Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

1. PEGDM synthesis and photoactive PEGDM macromer solution preparation

1. Weigh 2g of PEG (linear, 10,000Da) into a 40mL glass vial with a hard plastic cap.
2. Add approximately 308μL of methacrylic anhydride and loosely cap the vial.
3. Microwave the vial on high for 2 minutes in a standard domestic microwave. Wearing heat resistant gloves, thoroughly vortex the vial, then microwave on high for an additional 5 minutes.
4. Briefly allow the vial to cool enough to be handled with gloves. Uncap it and slowly add methylene chloride while swirling the vial. Add enough so that the solution appears clear and homogeneous, vortexing the sample as needed. The total volume of methylene chloride used should be 1.5 – 2 mL.
5. Precipitate PEGDM in 200 mL of cold, stirred diethyl ether by adding the solution dropwise.
6. Collect the PEGDM by vacuum filtration and allow it to dry.
7. Dissolve the PEGDM in an adequate amount of deionized water (typically 100 -150 mL).
8. Dialyze the solution against deionized water for 5 days in dialysis tubing with a molecular weight cut-off of 1,000 Da. Replace the water once daily.
9. Aliquot the solution into appropriate containers and freeze at -80°C overnight. Lyophilize the solution for 4 days to yield a purified white PEGDM powder. Store the powder at 4°C when not in use.
10. To prepare a photoactive PEGDM solution, first prepare a 0.5 weight percent photoinitiator stock solution by dissolving Irgacure 2959 in deionized water. Vortex the solution and store it in the dark.
11. Prepare a 10 wt% PEGDM and 0.025 wt% Irgacure 2959 solution in Hank’s Balanced Salt Solution (HBSS). Vortex thoroughly to ensure dissolution of the PEGDM. We typically prepare 1mL of this solution at a time.
12. Sterilize the solution by filtering it through a 0.2 μm syringe filter into a 1.8 mL microcentrifuge tube. Wrap the tube in aluminum foil and store at 4°C

2. Culture, infection, and aggregation of MIN6 cells

1. MIN6 cells are cultured in RPMI 1640 medium supplemented with 10% FBS, 7mM glucose, 100 units/mL penicillin/streptomycin, and 0.5 μg/mL amphotercin B in a humidified environment at 37°C and 5% CO₂.
2. To release the cells from the treated culture flask surface, aspirate the medium, rinse the cells with 37°C calcium/magnesium-free HBSS, aspirate the HBSS, then add 2 mL of 37°C trypsin-EDTA solution and allow the cells to incubate for 3 minutes.
3. Firmly jar the side of the flask to knock the cells free, then add 8 mL of 37°C medium and vigorously pipette the cell suspension up and down taking care not to introduce bubbles.
4. Pipette the cells into a sterile 15 mL centrifuge tube and perform a cell count using a hemocytometer or other cell counting procedure.
5. In preparing to infect the cells with the maker virus, first determine the total number of cells to be infected and calculate the volume of virus suspension required to achieve the desired multiplicity of infection (MOI) (50 to 100 infectious particles per cell).
6. Dilute the virus in HBSS by carefully pipeting the proper amount of virus suspension into a pre-aliquoted volume of HBSS in a 1.8 mL microcentrifuge tube. 50 μL of HBSS per well of aggregating cells is appropriate.
7. Disperse the cells by pipeting them up and down in their tube, then pipet the necessary volume to achieve 400,000 cells per well into the wells of a 6-well suspension culture plate.
8. Add the appropriate volume of diluted virus to each well to achieve the desired MOI.
9. Add medium to each well to bring the total volume up to 1.7 mL.
10. Place the plate on an orbital shaker inside the incubator. Set the shaker on a low setting so that the medium gently washes around the wells (˜100 rpm). Allow the cells to aggregate for 24-36 hours. Aggregates with an approximate diameter of 75-175 μm should be formed.

