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The CellRaft AIR system: A novel system enabling organoid imaging, identification, and isolation

Open AccessPublished:December 04, 2021DOI:https://doi.org/10.1016/j.slasd.2021.11.003

      Abstract

      Three-dimensional (3D) culture systems have been developed that can re-capitulate organ level responses, simulate compound diffusion through complex structures, and assess cellular heterogeneity of tissues, making them attractive models for advanced in vitro research and discovery. Organoids are a unique subtype of 3D cell culture that are grown from stem cells, are self-organizing, and closely replicate in vivo pathophysiology. Organoids have been used to understand tissue development, model diseases, test drug sensitivity and toxicity, and advance regenerative medicine. However, traditional organoid culture methods are inadequate because they are low throughput and ill-suited for single organoid imaging, phenotypic assessment, and isolation from heterogenous organoid populations. To address these bottlenecks, we have adapted our tissue culture consumable and instrumentation to enable automated imaging, identification, and isolation of individual organoids. Organoids grown on the 3D CytoSort Array can be reliably tracked, imaged, and phenotypically analyzed using brightfield and fluorescent microscopy as they grow over time, then released and transferred fully intact for use in downstream applications. Using mouse hepatic and pancreatic organoids, we have demonstrated the use of this technology for single-organoid imaging, clonal organoid generation, parent organoid subcloning, and single-organoid RNA extraction for downstream gene expression or transcriptomic analysis. The results validate the ability of the CellRaft AIR System to facilitate efficient, user-friendly, and automated workflows broadly applicable to organoid research by overcoming several pain points: 1) single organoid time-course imaging and phenotypic assessment, 2) establishment of single cell-derived organoids, and 3) isolation and retrieval of single organoids for downstream applications.

