The overall goal of this procedure is to monitor the cellular response to hypoxia in PEG encapsulated cell aggregates. In the first step, dispersed cells are infected with the marker virus and then cultured on a shaker to allow formation of aggregates. Next linear peg is functionalized and the PEG DM rimer is purified.
Then the cell aggregates are encapsulated in a PEG DM hydrogel. Finally, the hydrogels are cultured in various oxygen conditions while being fluorescently monitored for the hypoxia marker signal. Ultimately, the location and extent of hypoxia inducible factor activity within viable encapsulated aggregates of cells can be visualized through simple fluorescent microscopy.
This method can help answer key questions in the cell encapsulation and cell-based therapeutic delivery fields such as Where does hypoxia occur within 3D cultures, and how does it impact cell survival and function? The implications of this technique extend towards regenerative medicine approaches and therapy of hormone deficiencies such as stipend diabetes because of the major impact of hypoxia in aggregated and encapsulated tissues. Hypoxia inducible factor one activity has been shown to correlate with great reduced insulin secretion from beta cells, motivating attempts to dissociate primary eyelets and reform them into pseudo eyelet.
Aggregates of reduce diameter. Our marker can indicate hypoxia inducible factor activity with an aggregates at a cellular level within any loosened encapsulating material. PEG was chosen as a capsule material due to its favorable transfer properties, cyto compatibility, and the ease with which it can be functionally modified.
After weighing two grams of PEG in a 40 milliliter glass vial at approximately 308 microliters of meth, acrylic, anhydride and loosely close the vial with a hard plastic cap. Microwave the vial on high for two minutes in a standard domestic microwave. Then wearing heat resistant gloves thoroughly vortex the vial and microwave the solution on high for an additional five minutes.
Briefly allow the vial to cool enough to be handled and then uncap it while swirling the vial. Slowly mix in methylene chloride, vortexing the sample as needed until the solution appears clear and homogeneous. Next, add the solution dropwise to 200 milliliters of stirred cold dathyl ether to precipitate the PEG dm.
Then collect the PEG DM by vacuum filtration and allow it to dry. Once the PEG DM has dried, dissolve it in about 100 to 150 milliliters of deionized water. Then dialyze the solution against deionized water for five days in dialysis tubing with a molecular weight cutoff of 1000 Daltons after dialysis.
Eloquent the solution into appropriate containers and freeze them at minus 80 degrees Celsius overnight. Then lyophilize the solution for four days to generate a purified white PEG DM powder to release the cells from the treated culture flask surface. First, aspirate the medium, rinse the cells with 37 degrees CELs calcium, magnesium free HBSS, and aspirate the HBSS.
Now add two milliliters of 37 degrees Celsius trips and EDTA solution, and allow the cells to incubate at 37 degrees Celsius for three minutes. Then firmly jar the side of the flask to knock the cells free at eight milliliters of 37 degrees Celsius, medium to the flask and vigorously pipette the cell suspension up and down. Taking care not to introduce bubbles.
After transferring the cell solution into a sterile 15 milliliter centrifuge tube, count the cells by hemo cytometer to determine the total number of cells to be infected. Add the necessary volume of cell suspension to achieve 400, 000 cells per well into the wells of a six well suspension culture plate aliquot 50 microliters of HBSS per well of cells to be infected into a 1.8 milliliter micro centrifuge tube. Next, carefully pipette the proper amount of virus suspension into the prepared HBSS.
Now add the appropriate volume of diluted virus to each well to achieve the desired MOI, and then add medium to each well to bring the total volume up to 1.7 milliliters. Finally, place the plate on an orbital shaker Inside the incubator, set the shaker on a low setting about 100 RPM so that the medium gently washes around the well and allow the cells to aggregate for 24 to 36 hours. Begin by cutting the tapered tip off of a one milliliter plastic syringe and placing it opened end up in a rack to create a vessel in which the hydrogel will be formed.
Next, pipette 20 microliters of photo active PEG DM solution into the open end of the syringe onto the plunger, making sure the volume covers the entire plunger surface. Adjust the plunger in small increments to achieve a flat fluid surface. Now place the pipette back into the rack and position the rack underneath a UV lamp for approximately eight minutes to allow for hydrogel cross-linking.
During this time pipette volume containing the desired number of cells and aggregates into a 1.8 milliliter micro centrifuge tube. Cap the tube and then centrifuge the cells and aggregates for five minutes at 130 gs. Then using a micro pipetter, carefully remove the supernatant without disturbing the cell pellet at the tip of the tube.
As the media head approaches the pellet, use a finer 10 microliter pipette tip Evenly dispersing the cell aggregates throughout the gel precursor solution while ensuring that they're fully encapsulated. It's the trickiest part of this procedure. When performing this step, take care to resand and transfer the pellet gently.
Now, gently resuspend the cell pellet in 20 microliters of the photo active PEG DM solution. Using a wide mouth pipette tip and add the cell suspension to the syringe on top of the cross-linked hydrogel portion, expose the syringe to an additional eight minutes of UV light to complete the construction of the hydrogel. When finished, the cells should be fully encapsulated within the cylindrical hydrogel.
Finally, add one milliliter of HBSS into one well of a 24 well plate and one milliliter of media to another. Well then eject the hydrogel from the syringe into the HBSS to briefly wash the gel. Transfer the gel to the media and culture the plate under the desired conditions.
At any point during the culture, the cells can be imaged using fluorescent microscopy to track the initiation of the hypoxic signaling place, the 24 well plate onto the microscope stage, centering the well with the hydrogel under the objective. The signal is specific enough to identify individual signaling cells, but resolution may be inadequate for determining intracellular signal localization. In signaling cells, the signal is typically observed uniformly throughout the cytoplasm.
This figure shows a representative example of minx aggregates encapsulated in a PEG hydrogel within the gel cell. Aggregates should be fully enclosed in the matrix and homogenously distributed to allow for better nutrient transport as seen here. Note how the cross-link gel is solid throughout taking the shape of the vessel in which the reaction was performed.
In this figure representative fluorescent hypoxia signaling in aggregated min six cells in a PEG DM hydrogel is shown cells that were encapsulated and then placed in incubation at 20%oxygen for 44 hours. Pictured on the left do not display hypoxia signaling while cells that were encapsulated then incubated in 2%oxygen for 44 hours. As pictured on the right display, clear ubiquitous signal hypoxia can also be tracked in other cell types.
This figure shows representative hypoxic signaling in encapsulated mesenchymal stem cell aggregates. As seen in the leftmost figure fluorescent signal is not detectable at 12 hours in 2%oxygen then as seen in the center figure is more apparent after 24 hours and 2%oxygen. And finally, as seen on the right is highly expressed after 96 hours and 2%oxygen Following this procedure.
Other methods like Eliza, analysis of secreted proteins and cell viability assays can be performed in order to correlate hypoxic signaling with cell phenotype. With this development, this technique could pave the way for researchers in the fields of tissue engineering and cell-based therapeutic delivery to explore survival function and differentiation of cells within scaffolds and encapsulation materials.
