Name: Reproducible Manufacturing of SPOT as a High-throughput Scaffold-based Culture Platform (Video) | JoVE Journal
Uploaded: 2025-07-29T13:00:00.000+00:00
Duration: 6 min 22 s
Description: Learn reproducible manufacturing of SPOT, a high-throughput scaffold-based culture platform for organoid drug screening. This protocol enables automated liquid handling and improves disease modeling workflows.

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5 minutes

The authors acknowledge help from Jennifer Lam in image acquisition. Figure 1, Figure 2 and Figure 7A were created with BioRender.com. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant (grant #RGPIN-2021-03488) to A.P.M.; the Canada First Research Excellence (CFREF) Medicine by Design Grand Questions program to A.P.M.; the NSERC Canada Graduate Scholarships - Doctoral program to R.C.; the Peterborough K.M. Hunter Charitable Foundation Award to R.C.; the National Research Council (NRC) CRAFT Fellowship awarded to N.T.L.; the NSERC CREATE TOeP to N.T.L.; the NSERC Undergraduate Student Research Award to Z. K.; and the University of Toronto Excellence Award to C.M.T.

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</ol>

The authors declare no conflict of interest.

In this protocol, a detailed guide for the fabrication and use of 96- and 384-well SPOT is provided, covering assembly, manual, and liquid-handler-assisted cell-gel seeding. Unlike traditional hydrogel plug systems, SPOT eliminates meniscus-related artifacts, facilitating non-destructive, in situ imaging of organoids as described previously14,16,22. Additionally, the scaffold provides structural support, ensuring long-t...

