Organoids and spheroids are proving to be useful tools in drug target discovery and screening. The ability to scale up organoid production, culturing, and assaying is important in their widespread utility. Automation is key not only for increasing throughput, but also improving consistency and reducing the risk of human error during fabrication and use. We have previously demonstrated that, with the robotic BioAssembly™ Platform, adherent cells can be automatically passaged, cell suspensions can be dispensed into multiple well plates, and advanced 3D tissue models can be automatically fabricated, cultured, and assayed . Here, we further demonstrate an automated tumor spheroid workflow with the Platform. The workflow consists of forming domes of Matrigel® containing tumor spheroids into wells of a 96 well plate; incubating in an integrated, modular incubator (BioStorageBot™); performing daily culture medium changes; and performing daily imaging via an automated interface with the Molecular Devices ImageXpress® Confocal HT.ai High-Content Imaging System. In the analysis, we compared spheroid morphology in cultures managed by the automated system to those managed by expert, standard manual practices.
The BioAssembly™ Automation Platform
The BioAssembly™ Platform (Figure 1) consists of BioAssemblyBot® 400 (BAB400) by Advanced Solutions, containing a 6-axis robotic arm capable of utilizing a wide range of interchangeable bioprinting, fluid dispensing, material movement, and tissue fabrication tools. Additionally, our modular BioStorageBot™ incubator and the ImageXpress® Confocal HT.ai High-Content Imager from Molecular Devices were integrated to complete the manufacturing Platform for this application. For this specific spheroid workflow, the Platform employed a BioAssemblyBot Hand™ (BAB Hand™) | Pipette, a BAB Hand™ | Pick & Place, and modular tube, pipette tip, cooling, and pipetting stations. All these components and the operational tasks to complete the workflow, including automated communication between the different instruments, are controlled by our user-friendly BioApps™ software, a software platform that connects, controls, and instructs the Platform to perform each step in a workflow.
For this automated workflow (Figure 2), tumor spheroids were pre-formed manually by plating HCT-116 colorectal cancer cells (ATCC) into a non-adherent v-bottom plate at a concentration of 2,000 cells per well. After 24 hours, spheroids were suspended in 80% Matrigel® (a tumor cell-derived extracellular matrix frequently used to support spheroid cultures by providing a 3D environment and biological cues). 96 spheroids per 250 µl of cold Matrigel® was suspended in a sterile tube and placed within the cooling station within the Platform.
Using the BAB Hand™ | Pipette, BAB400 aspirated from the chilled tube and then dispensed 5 µl of the spheroid/Matrigel suspension into the center of wells of a 96 well plate to form discernable domes on the bottom of each well. Only the central wells of the plate were used to avoid known edge effects of the outer wells (the outer wells were filled with buffer). Successful operations consisted of forming discernable domes that had not spread out into a thin disc on the well bottom (Figure 3). From this, 81.25% of wells contained discernable domes when dispensed by BAB400, compared to 72.0% when manually performed by an experienced scientist.
After dispensing, a 10-minute wait period allowed the Matrigel® domes to gel before 50 µl of culture medium was added to each well followed by transfer to the BioStorageBot™ for culturing. BAB400 dispensed domes and added medium in 10’ 40” as compared to 15’ for the same number of domes when performed manually. Each day, as managed by the tumor-spheroid BioApp™, the BioAssembly™ Platform automatically performed culture medium exchanges and took transmitted light image acquisitions via the ImageXpress® Confocal HT.ai High-Content Imager (Figure 4). For medium exchanges, the plate is transferred to a modular tilt stage within the Platform that holds the well plate at an angle, analogous to someone holding the plate at a tilt when manually pipetting, allowing for more complete culture medium removal from each well without disturbing the Matrigel dome in the center.
From the daily images, the average size of individual spheroids was determined to be approximately half the diameter of those seeded manually as compared to those plated by BAB400 (the day 0 values in Figure 5). It was also noted that an average of 6.2 ± 2.8 spheroids were present in each dome formed manually in contrast to 2.0 ± 1.2 spheroids per dome formed by BAB400, the expected number based on seeding density. Combined, this suggests that the spheroids were fragmented by manual operations, perhaps via inconsistent pipetting rates, but not by the BioAssembly™ Platform. Over time, spheroids in both groups steadily increased their overall diameter (Figure 5).
Calculation of spheroid growth rates indicated that individual spheroids increased in size at similar rates for both workflows (Figure 6).
To assess organoid viability, ATP production (CellTiter Glo) was measured in spheroids cultured for three days by either the automation system or an experienced user. A viability index was calculated from luminesce values for each well, which were normalized to both spheroid number per well and average spheroid size (diameter) within that well (Figure 7). While there was an apparent higher viability index for the spheroids managed automatically as compared to those managed manually, this difference was not statistically significant.
Summary and Discussion
Here, we demonstrate an automated workflow in which cultures of colorectal cancer spheroids are established, maintained, and imaged autonomously via the BioAssembly™ Platform. The workflow entails all requisite operations, including the formation of Matrigel® domes housing the spheroids and interfacing with peripheral instruments (a BioStorageBot™ incubator and an ImageXpress® confocal scanner by Molecular Devices in this specific use case). Importantly, spheroid domes were not damaged during the workflow, including with daily media changes, resulting in consistently obtaining expected spheroid sizes and numbers per well.
At the heart of the automation Platform is the BioAssemblyBot® 400, a 6-axis robotic arm used routinely in high precision manufacturing, housed within a framework uniquely suited for the fabrication, manipulation, and manufacturing of living systems, including spheroid/organoid and advanced 3D tissue models. Via a universal interface, the arm can utilize a wide spectrum of end effector tools (BAB Hands™), to perform varied tasks and operations, depending on the application. This functional flexibility, managed by the user via an intuitive software interface called BioApps™, affords the user considerable freedom in performing automated or semi-automated workflows, regardless of complexity. While this current application involved plate management (to multiple operational locations/stations) and precise pipetting, other applications have included 3D bioprinting, cell patterning, and fugitive molding, for example.
The combined tasks leading to a prepared plate of spheroid domes including dispensing domes, gelation (10’ wait), dispensing media to domes, dispensing buffer to empty outer wells, and transfer to the BioStorageBot™ incubator took 26 min. 46 sec. The daily tasks of moving the plate of domes to and from the BioStorageBot™ for media exchanges and moving to and from the ImageXpress® scanner took 13 min. 40 sec. (not including scan time) for a total of 40 min 26 sec. When performed manually, this entire workflow took 41 min 24 sec (29 min + 12 min 24 sec). In addition to the time savings afforded by the automation, spheroids were not damaged, the expected number and size of spheroids per dome was attained, and there was improved consistency in dome formation.