      Keywords

      Introduction

      For decades, two-dimensional (2D) cell culture models have been used to study disease and advance drug development, as cell lines are typically inexpensive and easy to culture, making them convenient for high-throughput analysis. However, establishing cell lines, which are often tumor-derived or immortalized, involves extensive genetic and phenotypic adaptation to the culture environment, which decreases their relevance to normal cells and ultimately reduces their applicability as model systems [
      • Schutgens F.
      • Clevers H.
      Human organoids: tools for understanding biology and treating diseases.
      ]. In addition, 2D models lack important spatial arrangement and cell-to-matrix interactions, further limiting their predictive power [
      • Fang Y.
      • Eglen R.M.
      Three-dimensional cell cultures in drug discovery and development.
      ,
      • Langhans S.A.
      Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.
      ]. This lack of translation of 2D cell culture models to in vivo outcomes significantly impacts the drug discovery pipeline, where the probability of success is only 13.8% [
      • Wong C.H.
      • Siah K.W.
      • Lo A.W.
      Estimation of clinical trial success rates and related parameters.
      ]. While 2D models continue to provide significant value in research and development programs, there is considerable need for more advanced cellular models that provide better physiological relevance to tissues and organs.
      There are several three-dimensional (3D) models that offer increased complexity, and subsequent physiological relevance, over 2D models, including co-cultures, spheroids, encapsulated cells, and organoids [
      • Andersen T.
      • Auk-Emblem P.
      • Dornish M.
      3D cell culture in alginate hydrogels.
      ,
      • Booij T.H.
      • Price L.S.
      • Danen E.H.J.
      3D cell-based assays for drug screens: challenges in imaging, image analysis, and high-content analysis.
      ]. Organoids are self-organizing three-dimensional (3D) structures that can be grown from embryonic stem cells, induced pluripotent stem cells (iPSCs), or adult stem cells from humans or animal models [
      • Clevers H.
      Modeling development and disease with organoids.
      ,
      • Jamieson P.R.
      • Dekkers J.F.
      • Rios A.C.
      • et al.
      Derivation of a robust mouse mammary organoid system for studying tissue dynamics.
      ]. Organoids have seemingly infinite expansion potential, and when the cells within the 3D structure are differentiated, they exhibit tissue-specific physiological and diseased states that make them a more relevant and attractive in vitro model than 2D single cell type monolayer cultures for developmental research, drug discovery, personalized medicine, and toxicological studies [
      • Schutgens F.
      • Clevers H.
      Human organoids: tools for understanding biology and treating diseases.
      ,
      • Fang Y.
      • Eglen R.M.
      Three-dimensional cell cultures in drug discovery and development.
      ,
      • Booij T.H.
      • Price L.S.
      • Danen E.H.J.
      3D cell-based assays for drug screens: challenges in imaging, image analysis, and high-content analysis.
      ,
      • Clevers H.
      Modeling development and disease with organoids.
      ,
      • Jamieson P.R.
      • Dekkers J.F.
      • Rios A.C.
      • et al.
      Derivation of a robust mouse mammary organoid system for studying tissue dynamics.
      ,
      • Takasato M.
      • Er P.X.
      • Chiu H.S.
      • et al.
      Kidney organoids from human IPS cells contain multiple lineages and model human nephrogenesis.
      ,
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      • Hoffmann K.
      • Brinkmann V.
      • et al.
      The notch and wnt pathways regulate stemness and differentiation in human fallopian tube organoids.
      ,
      • Broutier L.
      • Mastrogiovanni G.
      • Verstegen M.M.A.
      • et al.
      Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.
      ,
      • Sachs N.
      • Papaspyropoulos A.
      • Zomer-van Ommen D.D.
      • et al.
      Long-term expanding human airway organoids for disease modeling.
      ].
      For 3D assembly, organoids require a source of extracellular matrix (ECM) to serve as a basement membrane. There are several commercially available basement membrane extract products, with Matrigel (which will serve as a reference moving forward) being one of the most commonly used. Traditional culture methods of organoids involve cells propagated between layers of or embedded in Matrigel domes. These culture methods are highly effective in supporting organoid assembly and growth but present several challenges in accurate assessment and throughput. First, standard culture methods result in a random arrangement of organoids in all three dimensions, with multiple structures of varying shape, size, and viability per well that are frequently overlapping. This increases the number of focal planes needed to capture the population and requires advanced instrumentation and computational methods for complex 3D image-based analysis to resolve and assess each organoid [
      • Booij T.H.
      • Price L.S.
      • Danen E.H.J.
      3D cell-based assays for drug screens: challenges in imaging, image analysis, and high-content analysis.
      ]. In addition, while the source materials for organoids are heterogeneous, which is advantageous in capturing the diversity of in vivo cellular material, traditional culture methods fail to capture the heterogeneity since responses are often homogenized across the population within each well [
      • Schutgens F.
      • Clevers H.
      Human organoids: tools for understanding biology and treating diseases.
      ,
      • Booij T.H.
      • Price L.S.
      • Danen E.H.J.
      3D cell-based assays for drug screens: challenges in imaging, image analysis, and high-content analysis.
      ,
      • Clevers H.
      Modeling development and disease with organoids.
      ,
      • Kim S.
      • Choung S.
      • Sun R.X.
      • et al.
      Comparison of cell and organoid-level analysis of patient-derived 3D organoids to evaluate tumor cell growth dynamics and drug response.
      ,
      • Sasaki N.
      • Clevers H.
      Studying cellular heterogeneity and drug sensitivity in colorectal cancer using organoid technology.
      ,
      • Sharick J.T.
      • Walsh C.M.
      • Sprackling C.M.
      • et al.
      Metabolic heterogeneity in patient tumor-derived organoids by primary site and drug treatment.
      ,
      • Sachs N.
      • de Ligt J.
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      • et al.
      A living biobank of breast cancer organoids captures disease heterogeneity.
      ]. For these reasons, there is a need for new culture techniques and automated instrumentation that can more efficiently and accurately evaluate heterogenous organoid populations through single-organoid imaging and recovery. Some recent technologies have leveraged image-guided aspiration for retrieval of individual 3D structures, but these tools have their own shortcomings – such as involving physical manipulation forces that can disturb the structure of organoids – that limit their utility to destructive endpoint analysis.
      We recognized this unmet need for automated tools that are specialized for 3D culture, image-based evaluation, and intact isolation of individual organoids. Currently, our technology, the CytoSort Array consumables and the CellRaft AIR instrument and software, enable compartmentalized microwell cell culture, single cell-resolution brightfield and fluorescence imaging, and automated analysis and isolation of cells of interest. While this technology was originally designed for 2D adherent cell culture, we endeavored to leverage its three key features – namely the ability to image, identify, and isolate cells in situ – to improve organoid workflows. Here we highlight changes to the consumable that allow for establishing and tracking large, compartmentalized organoids in culture; software upgrades that allow the system to image, analyze, and isolate 3D structures; and hardware upgrades in the CellRaft AIR System that enable faster, reliable single organoid isolation. We have adapted traditional Matrigel dome culture methods to facilitate a reliable, user-friendly workflow for the development and evaluation of hundreds of individual organoids on a single cell culture consumable. Our proof-of-principle experiments demonstrate the utility of this technology for high-quality brightfield and fluorescence imaging and temporal assessment of individual organoids, establishing single-cell derived organoids, and isolating individual organoids for downstream applications, such as subcloning and -omics. These workflows and their endpoints continue to increase in prevalence and value in developmental biology, drug discovery, and toxicology, demonstrating the broad potential of our technology to advance organoid research.