Despite advancements in biomedical research, developing physiologically relevant in vitro assays remains challenging. An ideal assay should accommodate a diverse range of cell types, treatment formulations, timescales, commercial instrumentation, and analytical readouts to meet the needs of researchers across various disciplines. However, due to the lack of suitable tools, researchers often rely on simplistic models, such as 2D monolayers of immortalized cell lines, which fail to recapitulate the complexity and clinical relevance, particularly in cancer research1,2. To address this, large-scale tumor biobanking and clinician collaboration have driven the development of high-fidelity in vitro models such as patient-derived tumor organoids (PDOs), which retain more patient-specific genetic and therapeutic profiles compared to cell lines3,4,5. Meanwhile, advancements in laboratory automation and drug discovery demand platforms that integrate seamlessly into high-throughput workflows. While existing platforms address either biological fidelity or scalability, few have been able to resolve both challenges simultaneously6,7,8. Developing automated, high-throughput models that replicate key disease features is crucial for advancing biomedical research and accelerating data generation for translational applications.
Numerous emerging models aim to address the challenges associated with culturing specialized cell types, such as PDOs, with high throughput (Figure 1). Standard platforms, where organoids are mixed with hydrogel in 96- or 384-well plates, present several limitations: (i) high cell-gel volume leads to inconsistent nutrient access, (ii) well meniscus curvature creates imaging artifacts and cellular gradients, (iii) hydrogel seeding lacks uniformity across batches and personnel, (iv) incompatibility with automated fabrication and automated microscopy, and (v) the use of extracellular matrix (ECM) that is typically soft and fragile risks sample destruction at physiological tissue densities or incorporation of contractile cell types1,9. Currently, the majority of plate-based PDO studies are conducted either by manually seeding the cell-gel into the plate10,11, or a gel-overlay method, where the hydrogel is diluted with a less viscous medium, and cells are centrifuged to the well bottom to place them in the appropriate optical plane12,13. Both approaches fail to provide mechanical support for long-term culture. Based on our previous observations (unpublished data), commercial hydrogel mixed with cells in standard well plates contracts from the surfaces of the well for 9 out of 12 wells over 12 days, while the gel-overlay method lacks physiological cell-matrix interactions. In addition, the resulting cultures cannot be maintained over long periods due to space limitations within the well and are incompatible with assays that require a high reagent-to-cell-medium ratio14,15,16. These practical barriers hinder the adoption of 3D models and limit experimental reproducibility across the field.
<img alt="Cancer research scalability diagram; models include organoids, lab-on-chip, and animal for throughput." class="xfigimg" src="/files/ftp_upload/68405/68405fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Assessment of culture models based on scalability and biological relevance. The relative positioning of various in vitro and in vivo culture systems along scalability (y-axis), defined by plate throughput, and biological relevance (x-axis), highlighting trade-offs between throughput potential and physiological fidelity. <a href="https://www.jove.com/files/ftp_upload/68405/68405fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Motivated by these challenges, the high porosity and mechanical reinforcement of a thin cellulose scaffold were leveraged to develop the Scaffold-supported Platform for Organoid-based Tissues (SPOT), available in both 96- and 384-well formats. SPOT incorporates a polymer pre-patterned scaffold using a previously developed infiltration method17 that matches the plate layout, enabling precise cell-gel droplet placement and providing structural support for the hydrogel phase (Figure 2A). Capillary-wicking into the scaffold guides the cell-gel solution, forming a mechanically enforced biocomposite network (Figure 2B). Due to the porous structure of the scaffold, the cell-gel infiltrates and forms a thin layer (approximately 70 µm thick), requiring ten-fold less amount of cell-gel to cover the bottom of the well14. The compatibility of SPOT with seeding pancreatic ductal adenocarcinoma (PDAC) PDOs was also demonstrated by assessing their growth using metabolic assays and image-based measurements. Since its development, SPOT has demonstrated significant versatility and usability for researchers engaged in organoid culture. Additionally, SPOT's design accommodates user-friendly scalability, with reduced reagent volumes and improved imaging capabilities through its flat, meniscus-free microgel geometry, ensuring uniform light penetration and accurate high-content imaging14 (Figure 2C,D). SPOT also supports co-culture with other relevant cell types, such as fibroblasts, to better recapitulate the tumor microenvironment18.
<img alt="Device fabrication: multi-layer well-plate, cell seeding, microscopy, fluorescence analysis diagram." class="xfigimg" src="/files/ftp_upload/68405/68405fig2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Overview of the 96-SPOT device and image analysis workflow. (A) Schematic representation of SPOT plate fabrication: the device is assembled by collating a commercial bottomless well plate (top), PMMA-patterned scaffold (shown in blue), and polycarbonate film (bottom) using layers of double-sided polyacrylic adhesive (shown in yellow). (B) Cell seeding into scaffold well: cells are suspended in a non-polymerized hydrogel (shown in pink) and pipetted into each well where it infiltrates the scaffold. The hydrogel is then allowed to gel to encapsulate the cells within the scaffold-supported hydrogel. (C) Microscopy: the resulting microtissue can be imaged using automated widefield microscopy. (D) Image-based analysis: Acquired images of fluorescent cells (green) suspended in gel can be analyzed using standard image-analysis pipelines. Representative epifluorescence microscopy image of GFP-expressing KP4 cells in one 96-SPOT well. Scale bar is 500 µm. This figure has been modified from14. <a href="https://www.jove.com/files/ftp_upload/68405/68405fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Another advantage of SPOT is that the system can be integrated with an open-source liquid handler, facilitating automated, reproducible fabrication of PDO cultures. This advancement reduces user variability and supports complex experimental designs, including high-throughput drug screening and personalized medicine approaches18. SPOT was also previously shown to be compatible with single-cell-based multiplexed characterization of cellular phenotypes in heterogeneous tumor environments and retrieval of the cells on demand from SPOT by digestion18. These features position SPOT as a versatile tool for screening applications, where longitudinal, high-resolution data are essential for evaluating drug efficacy and cellular behavior under physiologically relevant conditions14,18. By combining these technical innovations, SPOT addresses critical limitations in traditional 3D culture systems.
This protocol provides a comprehensive guide for fabricating SPOT components, assembling plates, and seeding methods for robust and reproducible 3D organoid cultures. It includes detailed instructions for manual plate seeding using a micropipette and automated seeding workflows with a liquid handler, which supports both 96- and 384-well formats. Additionally, this protocol outlines essential quality control measures, such as verifying uniform gel distribution and scaffold integrity, to maintain high experimental standards. Troubleshooting strategies are also provided to address common issues, such as incomplete gel infiltration, evaporation during seeding, and scaffold detachment, equipping researchers with practical solutions to optimize their workflows and achieve reliable outcomes.
SPOT is designed to fill the gaps that traditional organoid assays face, including challenges related to performing assays and media change while preserving gel integrity10,11,12,13. The overall goal of this method is to manufacture a scaffold-supported platform that supports organoid culture over a 12-day period. Additionally, this method also covers generating tissue arrays in SPOT through either manual or liquid handler-assisted seeding to allow users to perform any downstream perturbation, including drug IC50 assays and small molecule screening.

