The overall goal of this glass surface functionalization process is to allow for the gentle capture and release of cells of interest. This method can help answer key questions in the field of tumor angiogenesis, enabling screening of cell biomarkers that indicate drug resistance. The main advantage of this technique is that it is completely customizable, for virtually any cell type of interest.
As long as there is an antibody specific to the cell type, the surface can be adapted for its capture. Though this method can provide insight into cancer angiogenesis, it may also be applied to other diseases as purified samples are necessary for the development of more personalized treatment regimens. We first had the idea for this method when we were trying to develop a reversible release mechanism for capturing cells, using the interaction between desthiobiotin and the adivin family.
After cleaning a glass surface in an oxygen plasma machine for five minutes according to the text protocol, prepare 2.5 milliliters of 2%reconstituted 3-Aminopropyl triethoxysilane, or APTES solution, by combining 50 microliters of APTES and 2.45 milliliters of ethanol in a conical tube. Pipette the APTES solution onto the glass surfaces, then cover the surfaces and place them on a platform shaker at room temperature for 50 minutes to evenly distribute the APTES layer. In the meantime, preheat the oven to 55 degrees Celsius for the eight-and 24-well plates, or 90 degrees Celsius for the glass dishes.
After removing the plates from the shaker, use ethanol to rinse the glass surfaces according to the text protocol. With 100%nitrogen gas, dry the surfaces. Then place the eight-and 24-well plates in the oven for two hours, or the glass dishes in the oven for one hour.
Next, prepare 2.5 milliliters of d-Desthiobiotin, or DSB solution, by mixing 1.5 milligrams per milliliter of DSB in 37.5 microliters of DMSO, and adding five milligrams per milliliter of EDC to 2, 462.5 microliters of 0.1 Molar MES buffer, pH 6. Then combine the two solutions. After 15 minutes, add one microliter of BME to quench the reaction between DSB and EDC.
Remove the hot APTES functionalized glass surfaces from the oven, and allow them to cool for five to 10 minutes. Add MES buffer to the glass surfaces to rinse them by holding the pipette at a 70 degree angle so that the tip is not directly pointed at the surface. Then discharge and draw the buffer from a fixed point, such as the corner of the well.
Then, use the MES buffer to rinse two more times. Apply DSB solution to the glass surfaces. Transfer them to a damp paper towel inside a Petri dish, cover the dish, and incubate in the fridge for 18 to 24 hours.
After the incubation at four degrees Celsius, use PBS to rinse each glass surface three times, as demonstrated earlier in this video. Then, dilute Streptavidin, or SAV stock solution, to 0.4 milligrams per milliliter, and evenly apply it to the glass surfaces so that a thin layer forms. After incubating the glass for 18 to 24 hours, use 150 microliters of PBS to rinse each surface three times.
Then wet a paper towel with deionized water, and place it flat in a 14 centimeter Petri dish surrounding the plates to retain moisture in the wells. Cover the Petri dish with the glass surfaces, and place it in a four degrees Celsius biosafety Level 1 fridge until needed. To carry out cell capture, aspirate the medium from T175 flasks of cells.
Then use PBS to rinse the cells and aspirate the buffer. Add 10 milliliters of non-enzymatic lifting agent, such as Cell Dissociation Solution, to the flask. Then incubate at 37 degrees Celsius for six minutes.
After six minutes, add 10 milliliters of cold HBSS to dilute the lifting agent. Then pipette 20 microliters of the cells and use a hemocytometer to count them. Centrifuge the cell suspension at 500 x g, in four degrees Celsius, for five minutes.
And use HBSS to resuspend the cells to one times 10 to the sixth cells per milliliter. Pipette up and down to resuspend the cells in solution, and reduce cellular clumping that may reduce antibody binding. Then, split the cellular suspension into separate control and experimental solutions.
Add the biotinylated antibodies to the respective cells solutions and incubate on an end-over-end mixer at four degrees Celsius for 30 minutes. Use 150 microliters of HBSS to wash the functionalized glass in eight-well plates three times, as demonstrated earlier in the video. Add the cell solution to the wells, and incubate on ice on the shaker for 45 minutes.
In the meantime, prepare a 20 millimolar solution of biotin in sterile HBSS. Then, after the incubation, gently use 150 microliters of HBSS to rinse the cell solution from the glass. After repeating the rinse two more times, add 150 microliters of the biotin solution to each respective release well, and incubate for 20 minutes to allow the reaction to proceed.
Recollect nonspecifically bound cells by using HBSS to wash the cells. Then, proceed to fluorescence imaging and analysis according to the text protocol. Include images of live cells in a flask as a control.
In this experiment, MCF7GFP cells were exposed to the functionalized surface. 60%of the cells were captured using the HLA-ABC antibody. Upon exposure to 20 millimolar biotin, 80%of the captured cells were released.
As shown here when the MCF7GFP cells were mixed with RAW 264.7 cells, 50%of the RAW macrophages were captured, and the addition of 20 micromolar biotin subsequently released 80%of the captured RAW cells. This figure illustrates that the positive control RAW macrophages exhibit no fluorescence activity. However, the negative control MCF7GFP cells are positive for GFP fluorescence.
Because excess antibody can decrease cell capture, the antibody concentration was optimized by titrating from zero to 10, 000 nanograms per milliliter of HLA antibody. The results showed that the ideal antibody concentration was between 100 and 1, 000 nanograms per milliliter. In addition, cell optimization experiments determined that the ideal concentration of cells that can be captured is between one times 10 to the fifth, and one times 10 to the sixth cells, since quantities below that number are lower than the background.
Once mastered, this technique can be done in three days, with less than six hours of experimental time, not including the overnight incubations. While attempting this procedure, it's important to remember to collect the cell washes during the experiments, as they can give vital information about the effectiveness of the surface. Following this procedure, other methods like flow cytometry can be performed in order to answer additional questions, like what receptor expression levels the cells of interest may have.
After its development, surface functionalization has paved the way for researchers in the field of biomaterials to explore biosafe integration of materials, such as, implants in patients for a variety of diseases and injuries. After watching this video, you should have a good understanding of how to surface functionalize glass surfaces to capture and release cells of interest, allowing for downstream analysis. Don't forget that working with live cells in sodium azide can be extremely hazardous, and precautions such as proper training, PPE, and disposal, should always be taken while performing this procedure.
