure, it is important to remember that design of the device is so flexible that microchannel dimensions may be manipulated to achieve a desired degree of cell migration. After watching this video, you should have a good understanding of how to create a unique compartmentalizing microfluidic device suitable for culturing your cells of interest, and then performing long-term time lapse imaging to qualitatively and quantitatively define migratory characteristics of these cells Following this procedure.
Other methods like next generation sequencing or DNA microarray can be applied in order to answer additional questions such as how genetically different the highly migratory cancer cells are.