<table><tbody><tr><td>0.2mL PCR tubes (strips of 8)&amp;nbsp;</td><td>Sarstedt</td><td>72.985.002</td><td></td></tr><tr><td>1.5mL centrifuge tube</td><td>FroggaBio</td><td>LMCT1.7B</td><td></td></tr><tr><td>1000-&amp;mu;L pipette tips</td><td>Eclipse</td><td>4-1019-260-000</td><td></td></tr><tr><td>10-mL serological pipettes</td><td>FroggaBio</td><td>394010</td><td></td></tr><tr><td>10-mL syringe</td><td>BD, Canada</td><td>302995</td><td></td></tr><tr><td>10x Minimal essential medium (MEM 10x)</td><td>Gibco</td><td>11430030</td><td></td></tr><tr><td>150-mm dish</td><td>Corning</td><td>430599</td><td></td></tr><tr><td>16-gauge dispensing tips with Luer lock connection</td><td>McMaster-Carr, Illinois, USA</td><td>6699A2</td><td></td></tr><tr><td>20&amp;micro;L filter tips racks</td><td>Opentrons</td><td>999-00099</td><td></td></tr><tr><td>200&amp;micro;L filter tips racks</td><td>Opentrons</td><td>999-00081</td><td></td></tr><tr><td>200-&amp;mu;L pipette tips</td><td>Eclipse</td><td>4-1030-260-000</td><td></td></tr><tr><td>20-gauge dispensing tips with Luer lock connection</td><td>McMaster-Carr, Illinois, USA</td><td>6699A4</td><td></td></tr><tr><td>384-well no-bottom well plate (black)</td><td>Greiner</td><td>82051-544</td><td></td></tr><tr><td>3-mL syringe&amp;nbsp;</td><td>BD, Canada</td><td>309657</td><td></td></tr><tr><td>50-mL polypropylene conical centrifuge tube</td><td>VWR</td><td>CA21008-940</td><td></td></tr><tr><td>50-mL polypropylene conical centrifuge tube</td><td>VWR</td><td>CA21008-940</td><td></td></tr><tr><td>96-well no-bottom well plate (black)</td><td>Greiner</td><td>82050-714</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A18-1</td><td></td></tr><tr><td>Analytical balance</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>AxiDraw extension for Inkscape (https://wiki.evilmadscientist.com/Axidraw_Software_Installation)</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>AxiDraw Python API (https://axidraw.com/doc/py_api/#installation)&amp;nbsp;</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>AxiDraw V3 High Performance Personal Writing and Drawing Machine</td><td>Evil Mad Scientists, USA</td><td>N/A</td><td></td></tr><tr><td>Bovine collagen (PureCol 3 mg/ml)</td><td>Advanced BioMatrix</td><td>5005</td><td></td></tr><tr><td>Cell line of choice</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Clip Easel (included in AxiDraw V3 package)</td><td>Evil Mad Scientists, USA</td><td>N/A</td><td></td></tr><tr><td>Culture media of choice</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Dirt-Trapping Protection Tape</td><td>3M</td><td>6851 PN36851</td><td></td></tr><tr><td>Double-sided polyacrylic adhesive tape (800 mm x 1200 mm)</td><td>Adhesive Research, ARcare</td><td>90106NB</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>12483-020</td><td></td></tr><tr><td>Finum tea filters XL&amp;nbsp;</td><td>Riensch &amp;amp; Held GmbH &amp;amp; Co. KG, Hamburg, Germany</td><td>420.69.00</td><td></td></tr><tr><td>Flat caps for 0.2mL PCR tubes (strips of 8)</td><td>Sarstedt</td><td>65.989.002</td><td></td></tr><tr><td>Heavy duty plastic spring clamps</td><td>BENCHMARK</td><td>1024-554</td><td></td></tr><tr><td>HEPA module &amp;ndash; 110-130V 60Hz model</td><td>Opentrons</td><td>999-00109</td><td></td></tr><tr><td>Ice pack (frozen, 1400 mm x 1800 mm)</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Inkscape software</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Laser cutter</td><td>Universal Laser System</td><td>VLS3.75</td><td></td></tr><tr><td>Miniature ethyl vinyl acetate (EVA) tubing of 0.07-inch outer diameter</td><td>McMaster-Carr, Illinois, USA</td><td>1883T3</td><td></td></tr><tr><td>Multi-Channel electronic pipette P20 (Gen2)</td><td>Opentrons</td><td>999-00005</td><td></td></tr><tr><td>Multi-Channel electronic pipette P300 (Gen2)</td><td>Opentrons</td><td>999-00006</td><td></td></tr><tr><td>Nail polish</td><td>Sally Hansen</td><td>SBS-006861</td><td>Optional, for pigmentation of PMMA ink</td></tr><tr><td>Opentrons OT-2</td><td>Opentrons</td><td>999-00111</td><td></td></tr><tr><td>P1000 pipette</td><td>Gilson</td><td>FA10006M</td><td></td></tr><tr><td>P200 8-channel pipette</td><td>Gilson</td><td>F144072</td><td></td></tr><tr><td>P200 pipette</td><td>Gilson</td><td>FA10005M</td><td></td></tr><tr><td>Paper towels</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Parafilm</td><td>Bemis Company, Inc.</td><td>52859-079</td><td></td></tr><tr><td>Parchment paper&amp;nbsp;&amp;nbsp;</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Pasteur pipettes</td><td>VWR</td><td>14672-380</td><td></td></tr><tr><td>Penicillin-streptomycin (P/S)</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Poly(methyl methacrylate) (PMMA)</td><td>Sigma-Aldrich</td><td>182230</td><td></td></tr><tr><td>Polycarbonate film (760 mm x 1300 mm)</td><td>McMaster-Carr</td><td>85585K102</td><td></td></tr><tr><td>Polydimethylsiloxane monomer and cross-linker (PDMS, 1500 mm x 2700 mm)</td><td>Ellsworth Adhesives</td><td>4019862</td><td></td></tr><tr><td>Precision knife</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Rigid acrylic support (size fitted to bottom of well plate)</td><td>McMaster-Carr</td><td>8560K239</td><td></td></tr><tr><td>Rigid cardboard support</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Ruler</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Sodium bicarbonate (0.8M in distilled water)</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>Temperature deck with aluminum blocks (Gen2)</td><td>Opentrons</td><td>999-00097</td><td></td></tr><tr><td>Tracing wheel&amp;nbsp;</td><td>HEEPDD (Amazon)</td><td>B07YQWB284</td><td></td></tr><tr><td>Transparent tape&amp;nbsp;</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Trypan blue</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>Tweezers</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Vortex mixer&amp;nbsp;</td><td>N/A</td><td>N/A</td><td></td></tr></tbody></table>

