The overall goal of this experiment is to visualize the micro-structure of, and cell adhesion on, a non-transparent knitted titanium nucleus implant, using fluorescent imaging techniques. This method advances the analysis of tissue engineering with non-transparent scaffolds. One advantage of this technique is that one can trace the cell viability as well as the proliferation on the scaffold with defined culture ped-air.
Visual demonstration of this method is critical, as the fluorescent staining and, later, visualization is challenging. Individual scaffold properties such as the pore size may affect the timing, and/or the fluorescence of the scaffold may affect the choice of fluorophores that you are using. Place up to five scaffolds six or seven millimeters thick into a 50 milliliter tube, and wash them with water three times for 20 minutes per wash.
Do this at room temperature with gentle agitation. Next, wash the scaffolds in 30 milliliters of 1%Triton X-100 solution under the same conditions. Then, perform two more water washes.
Now, sequentially sonicate the scaffolds in reagent grade 99%acetone, 99%isopropanol, and 99%ethanol. Perform each sonication step twice for five minutes per sonication. Then, sonicate the scaffold three more times in water for a total of 15 minutes.
Finish up by placing the scaffolds on a lint-free tissue and air drying them overnight at room temperature. The next day, autoclave the scaffolds for 15 minutes at 121 degrees Celsius and 15 PSI. To confirm that the cleaning worked, use indirect fluorescence.
Load one well of a 12 well plate with 2000 microliters of the freshly made sulfur-Rhodamine B staining solution, and using forceps, transfer a scaffold into that well. Then, capture negative images of the scaffold using a fluorescence microscope equipped with an RFP LED cube filter set and look for impurities at high magnification. Using the bio-safety cabinet one, transfer the clean scaffolds to the wells of a 24 well plate.
Add 500 microliters of 37 degree Celsius culture medium to each well, and let the scaffold soak for 15 minutes. Meanwhile, prepare 500, 000 Ad-GFP infected SEP1 cells per 1 milliliter of medium. After 15 minutes, completely aspirate the medium from the scaffolds, and seat them with 100 microliters of the cell suspension.
Then, incubate them for 30 minutes at 37 degrees Celsius. After the brief incubation, add 500 more microliters of culture medium, and incubate the scaffolds for 24 hours. The next day, change the culture medium to remove non-adherent cells.
Then, evaluate the cells'adherence and spreading on the scaffolds, using a fluorescence microscope equipped with a GFP LED cube filter set. For a live-dead staining, first plate the SEP1 cells on the scaffold as before. After 24 to 48 hours, completely aspirate the culture medium and wash the scaffolds with DPBS for five minutes at room temperature.
Now, stain the cells using culture medium containing Calcein AM, Hoechst 33342, and Ethidium homodimer. These three fluorophores are all light-sensitive, so it is vital to keep the cells in the dark moving forward. Incubate the cells for 30 minutes with the fluorophores to get an even distribution throughout the scaffold.
Specific scaffold characteristics will affect this incubation time. After the incubation, wash the cells three times with DPBS using one milliliter per well. Execute each wash for five minutes at room temperature.
Immediately after the washes, document the scaffolds using fluorescence microscopy. To measure cell viability over time, use resazurin conversion measurements. First, completely aspirate the culture medium from the SEP1 cells seated in a 24 well plate and wash the cells with 500 microliters of DPBS per well.
Then, cover the cells with the required volume of sterile resazurin working solution and include one well with only resazurin. Then, incubate the cells at 37 degrees Celsius for about 30 minutes. After the incubation, transfer 100 microliters of the conditioned supernatant from each well into a 96 micro-well plate to measure its fluorescence as described in the text protocol.
For further cultivation, remove the resazurin from the well, and wash three times with one milliliter of DPBS. Perform each wash for five minutes at room temperature. After the third wash, add 500 microliters of culture medium to each well of cells, and continue their incubation for further time-course measurements.
Using indirect fluorescent staining, pre-cleaning impurities were documented to optimize the cleaning protocol. A significant reduction in substance load on the scaffold shows the efficiency of the described cleaning protocol. The successive implants used for arthroplasty treatment is determined by events that take place at the cell material interface.
After 24 hours of adherence with the scaffolds, the SEP1 cells had attached. Fluorophores were then applied to examine cell death and proliferation over a period of time on the scaffolds. Live-dead staining showed blue nuclear staining with red fluorescence in dead cells and green fluorescence in viable cells.
In addition, cell viability on the scaffold was quantified using the resazurin conversion assay. Over two weeks, data was collected on cells cultured with, or without, scaffolds. While attempting this procedure, it is important to remember to adjust the fluorophores you are using to the scaffold and microscope that you have available.
Following this procedure, other methods, like immunofluorescent staining, can be applied in order to visualize the presence of proteins, or the activation of signaling cascades. The implication of this technique extend towards the therapy of arthroplasty because the cell face interaction with the surrounding tissue in vivo will define its function and durability.
