Bioengineering
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Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation
Chapters
Summary July 2nd, 2018
We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.
Transcript
This method can help answer key questions in the cancer biology field, such as tumor formation, cell migration tracking, and cancer and healthy cell signaling. The main advantage of this technique is that it allows us to study tumorigenesis in a 3D environment along with stromal cells, such as fibroblasts, with low cost and high reproducibility. It is important to make sure the materials are both bioprintable and biofunctional, which requires quantitative study of its rheological properties and cell compatibility.
Visual demonstration of this method is critical as the hydrogel preparation and cell hydrogel mixing steps are difficult to learn because bubble formation interferes with the fidelity of the printed model. In a biosafety cabinet, mix three grams of alginate and seven grams of gelatin powder in a 250-milliliter beaker. Add a magnetic stirrer and 100 milliliters of DPBS.
Seal the beaker with sterile paraffin film and aluminum foil to avoid contamination. Then show the participant the unique and specific polar plots which correspond to their electromyography, or EMG, patterns. After this, continue the agitation at room temperature for two hours.
Heat the solution at 37 degrees Celsius until the gel undergoes a phase transition to the liquid state. Then transfer the solution into sterile 50-milliliter conical centrifuge tubes, and seal them. Centrifuge the tubes at 834 times g for five minutes to eliminate any gas bubbles.
Next, aspirate the hydrogel precursor into 10-milliliter syringes. Seal the syringes with caps and paraffin film. Store at four degrees Celsius until ready to use.
When ready to begin testing, place a syringe of prepared hydrogel precursor in a 37 degree Celsius water bath for one hour. While the syringe is warming, turn on and initialize the rheometer. After an hour, remove the syringe from the water bath.
Extrude approximately 0.5 milliliters of the precursor onto the rheometer platform. On the control panel, click the downward triangle button. Wait for the measuring tool to move down to the trimming position.
Using a spatula, trim any excess precursor that escaped the edge of the measuring tool, and discard the superfluous material. Then pipette mineral oil onto the edge of the measuring tool. Wait for the oil to fully seal the boundary.
On the control panel, click Continue. Then click the Start button. After the testing is complete, release the measuring tool, and click the upward triangle button.
Remove the measuring tool. Clean both the measuring tool and the platform with 70%ethanol. First, complete amplitude and temperature sweeps as outlined in the text protocol.
To begin an isothermal time sweep, click My Apps, and click the template for Isothermal time-temperature test. Click the Measurement step in the workflow. Then click the variable oscillation strain, and pull up the setup window.
Set the oscillation strain to 0.1%and set the oscillation frequency to one hertz. In the pull-up window, set the number of data points to 120. Set the data collection frequency to one point per minute.
Click Add button to add the temperature variable. Click the variable temperature, and set it to 25 degrees Celsius. Right-click the step Device, Move to measuring point in the workflow.
Click Delete. Then click the step Device, Set value. Uncheck the box Wait until value is reached, and set value to 25 degrees Celsius.
Click Picture, Title, Buttons at the bottom screen. Uncheck the box Continue. Then click the step Start, name the project, and save it.
Now load the sample onto the rheometer platform, and start the test following the previously described general testing protocol. After this, observe the G prime and G double prime versus the time. Find the crossover point of G prime and G double prime, and determine the time at the crossover point.
Then click Table, copy all of the data, and paste it into a text file. To measure the yield strength at various gelling times, click My Apps. Click the template for Yield and flow stress, Gel-like.
Name this project. Next, click on the Start step in the workflow. In the top menu, click Insert.
Then, in the drop-down menu, select Wait. Pull up the setup window, and set the waiting time to 50 minutes. Click Start, then click Insert again.
Select Device from the drop-down menu. Pull up the setup window, select Temperature, and set it 25 degrees Celsius. Uncheck the box Wait until value is reached.
In the workflow, select the Measurement step. Select the variable Shear Stress, and pull up the setup window. Set the initial and final shear stress to zero and 10, 000 pascal, respectively.
Then click Calculator, set the Point density to 0.2 point per pascal. In the Data points tab on the left, set one point per second. At the bottom of the pull-up window, select the Event Control tab, set the criterion Stop the measurement if the shear rate is greater than 100 per second.
Load the sample onto the rheometer platform, and begin the test following the previously described general testing protocol. After the test is complete, click Diagram to observe the strain-stress curve. Repeat this test for waiting times of 30, 40, and 50 minutes.