3. Encapsulation of cells in PEGDM

1. Cells may be encapsulated as a dispersion (step 2.4) or following aggregation (step 2.10). Since the hydrogel precursor PEGDM solution is a liquid, cells may tend to settle prior to crosslinking and hydrogel formation. If settling is expected to occur rapidly, such as with larger aggregates, it may be beneficial to produce the hydrogel in two steps so that the cells settle to a central plane of the gel. A one-step hydrogel synthesis procedure appropriate for dispersed cells is shown (Figure 2) and the two-step procedure is shown as well (Figure 3) and detailed below (3.2-3.9).
2. Create a vessel in which the hydrogel will be formed by cutting the tapered tip off of a 1 mL plastic syringe and placing it open end up in a rack.
3. Pipet 20 μL of photoactive PEGDM 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.
4. Place the pipet in the rack and position the rack underneath a UV lamp (365nm, ˜7 mW/cm²) for approximately 8 minutes to allow for hydrogel crosslinking. During this time, preparation of the cells may be begun
5. Pipet a volume containing the desired number of cells/aggregates into a 1.8 mL microcentrifuge tube, cap, and centrifuge the tube at 130 g for 5 minutes.
6. Using a micropipettor, carefully remove the media without disturbing the cell pellet at the tip of the tube. As the media head approaches the pellet, it may be helpful to use a finer, 10 μL pipet tip.
7. Gently resuspend the cell pellet in 20 μL of photoactive PEGDM solution (use a wide-mouthed pipet tip if working with aggregates) and add the cell suspension to the syringe on top of the previously crosslinked hydrogel portion.
8. Expose the syringe to an additional 8 minutes of UV light to complete construction of the hydrogel. When finished, the cells should be fully encapsulated within the cylindrical hydrogel (Figure 3).
9. Eject the hydrogel from the syringe and into 1 mL of HBSS in the well of a 24-well plate to briefly wash the gel. Transfer the gel to 1 mL of media in the well of a 24-well plate and culture under the desired conditions.

4. Hypoxia tracking

1. At any point during the culture, image the cells using fluorescent microscopy to track the initiation of hypoxic signaling. Depending on what is being imaged you can image in the multi-well plate or transfer the gel to a microscope slide prior to placement on the microscope stage. Peak excitation of Ds Red is achieved at 556nm and peak emission occurs at 586nm. Emission is fairly intense and thus photobleaching has not been observed to be problematic. Lag time between initiation of protein production and first signal detection is approximately 12 hours. Signal is specific enough to identify individual signaling cells, but resolution may be inadequate for determining intracellular signal localization. In signaling cells, signal is typically observed uniformly throughout the cytoplasm. Signal intensity can also increase with extended exposure to hypoxia, though we have not yet fully determined if this is due to increased protein expression, overall protein accumulation, or delayed protein maturation.

5. Representative results

A representative example of MIN6 aggregates encapsulated in a PEG hydrogel is shown in Figure 4. The crosslinked gel will be solid throughout, taking the shape of the vessel in which the reaction was performed. A gel with smooth outer surfaces is preferable for implantation to aid in prevention of a foreign body response. Within the gel, cell aggregates should be fully enclosed in the matrix and homogenously distributed to allow for better nutrient transport.

Representative images of hypoxic signaling in MIN6 aggregates are pictured in Figure 5. Identical MIN6 aggregates infected with the marker virus were cultured in either 20% O₂ (5a) or 1% O₂ (5b) for 44 hours before image capture.

Figure 1. Schematic of the activity of our hypoxia marker system. Adenoviral insertion of the Ds Red DR gene and upstream HRE promoter allows for hypoxia-induced production of the fluorescent protein under control of HIF-1.

Figure 2. Procedural flow chart for the single-step encapsulation of dispersed MIN6 cells in a crosslinked PEGDM hydrogel. For each gel, the dispersed cells are suspended in 40μL of the photoactive PEGDM macromer solution. This is placed in a decapitated 1mL syringe and the hydrogel is formed under 365nm UV light after 10-12 minutes. Upon completion, the hydrogel disk is removed from the syringe, washed and placed in a medium-filled well plate for incubation.

Figure 3. Procedural flow chart for the dual-step encapsulation of aggregated MIN6 cells in a crosslinked PEGDM hydrogel. For each gel, a half-gel is first formed by UV crosslinking of 20μL of the photoactive PEGDM macromer solution for 8 minutes. MIN6 aggregates are carefully suspended in an additional 20μL of photoactive macromer solution which is added on top of the pre-formed half-gel. The full gel is formed by an additional 8 minutes of UV exposure with aggregates fully encapsulated in the medial plane of the gel.

Figure 4. Image of a 40μL hydrogel under 20X magnification. MIN6 aggregates (~400,000 total cells) are clearly seen within the gel. Hydrogel diameter is approximately 6mm (bar = 1mm).

Figure 5. Fluorescent hypoxia signaling in aggregated MIN6 cells in a PEGDM hydrogel. Cells that were encapsulated and then placed in incubation at 20% O₂ for 44 hours do not display hypoxia signaling (a) while cells that were encapsulated then incubated in 2% O₂ for 44 hours display clear, ubiquitous signal. (bar = 100μm) (b).