The overall goal of fabricating elastic 3D macroporous microcryogels using cryogelation is to facilitate injectable regenerative therapy and in vitro high-throughput drug screening. This video presents the protocols to fabricate versatile 3D microtissues and apply them to regenerative therapy and drug screening. The regenerative therapy technique improves on stem cell retention, survival, and therapeutic functions after injection into a wound site.
Drug screening is also improved because of ease of use and potential for high-throughput screening. Generally, individuals new to 3D culture struggle because of the complicated protocols. This technique simplifies 3D culture and offers researchers greater control of the resulting 3D microtissues.
Assisting in the demonstration of these procedures will be Zhou Lyu, Liu Wei, Li Yaquian, and You Zhifeng, four graduate students from our laboratory. Wash the PMMA microstencil array chips with deionized water to remove the debris remaining from the laser engraving. Then, dry the microstencil array chips at 60 degrees Celsius.
Next, treat the dried chips in a plasma cleaner in groups of four. First, run the vacuum pump for two minutes. Then, use 18 watts of RF power for three minutes to increase the hydrophilicity of the chips.
Now, make one milliliter of gelatin precursor solution for every five chips. Dissolve the gelatin in a 60-degree Celsius water bath and incubate the solution on ice for five minutes. Once cooled slightly but still liquid, thoroughly mix glutaraldehyde into the gelatin precursor solution to a final concentration of 0.3%glutaraldehyde.
Now, pipette 200 microliters of prepared solution onto the upper surface of each chip. Distribute the solution evenly over the chips using a bent glass rod. Next, immediately transfer the solution-loaded array chips into a minus 20 degree Celsius freezer for 16 hours for cryogelation.
The next day, cool a lyophilizer to minus 40 degrees Celsius over about 30 minutes. Then, load the array chips, and lyophilize them for two hours under a vacuum. The ice will sublime under these conditions, and the chips will then be ready for use.
Begin by harvesting individual microcryogels from the chips. Start with overlaying the fabricated microcryogel array chip on top of the fabricated PDMS ejector pin array. Align each microcryogel with an ejector pin.
Then press the microcryogel array chip into the array to displace the microcryogels from their wells. Now, harvest the ejected microcryogels into a water bath, and collect them with the aid of a cell strainer. Use one strainer to collect all the microcryogels from one chip.
Next, wash the microcryogels with sodium borohydride on ice. Allow the non-crosslinked aldehyde residues to be quenched for about 20 minutes. Then, discard the sodium borohydride, and wash the microcryogels with five milliliters of deionized water for 15 minutes.
Perform a total of three to five water washes before proceeding. After removing the microcryogels from the last water wash, use tweezers to transfer them from the cell strainer to a 35-millimeter Petri dish. Each collection of microcryogels constitutes one cluster.
Now, add 50 to 70 microliters of deionized water to each cluster of microcryogels, and cover the dish. Then, gently tap the dish to level any stacked microcryogels. Then, freeze the microcryogels at minus 20 degrees Celsius for four to 16 hours.
Later, lyophilize them for two hours as before. The next step is to make the 3D microtissues. First, sterilize the microcryogels.
Then, harvest the cells with trypsin, quantify their density, and resuspend them at eight million cells per milliliter in growth medium. Next, pipette 60 microliters of cell suspension onto each cluster of microcryogels. Maintain the dishes in a humidified chamber, and incubate them at 37 degrees Celsius for two hours so that the cells will absorb into the gels.
After the incubation, add two milliliters of culture medium to each dish, and continue the culture, changing the medium every other day. In two days, 3D microtissues will have formed. To inject the 3D microtissues into a mouse, first transfer them into a five-milliliter pipette, and then load them into a cell strainer to filter the culture medium.
Then, resuspend the 3D microtissues in a 15%gelatin solution. After preparing the animal for the implantation, intramuscularly inject the microtissues into three locations of the gracilis muscle. Each injection should contain 150 microcryogels in 150 microliters of solution.
Use a one-milliliter syringe with a 23 gauge needle. After assembling the microcryogel array for on-chip cell culture, carefully place them into a preheated humidity chamber without getting them wet. Then carefully aliquot three microliters of thoroughly-mixed cell suspension directly onto each microcryogel well.
Allow the pipette tip to lightly touch the surface of the microcryogel before expelling the solution. Do not seed the peripheral wells. After loading the wells, add 10 microliters of selective medium to each well, and add medium to the peripheral wells to slow evaporation using a 96-channel liquid dispenser.
Then, culture the chips in the humidity chamber for 24 hours to form 3D microtissues. The next day, using the 96-channel liquid dispenser, add 10 microliters of drugs to each well in a concentration gradient four times to complete addition of all 384 wells, including DMSO controls. Then, continue the culture for 24 hours.
After 24 hours of drug treatment, add four microliters of resazurin stock solution to each well, and incubate the plate for two hours, allowing the cells to metabolize the resazurin. To assay the cells'metabolism of resazurin, use a plate reader. Harvested gelatin microcryogels had predefined shapes and sizes.
Analysis by SEM showed that the microcryogels contained interconnected macroporous structures with pore sizes in the range of 30 to 80 microns. The injectability of the gelatin microcryogels was quantitatively assessed by culturing them post-injection. 1, 000 per milliliter were injected at six newtons less than the clinically acceptable force of 10 newtons.
The stem cells in microcryogels retained high viability and great proliferative capacity after injection during five days of culture. Using such cells, the mouse limb ischemia model was therapeutically treated, and the physiological status of the ischemic limbs was examined 28 days after surgery. Microtissue treatment with 100, 000 stem cells improved limb salvage.
Only 25%of mice showed spontaneous toe amputation after 28 days. In a different application, the 3D microtissue array was used for high-throughput drug testing. Hepatocellular carcinoma cells were treated with doxorubicin, and non-small cell lung cancer cells were treated with IMMLG-8439.
In both cases, drug resistance was increased by 3D culturing. After watching this video, you should have a good understanding of how to fabricate elastic 3D microporous microcryogels for off-chip 3D culture using injectable regenerative therapy or on-chip 3D cell culture arrays for in vitro high-throughput drug screening. This technique paved the way for researchers in the field of cell-based regenerative therapy and drug screening, to make use of 3D cell culturing, thus advancing both research fields.
Don't forget that working with glutaraldehyde and sodium borohydride can be extremely hazardous, and precautions such as wearing proper protective garments and working under a laboratory hood should always be taken while performing this procedure.