The following steps must be performed in sterile conditions. To begin, sterilize the bioprinter by spraying it thoroughly with 70%ethanol and then exposing it to UV light overnight. Remove a syringe of hydrogel precursor from storage, and warm it in a 37 degree Celsius water bath for one hour.
Transfer a T-flask of previously cultured cells from a CO2 incubator to the biosafety cabinet. Discard the culture medium, and then rinse the cells twice with DPBS. Add a warmed trypsin-EDTA solution to the cells.
Incubate at 37 degrees Celsius for six minutes. After this, inactivate the trypsin by adding the same volume of FBS. Remove the syringe of hydrogel precursor from the water bath, and clean it.
Sterilize the syringe with 70%ethanol before transferring it into the biosafety cabinet. Extrude approximately three milliliters of hydrogel precursor into a 10-milliliter printing cartridge. Mix precursor with MDA-MB-231-GFP cells at a concentration of one million cells per milliliter by slowly pipetting it to avoid producing bubbles.
Cover the cartridge with the end and top caps, and seal it with paraffin film. Then centrifuge it at 834 times g for one minute to eliminate any gas bubbles. Repeat this entire process for the other precursors.
After this, sterilize all three cartridges with 70%ethanol. Load the sterilized cartridges into the chambers of the bioprinter. In the printer's control software, click manual.
Then expand the Tool head tab in the printer's control software. Click on one cartridge at the graphic interface at the left side, set the cartridge temperature to 25 degrees Celsius, and wait 35 minutes to allow the precursor to reach printable conditions. Repeat this for the other two cartridges.
In the printer's control software, expand the Tool head tab. Click on one cartridge in the graphic interface, then click the measure short button to measure the position of the nozzle tip. Repeat this for the other two cartridges.
Next, position a clear polystyrene microplate on the printer stage. Open the G-code file, and change the pressure to 200 kilopascals for all cartridges. Return to the control software, and click the Scaffolder Generator tab.
Select a point to start printing, and then click the Run button. Repeat this process to obtain three replicates. After this, add a 100-millimolar calcium chloride solution for one minute to cross-link the models.
Rinse twice with DPBS. Then use a spatula to carefully transfer the models onto an agarose-coated, six-well plate. Add five milliliters of DMEM to each well, and incubate at 37 degrees Celsius and 5%carbon dioxide, replacing the cell culture medium every three days.
In this study, three-dimensional hydrogel systems are fabricated to recapitulates in vivo tumor microenvironments and facilitate the formation of multicellular tumor spheroids. A temperature sweep of the A3G7 precursor shows G double prime to be greater than G prime at 37 degrees Celsius, which validates the known properties of the precursor. As the temperature decreases, the precursor undergoes physical gelation due to the spontaneous physical entanglement of the gelatin molecules into a tri-helix formation.
Both the G prime and G double prime increases the temperature decreases and converge at 30.6 degrees Celsius, indicating a sol-gel transition. Confocal microscopy of the printed propeller model shows that the MDA-MB-231-GFP cells begin to develop spheroids 15 days into the culture period, and the spheroids continue to increase in size and numbers until day 30. Some of the IMR-90-mCherry cells are also seen to form agglomerations.
After 30 days of culture, IMR-90-mCherry cells could be observed in the region initially occupied by the MDA-MB-231-GFP cells, while MDA-MB-231-GFP cells could be seen in the IMR-90-mCherry-dominated region. This implies possible migration events in the model. Confocal microscopy of the disk model reveals that MDA-MB-231-GFP cells behave similarly to how they did in the propeller model, resulting in a spheroid-laden hydrogel after 21 days of culture.
Once mastered, this technique to print models can be done in two hours, if it is performed properly. While attempting this procedure, it's important to remember to keep hydrogel and cells fresh as possible, do not mix old hydrogel batches with new ones, and to work under a sterile condition to avoid misleading results. Following this procedure, other methods like scanning electron and time-lapse microscopy can be performed in order to answer additional questions, like material porosity, cell migration rate, and matrix remodeling.
The implication of this technique extend toward cancer therapy because this in vitro model allows systematically controlled studies of tumor formation and behavior in co-culture systems. Besides breast cancer cells, the model can also be readily applied to systems using other types of cells at various initial locations. After watching this video, you should have a good understanding of how to create 3D heterogeneous disease models by properly preparing hydrogels, embedding cells into and printing the material within the optimal printing window.
Don't forget that working with biological samples can be extremely hazardous and precautions such as wearing personal protective equipment, such as gloves, lab coat, and safety goggles while performing this procedure.
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