      Materials and methods

      Array manufacturing materials

      Sylgard 184 Polydimethylsiloxane (PDMS) was prepared from a silicone elastomer kit from Ellsworth Adhesive Co (Germantown, WI). Octyltrichlorosilane (97%) and gamma butyrolactone were purchased from Sigma-Aldrich (St. Louis, MO). Poly(acrylic acid), 30% solution in water (MW∼30 kDa) was purchased from PolySciences, Inc. (Warrington, PA). Custom cassettes were injection molded using polystyrene material and were purchased from Protolabs (Maple Plain, MN). Custom dip-coating solution was prepared at Cell Microsystems, Inc. (Durham, NC).

      3D CytoSort array fabrication

      3D CytoSort Arrays were manufactured for organoid culture and recovery utilizing the following protocol. A SU-8 photoresist master template consisting of 80 µm tall, 500 × 500 µm pillars separated by 30 µm spaces was fabricated by deep reactive-ion etching (Alcatel AMS 100) at the Chapel Hill Analytical and Nanofabrication Laboratory (UNC-Chapel Hill, NC). The master was covalently modified through chemical vapor deposition with octyltrichlorosilane to reduce adhesion to PDMS. Sacrificial rigid substrates to ensure efficient dip-coating on the microarrays, as well as minimal PDMS deformity/sag, were prepared by spin-coating (H6-23 Spin Coater, Laurell, North Wales, PA) a thin layer of 7.5% poly(acrylic acid) (PAA) onto glass slides (75 × 50 mm, Corning, Corning, NY) at 500 rpm for 10 seconds and then 1500 rpm for 30 seconds. PDMS was poured onto the silica master template and degassed for 10 minutes at -710 torr. The master was then placed on the spin-coater for 30 seconds at 225 rpm and cured at 100°C for 60 minutes. Demolding the glass-backed PDMS from the silanized master template resulted in a microwell array (80 µm deep, 500 × 500 µm). Each array was dip-coated in a solution of 20% poly(styrene-co-acrylate) (weight percentage) in gamma butyrolactone (GBL) containing 1% γFe2O3 nanoparticles. Polymer solution was isolated in each individual microwell through discontinuous dewetting from the hydrophobic PDMS. CellRafts were formed after baking off the GBL solvent for 18 hours at 100°C. The CellRaft array was bonded to an injection-molded polystyrene cassette using PDMS glue cured at 70°C for 60 minutes, then oxygen plasma treated (Harrick Plasma, Ithaca, NY) for 2 minutes. Sacrificial glass backings were removed by soaking the backing in DI water at 70°C for 2 hours to dissolve the PAA. Each array underwent 2 additional minutes of oxygen plasma treatment then were coated with an anti-bubble solution for 30 minutes. After treatment, extra solution was aspirated and the array was topped with a polystyrene lid and packaged in a self-sealing sterilization pouch. Packaged arrays were gamma sterilized at 10-15 kGy for 130 minutes (Steris Applied Sterilization Technologies, Libertyville, IL) before use for cell culture.

