Our protocol helps imaging oxygenation of multicellular spheroids using a conventional fluorescence microscope. Using this method, one can produce thousands of oxygen probe-stained spheroid microtissues and subsequently use them in 3D printing applications. For instance, vascularized bone tissue grafts.
near-infrared oxygen probe is compatible with the use of other dyes such as green fluorescence staining of Ultimately, this allows for live and multi-parameter long-term analysis. Transfer micro-patterned PDMS stamps from a storage vial to a sterile Petri dish and air dry with the smooth surface up under the sterile laminar airflow conditions for 10 minutes. Put each stamp in the middle of the well of a 12-well sterile tissue culture plate.
Air dry for one to two minutes with an open lid. Prepare 50 milliliters of 3%homogenous agarose solution and immediately add approximately two milliliters of hot agarose solution to each well of the 12-well plates to cover the inserted PDMS stamp. Solidify the agarose for 20 minutes by incubating it under sterile airflow.
Using a sterile spatula, turn the agarose with the embedded PDMS stamp upside down in each well. Add approximately 200 microliters of sterile water on the smooth top side of the stamp. Detach it from the agarose with a spatula and remove it from the well.
Add one milliliter of corresponding sterile cell culture media to the agarose stamps for direct use. Cover the plate with the lid and incubate overnight at four degrees Celsius. Before use, warm the agarose micro-patterned plates for one hour at 37 degrees Celsius in a carbon dioxide incubator.
Rinse 70%to 90%confluence cell culture with pre-warmed PBS. Add dissociating enzyme solution and incubate for three to five minutes at 37 degrees Celsius. Later, neutralize trypsin with complete cell culture media containing 10%FBS.
To obtain a single cell suspension, dissociate cell aggregates using a 1, 000 microliter pipette tip on the top of the serological pipette. Using a counting chamber, count the number of cells per milliliter of the cell suspension. Dilute the cell suspension to 500, 000 cells per milliliter and add concentrated oxygen probe solution to it.
Remove old media from micro-patterned wells of the 12-well agarose-coated cell culture plate and add one milliliter of the prepared cell suspension to the wells. Culture the spheroids in a carbon dioxide incubator for two to five days in the continuous presence of the oxygen probe to ensure it's loading during spheroid formation and compaction. Close the end of a sterile three cubic centimeter cartridge with a tip cap and fill with bio ink by pipetting.
Insert a plunger in the cartridge and hold it upside down. Remove the tip cap and push the plunger toward the bio ink until all air from the cartridge is removed. Cool the bio ink in the heating mantle of the bioprinter at 23 degrees Celsius.
Spray the bioprinter with 70%ethanol and dry it with absorbing paper. Screw in a pressure adapter on the top of the cartridge. Mount a 22 gauge conical polyethylene needle on the cartridge.
Install the cartridge in the heating mantle on the extrusion-based print head. Plug the pressure inlet and open the clip on the inlet. Load the G-code file of your design into the printing software.
Start needle length measurement. Regulate the printing pressure by dispensing a little bio ink in a sterile Petri dish. Install a six-well plate, open the well plate, close the hood and print.
After printing, let the scaffolds be physically crosslinked for 10 minutes at five degrees Celsius. Irradiate the printed scaffolds for 60 seconds with an ultraviolet LED lamp. Immediately add the corresponding growth media containing dual antibiotic solution to all wells with bioprinted grids.
Culture the scaffolds at 37 degrees Celsius in a carbon dioxide incubator. For microscopy observation, transfer a printed waffle grid into a microscopy dish cover with imaging media. Using a one milliliter pipette, gently wash out the oxygen probe pre-stained spheroids from the agarose micro wells and transfer them into a 15 milliliter conical bottom tube.
To ensure the collection of all spheroids from a micro-patterned well, rinse the well one to three times with the additional one milliliter of culture media and combine all spheroid suspensions in one vial. Leave the vial vertical for up to 10 minutes to let the spheroids settle down on the bottom. Carefully remove the media from the tube leaving the spheroids undisturbed.
