Our protocol facilitates the printing of preformed 3D cellular structures such as breast epithelial spheroids enabling the rapid formation of biologically-relevant models and a variety of bio-inks. The main advantage of this technique is that it facilitates the bioprinting of 3D tumor spheroids onto a vascular network that can be used almost immediately for any experimental study. These 3D bioprinted tissue models provide a rapid in vitro platform that can be expanded for personalized precision medicine.
This highly versatile 3D bioprinting method can be applied to other physiological systems in which 3D interactions with the vasculature are important. For breast epithelial spheroid formation, first freeze 200 microliter pipette tips for 30 minutes and place growth factor reduced matrix solution on ice. When the tips have frozen, using one new ice cold pipette tip per well, add 30 microliters of the ice cold matrix solution onto the side and along the corners of each well of an eight-well chamber slide, adding a final drop to the center to ensure an even coating.
When all of the wells have been coated, incubate the slide at 37 degrees Celsius and 5%carbon dioxide for 15 to 20 minutes. While the matrix is polymerizing, resuspend MCF 10A cells in MCF 10A assay medium at a two times 10 to the five cells per milliliter concentration. At the end of the incubation, add 50 microliters of cells and 450 microliters of MCF 10A spheroid growth medium to each well and immediately return the slide to the incubator.
Every four days, tilt the chamber slide to a 45 degree angle to allow 200 microliters of supernatants to be replaced with fresh spheroid growth medium per well. For HUVEC network formation, prepare a matrix solution coated eight-well chamber slide as demonstrated and pre-stain a 10 centimeter dish of HUVECs with 10 microliters of red cell tracker die for 30 minutes in the cell culture incubator. At the end of the incubation, wash the endothelial cells with 10 milliliters of warm PBS before treating them with two milliliters of 0.05%trypsin EDTA.
After five minutes at 37 degrees Celsius and 5%carbon dioxide, neutralize the trypsin with five milliliters of endothelial growth medium two and transfer the cells to a 25 milliliter conical tube for centrifugation. Resuspend the pellet in five milliliters of serum-free endothelial basal medium two for counting and dilute the cells to a one times 10 to the six cells per milliliter concentration. Then add one times 10 to the five HUVECs to each well and place the plate in the cell culture incubator for six hours.
After five to eight days of culture, count the number of spheroids in each well in representative phase contrast microscopy images and use a 1, 000 microliter pipette tip with the last 0.5 centimeters of the tip removed to carefully collect all of the spheroids from each well of the eight-well chamber slide into a 50 microliter tube. Resuspend spheroids in an appropriate bio-ink at a 100 spheroids per 100 microliters of ink concentration and load the pooled spheroids into a 10 milliliter sterile syringe. Equip the syringe with a 25 gauge sterile needle and attach the syringe to the biodeposition system.
Remove the medium from each well of HUVEC network and extrude 100 microliters of breast epithelial spheroids into each of six wells of the HUVEC network chamber slide at a flow rate of one milliliter of spheroids per minute. Then add 400 microliters of MCF 10A spheroid growth medium to each well and place the co-cultures in the cell culture incubator for 24 to 96 hours. After five to eight days of culture as demonstrated, non-tumorigenic MCF 10A breast epithelial spheroids should appear round with a hollow center with Integrin alpha-6 polarized to the outer edge of the spheroid.
Highly invasive MDA-MB-231 breast cancer epithelial cells form irregular spheroids that may show cells migrating out of the spheroids if maintained in the Matrigel culture for too long. After six to eight hours of sparse serum-free culture as demonstrated, HUVEC networks can be imaged by phase contrast or confocal microscopy. After breast epithelial spheroid bioprinting onto the HUVEC networks, both the spheroids and the networks should maintain their original morphology for at least 24 hours.
The main thing to remember while attempting this protocol is to handle the spheroids carefully before printing to avoid clustering. Following bioprinting and co-culture, tumor cell viability, proliferation, migration, and other biochemical interactions with the endothelial network can be measured with and without a pharmacological treatment. we have demonstrated that drug testing can be initiated as early as two hours after co-culture bioprinting and have tested spheroid addition to endothelial networks with the anti-cancer drug paclitaxel.