reproducible manufacturing of spot as a high-throughput scaffold-based culture platform

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

Patient-derived organoids (PDOs) are becoming increasingly used in the field of cancer research to model tumors. They combine the benefits of in vivo and traditional in vitro models, recapitulating tissue complexity and heterogeneity while still consisting of human cells. The rise of physiologically representative PDO models has prompted a need for devices that enable straightforward analysis of organoid behavior. This protocol explains how to assemble and use the Scaffold-supported Platform for Organoid-based Tissues (SPOT), a high-throughput, three-dimensional (3D) organoid culture device. This platform eliminates the meniscus typically formed by seeded hydrogels to allow accurate, quick imaging, similar to the imaging process for two-dimensional (2D) cultures. As a result, organoids and co-cultures seeded in SPOT can easily be imaged using high-throughput microscopy. Designed for off-the-shelf use, SPOT is accessible to researchers with basic tissue culture experience and is compatible with both manual and automated seeding workflows. This protocol details the fabrication of SPOT in 96- and 384-well formats, including step-by-step instructions for assembly, seeding, quality control, and troubleshooting. While fabrication requires 3-4 h, excluding idle time, multiple plates can be produced simultaneously, improving scalability. By streamlining PDO culture and analysis, SPOT provides a robust tool for high-throughput drug screening and translational cancer research.

PMMA-patterning of the paper scaffold serves as a physical barrier to prevent content exchange across wells. This design is critical for ensuring that adjacent wells remain chemically and biologically isolated, preventing unintended diffusion of media, drugs, secreted factors, or even cells14. The incorporation of PMMA allows for precise compartmentalization, making SPOT a reliable platform for high-throughput screening and co-culture studies where cross-contamination must be minimized<sup class="...

This protocol documents the fabrication and assembly of SPOT, a high-throughput scaffold-based culture platform, in 96- and 384-well formats. It also details the seeding process for SPOT, both manual and using a commercially available liquid handler.

Patient-derived organoids (PDOs) are becoming increasingly used in the field of cancer research to model tumors. They combine the benefits of in vivo and ...

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Research

JoVE Journal

Bioengineering

In the past many in vitro culture systems --  mainly monolayer cultures  -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

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A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

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Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

A High-Throughput Platform for Culture and 3D Imaging of Organoids

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

This protocol presents a standard high-throughput screening workflow for industrial microorganisms. It involves constructing a mutant library via ARTP mutagenesis, performing positive droplet screening with automated microfluidics, and subsequently verifying the results. The workflow’s reliability was validated by screening high-yield B. coagulans strains.

High-Throughput Screening of L-Lactic Acid-Hyperproducing Bacillus coagulans Mutants Using an Automated Droplet Microfluidic Platform

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Learn reproducible manufacturing of SPOT, a high-throughput scaffold-based culture platform for organoid drug screening. This protocol enables automated liquid handling and improves disease modeling workflows.

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

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