      Cells, media, and 3D culture matrix

      Mouse pancreatic organoids and mouse hepatic organoids (cat # 70933, cat # 70932, StemCell Technologies, Inc., Vancouver, BC) were cultured and maintained in a 37°C, 5% CO2 incubator in PancreaCult Organoid Growth Medium (Mouse) or HepatiCult Organoid Growth Medium (Mouse) (cat # 06040, cat # 06030, StemCell Technologies, Inc.) supplemented with 1% penicillin/streptomycin (cat # 15140-122, Gibco Biosciences, Dublin, Ireland) per the manufactures’ guidelines for growth and expansion in Matrigel domes (Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-free, LDEV-free, cat # 356231, Corning, Inc., Corning, NY).
      Mouse pancreatic and hepatic organoid suspensions were prepared for cell seeding as fragments from 24-well Matrigel dome culture as described in the manufacturer's protocols using the standard complete media as described above. Mouse hepatic organoids were enzymatically dissociated for single cell suspension using a DNase I with TrypLE solution prepared by mixing 50 µL of 1mg/mL DNase I Solution (cat # 07469, StemCell Technologies, Inc.) with 5 mL TrypLE Express Enzyme (cat # 12605010, Gibco Biosciences). Pelleted fragments were resuspended in 1mL of the DNase I with TrypLE solution for 10 minutes in a 37°C water bath, mixing the suspension every 2.5 minutes by pipetting to ensure fragments dissociated into single cells. For single cell-derived organoid culture only, complete HepatiCult Growth Medium was supplemented with 10µM Y-27632 (cat# ACS-3030, ATCC, Baltimore, MD).

      Cell seeding on the 3D CytoSort Array

      Cell seeding procedures were adapted from the traditional Matrigel dome culture methods to facilitate seeding within the microwells of the 3D CytoSort Array and the release and collection of the CellRafts for single-organoid recovery. To prepare for cell seeding, the array was washed three times (3mL each, 3 minutes per wash) with sterile pre-warmed (37°C) Ca Mg PBS (cat# 10010-023, Gibco Biosciences). After the final wash was aspirated, 3mL of fresh PBS was added to the reservoir and the array was placed on ice for 1 hour to cool the array prior to cell seeding. The organoid fragment or single cell suspension was prepared as previously described. Fragment suspensions were counted by light microscope in n=3 10µL droplets. Volume needed for seeding the array was calculated using the following equation:
      Volumeneededforseeding(μL)=Desirednumberoffragments(Averagefragmentcount/10μL)
      (1)


      For single cell suspension, cells were counted using the Countess II Automated Cell Counter (Invitrogen, Waltham, MA) and volume needed for seeding was calculated by the following equation:
      Volumeneededforseeding(mL)=DesirednumberofcellsCellconcentration(cells/mL)
      (2)


      Cells or cell fragments were seeded at a 1:1 ratio of cells:CellRafts. The desired volume of cell suspension was added to a 15 mL conical tube with 1 mL of cold Advanced DMEM/F-12 (cat# 12634010, Gibco Biosciences) and centrifuged at 300 x g to pellet the cells. To prepare dilute Matrigel media for cell seeding, 100 µL of Matrigel was added to 5 mL ice cold complete PancreaCult or HepatiCult growth media for a final Matrigel concentration of 0.24 mg/mL Matrigel (1.2 mg Matrigel in total seeding volume). After centrifugation, the supernatant was carefully removed, and the cell pellet was resuspended in 1 mL of ice cold dilute Matrigel media. The 3D CytoSort Array was removed from ice, PBS was aspirated off the array, and the cold dilute Matrigel cell suspension was added to the reservoir. After cell seeding, the remaining 4mL of cold dilute Matrigel media was slowly added to reach 5 mL total volume within the array reservoir, and the array was returned to ice for 20 minutes. This cold incubation is essential to ensure the dilute Matrigel and cell suspension successfully wick into the microwells prior to polymerization of Matrigel at 37°C. Cells, or clusters of cells, settle into the microwell footprint of the CytoSort Array in a Poisson-like distribution and the dilute Matrigel allows for a loose attachment of developing organoids to the CellRafts. After the cold incubation, the array was placed in a 37°C, 5% CO2 incubator. The 3D CytoSort Array was scanned in brightfield using the CellRaft AIR System 4 hours and every 24 hours after seeding to monitor organoid growth and development.

