3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

We offer a mild bioprinted process for preparing microcarriers with a good controllability and biocompatibility. After encapsulation by cells, the carriers can also be used in in vitro cell expansion and functional microtissue construction. Limited displacement, rather than severe forces under similar conditions act on the nozzle during the printing process, maintaining the original physical/chemical properties of the bio-ink to the maximum extent.

In addition to conventional microcarriers, units with internal cell distribution or tritosin-encapsulated capsule structures can be constructed and applied to the assembly of different tissue structures. For the nozzle preparation, load glass micropipettes onto the puller according to the manufacturer's instructions and set the pulling parameters for the puller. Use a microforge to cut off the nozzle at the designated diameter to obtain the specific tip diameter for the experiment, and sterilize the nozzle in alcohol for five minutes.

Then, rinse the nozzle three times with sterile water to remove any residual alcohol. To prepare the hydrogel bio-ink, dilute sterilized 4%sodium alginate stock solution in 0.9%sodium chloride at 0.5, 1, 1.5, and 2%weight-by-volume concentrations. For microdroplet formation, load five milliliters of bio-ink into a disposable sterile syringe and install the syringe onto a syringe pump.

Using a one-millimeter inner-diameter hose, connect the syringe and printing nozzle. Tighten the clamping screw to secure the nozzle into place and rapidly depress the syringe pump to load the bio-ink into the nozzle. Set the signal generator parameters and preset the motion path and trigger mode of the vibration to drop on demand.

Then print the droplets following the pre-designed patterns. For microcarriers formation, add five milliliters of crosslinking solution to a Petri dish, and place the dish under the printing nozzle as the substrate. After printing, crosslink the microcarriers for three minutes before transferring the microcarrier suspension to a centrifuge tube.

Then, enrich the microcarriers by centrifugation and resuspend them in an appropriate culture medium at approximately 600 microcarriers per milliliter. Before their inoculation, replace the supernatant of an A549 cell culture with five milliliters of serum-free medium supplemented with 10-micromolar cell tracker green CMFDA dye for a 30-minute incubation in the cell culture incubator. At the end of the incubation, replace the dye solution with fresh medium and resuspend the cells at a density of 1.6 times 10 to the six cells per milliliter of medium.

Add one milliliter of microcarrier suspension to the cells and one milliliter of A549 cells to each well of a low-adherent six-well culture plate. Place the plate onto a shaker in the cell culture incubator at 30 revolutions per minute. At the end of the incubation, remove the plate from the shaker and allow the microcarriers to settle for 30 minutes before observing and measuring the printing nozzle, Petri dishes, and microcarrier-inoculated cells by bright-field and confocal fluorescence microscopy.

Using a 30-micrometer-tip nozzle, multiple types of bio-ink, including PBS, 1.5%alginate, and 1.5%gelatin can be stably printed onto Petri dishes. As the inks increase in viscosity, the printing process becomes increasingly more difficult and greater driving forces are needed to obtain larger droplets. The printing parameters can be set to print the droplets in specific 2D arrangements, as observed in this printing experiment.

The diameter of the nozzle tip has a direct impact of the size of microcarrier that can be printed. As observed in these bright-field and confocal images, A549 cells adhere to the alginate-collagen microcarriers after two days of culture. After six days, the A549 cells almost fully cover the microcarrier surfaces.

Following this procedure, we have coated the microcarrier surface with chitosan, and used gluconate to dissolve the alginate gel to form a cystic structure that can be applied to construct hollow structures such as alveoli. The printing process for preparing microcarriers is quite gentle, thus, this method can be widely used for printing cell-containing microcarriers.