Bioprinting technology has grown along with stem cell research and has become a vital tool with diverse applications in the biological and medical fields. In vitro 3D cell culture techniques create an accurate in vitro environment and provide an alternative to in vivo models used for fundamental cell biology and physiology research. However, these applications are currently hampered by organoid variability, low throughput, and limited scale. Although bioprinting technology is expected to be applied in regenerative medicine and drug discovery, there are enduring concerns regarding the impact of cell dispensers on cell integrity.
Organoid Fragments
The Corning™ Matribot™ bioprinter exhibited cell-friendly dispensing performance similar to that of the manual dispensing operation. The cultures dispensed by the Corning™ Matribot™ bioprinter were suitable for further molecular and protein analysis as well as downstream applications including high throughput drug screening.
The Corning Matrigel™ matrix was pre-thawed and aliquoted according to the manual. All the tips and syringes used for handling spheroids or organoids were pre-chilled. The media were equilibrated to room temperature (15°C to 25°C) before use. A549 and MDCK cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10 percent fetal bovine serum (FBS) until they reached 90 percent confluence.
The medium was removed by aspiration and the cells were washed with phosphate-buffered saline (PBS) and dissociated into single cells using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). An equal volume of complete medium was then added to the cells and mixed. The suspension was centrifuged at 300 x g for 3 min, the supernatant was discarded, and the pellet was resuspended in undiluted Corning Matrigel™ matrix.
An equal volume of complete medium (DMEM + 10 percent FBS) was then added to the cells and mixed. The suspension was centrifuged at 300 x g for 3 min, the supernatant was discarded, and the pellet was resuspended in undiluted Corning Matrigel™ matrix. The final cell density was adjusted to 50cells/μL and the mixture of the Matrigel™ matrix and cells were transferred to a syringe, inserted into the printhead of the Corning Matribot™ bioprinter, and dispensed using the parameters listed in Table 1. Manual dispensing was performed using a single-channel pipette. The plates were incubated at 37°C for 15 min., followed by the addition of complete medium (1mL) to each well. The plates were incubated at 37°C under 5 percent CO2, and the medium was changed every 2 to 3 days.
"We demonstrated that 3D cultures dispensed by using the Corning™ Matribot™ bioprinter were comparable with those obtained via manual operation. Both methods had brought similar morphology, component cell types, and gene expression profile."
Mouse intestinal organoids (MIOs) were thawed, cultured, and passaged in complete IntestiCult™ organoid growth medium, according to the manufacturer’s instructions, until a density of approximately 150 organoids/well was achieved. A pre-wetted 1,000μL pipet was used to break up the organoids by pipetting up and down 20 times, followed by centrifugation at 290 x g for 5 min at 4°C. The supernatant was carefully discarded and the pellet was resuspended in undiluted Matrigel™ matrix by pipetting up and down 10 times. The mixture of Matrigel™ matrix and MIO suspension was transferred to a syringe, inserted into the printhead of the Corning Matribot™ bioprinter, and dispensed. Manual dispensing was performed using a single-channel pipette. The plates were incubated at 37°C for 15 min, followed by the addition of complete IntestiCult™ organoid growth medium (1 mL) to each well. The medium was changed three times per week.
Results and Discussion
The morphology of A549 spheroids generated from bioprinter-dispensed and manually dispensed samples seemed to have normal shapes. After culturing for 7 days, the bioprinter-dispensed and manually dispensed MDCK spheroids showed obvious and typical cystic structures. Complex, multilobed MIO structures were also observed after 5 days of culture. Moreover, the densities of the formed 3D cultures were comparable between the bioprinter-dispensed and manually dispensed samples.
Immunofluorescence staining was performed to investigate the presence of specific cell type markers in the 3D cultures. Significant junctional F-actin was observed at the cell-cell contact points within the A549 spheroids, indicating that stable and strong intercellular adhesive interactions were formed. Moreover, 3D cultures of A549 cells showed bright and continuous E-cadherin labeling on the cell surface and at cell-cell contact sites. Hence, immunofluorescence staining confirmed that F-actin and E-cadherin were highly expressed in A549 spheroids generated by both bioprinting and manual dispensing.
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