3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer

Hypoxia is a key driver of cancer development that induces genomic instability and tumorigenesis. We demonstrate a method for generating central hypoxia in a solid cancer in vitro based on 3D bioprinting technology. Using this method, a radio hypoxic gradient can be reproduced using a simple strategy that combines a 3D printed gas permeable barrier and a glass cover.

This hypoxic cancer on-a-chip technology can be used to predict drug efficacy to facilitate patient specific anticancer drug prescription. And it's also expected to enable a quick diagnosis of aggressive cancers. To assist in the preparation of a three milliliter neutralized collagen pre-gel solution, cut 30 milligram collagen sponges into a five by five millimeters squared pieces.

Place the pieces into a sterile 10 milliliter glass vial and add 2.4 milliliters of 0.2 micron syringe filter 0.1 normal hydrochloric acid to the vial for a three-day incubation at four degrees Celsius and 15 revolutions per minute. After the digestion, strain any undigested collagen particles through a 40 micron cell strainer and store the acidic collagen solution at four degrees Celsius for up to seven days. To adjust the pH of the 1%neutralized collagen pre-gel solution, add 30 microliters of phenol red solution to a final concentration of 1%and 300 microliters of 10X PBS to a final concentration of 10%After mixing and centrifugation, use one normal sodium hydroxide to neutralize the pH to seven, verifying the color change and add distilled water to obtain a total volume of three milliliters.

Then store the pH adjusted 1%neutralized collagen pre-gel solution at four degrees Celsius for use within three days. To precheck the gelation of the neutralized collagen pre-gel solution, use a positive displacement pipette to add 50 microliters of collagen droplets to a small dish and incubate the droplets in a 37 degrees Celsius incubator for one hour. At the end of the incubation, check whether the collagen has changed from a transparent color to an opaque white.

Tilt the container to confirm that the collagen is adhere to the bottom of the container. And pour PBS onto the droplet to confirm that the collagen construct does not break in solution. For 3D printing of a sacrificial PEVA mold, click File and save file type as STL to convert the 3D CAD file into an STL file and click Option and output form as ASCII to generate the G-code.

To import the generated STL file, click File and open STL file and select the saved STL file. To automatically generate the G-code of the sacrificial PEVA mold, select the slice model of the STL-CAD exchanger. Then to generate printing paths for the chip fabrication, use a 50 gauge precision nozzle at a pneumatic pressure of 500 kilopascals at 110 degrees Celsius to print the sacrificial PEVA mold onto a sterile adhesive hydrophilic histology slide.

To cast the PDMS barrier, mixed six milliliters of PDMS base elastomer and 600 microliters of curing agent for five minutes in a plastic reservoir and load the homogeneously blended solution into a 10 milliliter disposable syringe. Equipped the syringe with a 20 gauge plastic tapered dispense tip and fill the sacrificial PEVA mold with the blended PDMS solution. The blended PDMS will fill this sacrificial PEVA mold with a convex surface and the barrier will be higher than that of the mold.

After curing the PDMS barrier in a 40 degrees Celsius oven for over 36 hours to avoid melting the PEVA, use a pair of precision tweezers to detach the sacrificial PEVA mold and sterilize the gas permeable barrier at 120 degrees Celsius in an autoclave. To mix the solution with cancer cells, resuspend each type of collected cell pellet in 20 microliters of culture medium and add one milliliter of 1%neutralized collagen pre-gel solution into each of the resuspended cell suspensions on wet ice with gentle mixing. Use a positive displacement pipette to mix each cell suspension.

When homogeneous solutions are obtained, use a positive disposable pipette to transfer the cell encapsulated collagen bioinks into individual three milliliter disposable syringes and store the syringes at four degrees Celsius until 3D cell printing. For 3D printing of cancer-stroma concentric rings, convert the appropriate 3D CAD file into an STL file format and use an STL-CAD exchanger to generate a G-code of the cancer-stroma concentric rings. Load the cell encapsulated collagen bioinks into the 3D printer head and set the temperature of the head and plate to 15 degrees Celsius.

Load the generated printing path on the control software of the 3D printer and click Start to print the collagen bioinks onto the gas permeable barrier according to the loaded G-code with an 18 gauge plastic needle at a pneumatic pressure of approximately 20 kilopascals at 15 degrees Celsius. At the end of every printing operation, manually place a sterilized 22 by 50 millimeter glass cover on top of the gas permeable barrier to generate the hypoxic gradient. After generating three hypoxic cancer-on-chips transfer the chips to a 37 degrees Celsius incubator for one hour to crosslink the collagen bioinks.

When all of the hypoxic cancer-on-a-chips have been printed, gently rubbed the cover glasses on top of the gas permeable barriers with a cell scraper for tight bonding. To refresh cell culture medium to the chips without detaching the cancer construct, tilt the chips and use a pipette to introduce 1.5 milliliters of endothelial cell growth medium to the side of each chip. The hypoxic cancer-on-a-chip was designed in the form of concentric rings to mimic the radial oxygen diffusion and depletion observed in cancer tissues.

After defining the control volume of a space in which oxygen is diffused and consumed by cells, an appropriate cellular density for central hypoxia generation can be determined through computational finite element analysis. Upon 3D cell printing, a compartmentalized cancer-stroma concentric ring structure can be created to reproduce the anatomical features of the solid cancer. Quantitatively, the post-printing cell viability is typically greater than 96%confirming that the manufacturing conditions are appropriate for cancer and stromal cells.

In this representative analysis, two groups were compared according to the presence and absence of the oxygen gradient to verify the effects of a hypoxic gradient on cancer progression. Under both conditions mature CD31 positive endothelial cells were present within the peripheral regions, indicating that spatially patterned living constructs were produced using 3D bioprinting technology. Compared to the oxygen gradient negative condition the gradient positive condition demonstrated a hypoxic gradient, indicating the gradual expression of HIF1 alpha.

SHMT2 positive pseudopalisaded cells and SOX2 positive pluripotent cells were also observed, indicative of the presence of aggressive pathophysiological features of solid cancer. The hypoxic cancer-on-a-chip is a useful tool for investigating the pathophysiological characteristics of solid cancers and the dynamic crosstalk between cancer cells and tumorigenesis promoting microenvironment. As the methodology can be adapted for a patient-specific drug design in a reasonable timeframe, this approach is expected to bridge the gap between in vivo and in vitro models of cancer.