Methods

VitroGel and cell suspension for spheroids

4IC cells¹ were cultured with advanced DMEM supplemented with glucose, NEAA, 2mM glutamine, and insulin 120μg/L, 10% FBS (Gibco 12491-015). For assays, spheroids were cultured with DMEM + 10% dialyzed serum (2mM glutamine, 5mM glucose, without phenol red).

The patient-derived triple-negative breast cancer cells were harvested using trypsin and suspended in a medium with 30% FBS at a concentration of 5 X 10⁵ cells/mL. The 3D cell culture was performed in VitroGel Hydrogel Matrix (SKU: VHM01) with the following steps:

1. Bring the VitroGel Hydrogel Matrix to room temperature or warm up to 37°C.
2. Mix the hydrogel solution and the cell suspension at a 2:1 (v/v) ratio.
3. Add 10 μL of the hydrogel-cell mixture to each well of a 96-well plate (Non-Tissue Culture Treated)
4. Wait 10–15 minutes at room temperature for hydrogel formation. Do not disturb the hydrogel by tilting or shaking the well plate during the formation process.
5. Carefully add 90 μL cell culture medium on top of the hydrogel.
6. Place the well plate in an incubator and change the cover medium every other day.

BioAssemblyBot 400 for dispensing (pipette tool), media exchange, drug screening, and live and dead assays

The BAB400 provides a sterile environment with a HEPA air flow system. Spray and wipe the surfaces except for tools and any electronics and turn on the HEPA filter functionality at least an hour (overnight preferred) prior to use. Using BAB400’s human machine interface (HMI) and a joystick controller, different paths can be designed for various steps. The paths are set for each workflow desired using the console to record the coordinates of the reservoir, plate, tubes, trash, etc. The media and cell suspension in the VitroGel are prepared beforehand in the BAB400 and transferred to the reservoir.

Gripper (PnP tool) Sequence can be used to de-lid and lid the plates, and to move the plates to the ImageXpress Micro Confocal system for imaging once dispensing is completed using BAB400. BAB400 has the option of running multi-step processes without human interaction. Experiments can be turned into automated workflows by stitching together custom experimental protocols to build 3D cellular models. BioApps™ Maker is an easy-to-make and easy-to-automate solution that was used to automate a sequence of steps for independent dispensing, maintenance, and imaging of the 3D models.

For example, the hydrogel dispenses sequence steps involved are laid out:

Get tip → Pipette tool moves to the hydrogel reservoir → down (into the reservoir) → Mix Y number of times (aspirate followed by dispense at defined speed) → Move to the plate (left/right/directions – then well center) → Dispense (define volume and dispense speed) → Trash the tips (into a biohazard bag in BAB400). A similar path could be designed for media aspiration and dispense – the tip goes to the edge of the wells to keep the VitroGel domes intact. Media with select drugs and Live/dead stain addition can also be used for further treatments and imaging assays of the spheroids. Optional step: Precooling the temperature-controlled stage of BAB400 to 10°C for faster gelation of dispensed VitroGel droplets in the 96-well.

ImageXpress Micro Confocal Imaging and Analysis

After the BAB400 moves the microwell plates to the ImageXpress Micro Confocal imager (Molecular Devices), transmitted light (TL) or fluorescent images were acquired using MetaXpress® High-Content Image Acquisition & Analysis Software. Z-stack images for the spheroids were acquired with the 4X or 10X objectives using confocal mode. MetaXpress Image Analysis Software was used for all analyses. BioApps™ can pull up and execute the right preselected imaging acquisition and analysis files for a particular sequence or experiment.

Results

Multiple 96-well plates were plated with 4IC breast cancer cells in VitroGel domes were set up by both the BAB400 and manual dispenses. These were observed on day 3 using the Cyto3D® Live-Dead Assay Kit (TheWell Bioscience, SKU: BM01) stain for live and dead counts, and when analyzed by using imaging method as described above. Live-dead analysis was used for counting live and dead cells. Apparently, there wasn’t a significant difference in cell viability between cells plated manually or automatically. The BAB400 workflow had an average viability of 93%, whereas the manually dispensed well plates had a viability of 92.4% (Figure 2). Therefore, automated cell printing did not impact cell viability.

To test the workflow for drug treatment, the drugs Trametinib and Idarubicin were added to the multiwell plates on day 3 to make up a final concentration of 4μM and their effects were evaluated on day 5 after 48h of treatment. We observed a significant decrease in cell viability in both drug treatments when compared to the control (Figure 3). Cell viability was determined by imaging methods after staining with viability dyes as described above. The dead cells percentage in idarubicin 4μM was the highest with 97% of cells in spheroids dead, whereas in the case of trametinib it was 66.4% and the control was 7.5% (Figure 3).

Conclusion

The 3D cellular model workflow can be automated by integrating the BAB400 and ImageXpress Micro Confocal imager with an easy-to-handle and tunable ECM matrix (VitroGel) for 2D/3D cell culture, maintenance, and differentiation of 3D cellular models that can be used for compound screening and a variety of assays. This will effectively contribute to the ongoing basic, pharmacological, and biomedical research and product development by saving time and cost and reducing repetitive steps.

Acknowledgment

Grateful for the patient-derived cell lines from Dr. Matthew E. Burow; Associate Professor of Medicine and Tulane Cancer Center Program Member

Reference

1. Cromwell EF, Sirenko O, Nikolov E, Hammer M, Brock CK, Matossian MD, Alzoubi MS, Collins-Burow BM, Burow ME. Multifunctional profiling of triple-negative breast cancer patient-derived tumoroids for disease modeling, SLAS Discovery 2022, 27(3): 191–200., https://pubmed.ncbi.nlm.nih.gov/35124274/