Automating a Magnetic 3D Spheroid Model Technology for High-Throughput Screening

Abstract

Affordable and physiologically relevant three-dimensional (3D) cell-based assays used in high-throughput screening (HTS) are on the rise in early drug discovery. These technologies have been aided by the recent adaptation of novel microplate treatments and spheroid culturing techniques. One such technology involves the use of nanoparticle (NanoShuttle-PL) labeled cells and custom magnetic drives to assist in cell aggregation to ensure rapid 3D structure formation after the cells have been dispensed into microtiter plates. Transitioning this technology from a low-throughput manual benchtop application, as previously published by our lab, into a robotically enabled format achieves orders of magnitude greater throughput but required the development of specialized support hardware. This effort included in-house development, fabrication, and testing of ancillary devices that assist robotic handing and high-precision placement of microtiter plates into an incubator embedded with magnetic drives. Utilizing a "rapid prototyping" approach facilitated by cloud-based computer-aided design software, we built the necessary components using hobby-grade 3D printers with turnaround times that rival those of traditional manufacturing/development practices at a substantially reduced cost. This approach culminated in a first-in-class HTS-compatible 3D system in which we have coupled 3D bioprinting to a fully automated HTS robotic platform utilizing our novel magnetic incubator shelf assemblies.

Keywords: 3D printing; HTS; magnetic bioprinting; organoid; spheroid.

Figure 1.

(A) 384-well n3D Bioscience magnetic spheroid drive. (B) 384-well n3D Bioscience magnetic spheroid drive with Greiner ULA surface treated microtiter plate (side view). (C) 384-well n3D Bioscience magnetic spheroid drive with Greiner ULA surface treated microtiter plate (top view). (D) OEM GNF incubator shelf. (E) First-generation spheroid drive adapter shelf. (F) First-generation spheroid drive adapter shelf with 384-well n3D Bioscience magnetic drive installed.

Figure 2.

(A) Second-generation HTS incubator 384-well alignment jig. (B) Second-generation 384-well spheroid drive. (C) Second-generation HTS incubator 1536-well alignment jig. (D) Second-generation 1536-well spheroid drive shown partially loaded with magnets.

Figure 3.

(A) Third-generation n3D Bioscience drive with 3D printed adapter wings installed. (B) Third-generation HTS incubator 1536-well spheroid drive with wings uninstalled and alignment bracket for y-axis positioning of wings.

Figure 4.

GNF shelf stack installed into GNF robotic carousel.

Figure 5.

(A) Scatterplot of plate statistics. (B) Randomized scatterplot of just the controls form the 150K n3D-KRAS pilot assay. The bottom series of data points near the 0% response point on the chart are low control wells containing cells and DMSO, while the top data points near the 100% response point are high control wells containing medium and DMSO. (C) Randomized scatterplot of the 150K n3D-KRAS pilot assay data shown as normalized activity per the controls shown in panel B. Each sample tested is represented by a single black dot.

Figure 6.

(A) Concentration-response curves for 3 control compounds (doxorubicin, 5-fluorouracil and sn-38) vs. hT1 in 3D formats. CRC results in offline, non-robotic validation run. (B) CRC results in online robotic validation run. The curve represents the mean and standard deviation of 16 replicates per each concentration. (C) CRC results of hT1–2D cells without NanoShuttle in an offline, non-robotic run. (D) CRC results of hT1–2D cells with NanoShuttle in an offline, non-robotic run. All CRC data are shown in Table 1.