Reproducible Manufacturing of SPOT as a High-throughput Scaffold-based Culture Platform

Our research focuses on developing high-throughput, scaffold-supported platforms for robust, reproducible, patient-derived orgonomic cultures to improve drug screening assay workflow and help advanced disease modeling.

Automated liquid handlers, scaffold-supported hydrogel platforms, organ-on-a-chip technologies, and single-cell assays like flow cytometry and CYTOPs are some of the current available technologies.

Current challenges include preserving hydrogel integrity, overcoming gel meniscus for real-time imaging, and ensuring uniform cell distribution in long-term organoid-based assays.

Our protocol enables consistent, scaffold-supported hydrogel loading, minimizes meniscus issues, supports long-term organoid culture, and integrates smoothly with automated liquid handlers and high-content imaging system.

We are most excited about using our disease modeling platforms to explore disease progression mechanisms in cancer and the development of obesity.

[Instructor] To begin, take a PMMA pattern scaffold for either 96 or 384 SPOT. Trim the excess paper around the blue region of the scaffold, leaving a five millimeter white margin around the perimeter. Ensure the resulting sheet measures approximately 760 millimeters by 1,300 millimeters. To prepare a 96 SPOT scaffold for assembly, lay a piece of polyethylene film over a rigid acrylic support. Place this film-covered support onto the work surface and lay the scaffold on top of the polyethylene film. Align the edge of a ruler between the first two rows of the scaffold to avoid tracing into the wells. Place the tracing wheel at the bottom of the scaffold and roll it upward to the top, guided by the ruler. For a 384 SPOT scaffold, do not perform perforation. Next, lay the scaffold with the rough side facing up on a PDMS slab, ensuring rows run horizontally. Use the priming line to determine which side is rough, then smoothen the scaffold onto the slab to remove wrinkles or bubbles. Remove the protective layer from the non-engraved side of the double-sided tape. Slightly buckle the tape in the middle and align the middle two columns of the tape with the scaffold. Now, press the middle of the tape onto the scaffold to secure alignment. Smooth the tape outward from the center to affix it evenly. Using tweezers, gently lift the scaffold from the PDMS slab. Store the slab for future use. To attach the scaffold to the second well tape, flip the scaffold so the smooth, non-taped side faces up on the workbench. Remove the middle protective layer from the engraved side of the second tape to expose two central columns. Slightly buckle the middle of the second tape and align it to the middle columns of the scaffold, then press the tape center to fix it in place. If engraved tape is used, remove the remaining protective layers adjacent to the exposed center. Smoothen the tape, starting from the middle and moving outward. To attach the scaffold to the bottomless plate, remove the protective layer. Align the scaffold under the bottomless plate with a lid. Once aligned, pressed down its middle onto the plate. If using engraved tape, remove adjacent layers and smooth the scaffold onto the plate outward from the center. To attach the scaffold to the polycarbonate film, use tweezers to carefully loosen the protective layer from one side of the polycarbonate film, then slowly peel off the protective layer without causing any indentations in the film. Repeat the process for the second side, then set the film aside on the workbench. Peel the protective tape layer from the plate bottom and press the polycarbonate film onto the scaffold from the center outwards. Then firmly press the polycarbonate film onto the scaffold to ensure each well has a defined border. Using a precision knife, carefully cut off any excess material from the bottom of the plate. After full assembly, place the 96 SPOT plate into a clear, resealable plastic bag and take it outside the biosafety cabinet. Place a rigid acrylic support underneath the plate and clamp both at opposite corners. Store the plate in a clean, dry space until further use. No media leakage or exchange was observed when alternating wells in the 384 SPOT plate were filled with fluorescein and PBS during a 30-day test period, confirming the efficacy of the PMMA barrier in maintaining well-to-well isolation. GFP-expressing pancreatic tumor organoids seeded into 96 SPOT plates remained viable and expanded progressively from day zero through day 12 of culture. On day 12, SPOT-grown organoids displayed strong cytokeratin 19 and Zonula Occludens-1 expression, along with the presence of internal lumen structures. Suboptimal and optimal tissue seeding outcomes in 384 SPOT plates were visually distinguishable by edge definition and cell distribution uniformity. Consistently uniform seeding was achieved across the entire 96 SPOT and 384 SPOT plates using the automated liquid handler.