      Live Cell Staining Organoids for phenotypic characterization

      Mouse hepatic organoids were stained on the 3D CytoSort Array with Hoechst 33342 (cat # R37605, Molecular Probes, Eugene, OR) and a directly conjugated (FITC) primary antibody for epithelial cell adhesion molecule (EpCAM, cat# 11-5791-82, eBioScience, San Diego, CA). Briefly, a 50% media exchange was performed 5 times (2 mL each) using Fluorobrite DMEM (cat# A1896701, Gibco Biosciences) being careful not to dislodge organoids from the microwell footprint. A 2X staining cocktail was prepared as follows: 1.6 mL Fluorobrite DMEM with 400uL of 10% BSA in PBS (cat# 37525, ThermoFisher Scientific, Waltham, MA), 1:400 (20 µL) of the EpCAM primary antibody, 8 drops of Hoeschst 33342. After the final wash with Fluorobrite DMEM, media was removed, leaving approximately 2 mL in the reservoir. The total volume of the staining cocktail was added to the reservoir for a final concentration of 1% BSA, 1:200 EpCAM-FITC primary antibody, and 2 drops per mL Hoechst 33342. The array was placed in a 37°C, 5% CO2 incubator for 45 minutes, followed by five 50% media changes (2 mL each) with Fluorobrite DMEM to wash away excess antibody before imaging. Immediately following staining, the array was scanned using the CellRaft AIR System in brightfield, blue fluorescence (390 / 432nm), and green fluorescence (475 / 522nm). Two organoid-containing CellRafts were selected for additional z-stack imaging with the CellRaft AIR System, as described in the Results and Discussion.

      RNA purification and quantitation

      Individual mouse hepatic organoids ranging in size from approximately 200µm to 700µm were isolated using the CellRaft AIR System for RNA purification using the Qiagen RNeasy Plus Micro kit (cat# 74034, Qiagen, Hilden, Germany). Organoids were released from the 3D CytoSort Array and collected using the system's PCR-style wand into PCR strip tubes with 25 µL of RLT Plus Buffer. After isolation was performed, the cell lysate was transferred to a 1.5 mL microcentrifuge tube and 325 µL of RLT Plus was added to each sample to reach the final volume of 350 µL. RNA purification was performed per the manufacturer's guidelines with a final elution volume of 14 µL. Purified RNA was quantified using the Agilent 2100 Bioanalyzer and RNA 6000 Pico Kit (cat# 5067-1513, Agilent Technologies, Santa Clara, CA) using the standard protocol.

      Results and Discussion

      The utility of organoids in bridging the gap between traditional 2D in vitro assays and clinical applications is clear due to their ability to recapitulate key aspects of in vivo biology. However, traditional methods of organoid culture present challenges in throughput, image-based phenotypic assessment, and recovery of intact, viable organoids for downstream expansion and analysis. Our work aimed to fill these unmet needs in organoid workflows by leveraging the CytoSort Array and CellRaft AIR System to enable organoid protocols that offer greater throughput, temporal image acquisition, and data cataloging of individual organoids, while maintaining the automated isolation and transfer benefits of the CellRaft AIR System. After recognizing the potential for our technology to provide an automated solution to organoid workflows, it became apparent that modifications to the current consumables, software, and hardware would be necessary for success.

      Adaptation of the CytoSort Array and CellRaft AIR system to enable 3D cell culture