Add a fresh culture medium that will be sufficient for at least 20 spheroids per sample dish and gently resuspend the spheroids by pipetting. Immediately add an equal volume of spheroid suspension to each pre-coated microscopy well. Leave the spheroids for one hour at 37 degrees Celsius to attach to the surface of the microscopy well.
Remove the medium from the microscopy well and rinse once with imaging media. Add the exact amount of imaging media to the sample. Set up the microscopy well with stained spheroids on the microscopy stage.
Using the 10X magnification objective, preview the sample in transmission light. Do preliminary focusing on spheroids, locate them in the center of the image and bring the objective with the required high magnification to the working position. Focus on transmission light mode in the equatorial cross-section of the spheroid.
Adjust settings for collecting fluorescence or phosphorescence signals of reference and sensitive spectral channels of the oxygen probe and start the imaging process. Open the vsi file with oxygen probe intensity data from reference and sensitive spectral channels in merged mode. Open the ratio analysis function window from the measure menu.
Choose the intensity of the reference channel as the numerator and the intensity of the sensitive channel as a denominator for R calculation. In the ratio analysis window, apply corresponding intensity threshold settings to each spectral channel to subtract the background from the merged intensity image. ROI masks applied to spheroid images determines spheroid borders.
Increase the threshold values until the image background is uniformly black. In the ratio analysis window, adjust the scaling factor to the ratio image until the preview image provides the desired resolution of the R gradient. The image of intensity ratio distribution is added as a layer to the original multichannel image.
In the window of the multichannel image, activate the layer with a false color bar. Manually determine the linking and unlinking limits of a ratio distribution histogram using the fixed scaling option in the adjust display window. Open the corresponding transmission light image of spheroids.
Choose linear ruler function and measure the diameter of spheroids. Export data as a spreadsheet table file. Choose the ROI of the required size and shape and apply it to the periphery and hypoxic core of the spheroid.
Transform the ROI to the measurement object to analyze the average R inside the chosen ROI. Export data in a spreadsheet compatible table format. Apply measurements to each microscopy section of spheroids to obtain the dataset of spheroid diameters RC and RP to perform further calculations and statistical comparison.
Combine all data in one spreadsheet file. MMIR1 probe has low photobleaching and is suitable for real-time study of rapid respiratory response in hDPSC spheroids to different mitochondria stimuli. FCCP displayed only a mild uncoupling effect in some areas and then slightly decreased cell respiration.
On the other hand, rotenone strongly inhibited respiration leading to spheroid reoxygenation and dissipation of the periphery-to-core oxygen gradients within approximately 80 seconds after stimulation. Using the automated protocol of pixel by pixel intensity ratio calculations provided by the imaging software, real-time detected periphery-to-core oxygen gradients in all spheroid types are visualized with the oxygenated periphery and hypoxic niches in the center. The graphs herein depict ratio profiles of the equatorial spheroid cross-sections for different spheroid types.
Compared to homocellular hDPSC spheroids, heterocellular hDPSC to HUVEC spheroids had significantly steeper gradients. The periphery-to-core oxygen gradient in heterocellular spheroids is likely generated by hDPSC in their composition according to hDPSC spheroids oxygenation having strong respiration activity. The graphs show that bioprinted hDPSC spheroids had a significantly oxygenated periphery than spheroids measured before bioprinting while their core oxygenation had similar values.
If occasional probe precipitation disturbs uniform spheroid formation, filter or centrifugate the probe solution at high speed before use. Correcting needle length measurement and pressure regulation are essential steps to match the bioprinting rate and strut diameter and bioprint microaggregates in a porous scaffold. Choose the microscopy settings for oxygen sensitive probe imaging, considering their effect on probe photostability.
Oxygen imaging is compatible with many types of live microscopy measurements. After imaging, one can also perform immunofluorescence, extract proteins for Western blotting, or perform gene expression analysis.