      Development of a microwell array for 3D cell culture

      Standard cell culture consumables used for organoid growth and maintenance present pain points in throughput due to the manual manipulation of cellular material in ECM, image acquisition and analysis of multiple, overlapping structures per well, and the collection of intact individual organoids of interest for further analysis. Newer consumable technologies have been developed that focus on multiple microwell positions that house individual organoids in standard tissue culture consumable formats; however, these technologies still require extensive, manual upstream effort and do not offer retrieval capabilities. The CytoSort Array is uniquely suited to address these bottlenecks and inefficiencies of standard consumables.
      CytoSort Arrays enable the isolation of cells in a viable and unperturbed state, while simultaneously providing a culture environment that replicates standard in vitro conditions in tissue culture dishes. The CytoSort cell culture consumables comprise elastomeric microwell arrays that feature a releasable, optically transparent, polystyrene microscale cell culture surface, dubbed a CellRaft, within each well. These micron-sized CellRafts facilitate growth of a wide range of adherent and suspension cell types. A cell sample of interest is seeded on the array, and the cells randomly distribute by gravity into microwells following a Poisson-like distribution. After imaging and identification, individual cells of interest can be isolated through a stress-free methodology that utilizes mechanical forces to release the chosen CellRaft from its microwell without disturbing the attached cell layer and gently transfers it to a 96-well tissue culture or PCR plate using a magnetic wand.
      We have previously developed, and grown organoids on, arrays with smaller CellRafts, but it was apparent that the surface area limits the size of organoids that can be cultured. To enable the growth of larger organoids, 500 × 500µm CellRafts were designed. Each CellRaft is separated by 30µm PDMS walls to deter nonspecific binding and undesired growth of single cells/organoid fragments on the PDMS material and to support the growth of 3D structures up to 1mm in diameter (Fig. 1A). The array itself is comprised of 46 × 46 CellRafts in a single reservoir, yielding more than 2,100 positions for segregated organoid growth and the potential to interrogate, characterize, and recover as many organoids from a single consumable as seeding twenty-two 96-well or six 384-well plates.
      Fig 1
      Fig. 13D CytoSort Array. (A) CellRaft layout and dimensions of individual CellRafts and PDMS walls. (B) Fully fabricated 3D CytoSort Array. (C) AIR System image of a released CellRaft from a 3D CytoSort Array that was incubated at standard cell culture conditions, 37°C 5% CO2, for 4 weeks.
      The 3D CytoSort Array is attached to a 65mm-diameter injection molded polystyrene cassette that provides exterior borders of a contiguous media reservoir that facilitates a highly viable culture environment (Fig. 1B). When housed in its AIR System adapter plate, each cassette is within ANSI/SLAS tolerance ranges for height, width, and length to ensure compatibility with standard microscopy equipment, liquid handlers, and the AIR System. Because establishment of organoids and organoid assay times are generally extended compared to 2D culture methods, integrity of the arrays was evaluated after 4 weeks of culture time to mimic use-case scenarios. At the end of the test period, no leaks or structural disruptions nor negative impact on CellRaft releasability were observed (Fig. 1C), validating the robustness of the 3D CytoSort Array for 3D culture.

      Two-dimensional organoid detection and characterization with the AIR System software

      Once cells are seeded onto the 3D CytoSort Array, the CellRaft AIR System software, and its companion Off the AIR data analysis software, provide the interface that powers the automated imaging, identification, and isolation of organoid-containing CellRafts. Full array scans were acquired using the AIR System and software 4 hours after cell seeding to identify CellRafts containing single cells and daily thereafter to monitor organoid development and growth. Using the software's Raft Cytometry tool, a population was built to identify CellRafts with single cells at the initial scan by setting parameters for the number of cells or fragments detected on the CellRaft (AOI count) as well as area, aspect ratio, solidity, and circularity of each object.
      Raft Cytometry was subsequently applied to each serial scan to automatically detect organoids within the population of single-cell CellRafts and estimate their growth by characterizing their diameter, roundness, and fluorescence intensity. Using brightfield image segmentation, each field of view is processed by generating a black and white mask used to identify and characterize organoids present in the array (Supplemental Fig. 1). From the created mask, the central point is identified, which assigns the organoid to a specific CellRaft, a circle is fitted, and its diameter is calculated.
      Using these tools, a population was built to identify organoids of interest based on the criteria of a single organoid present on the CellRaft and a diameter greater than 150 microns. For non-clonal workflows, software-guided selection of CellRafts containing a single organoid was used, without filtering for single cells at the initial scan. Additional filters for fluorescence intensity can be added when necessary. This two-dimensional imaging analysis rapidly identifies organoids of interest for further 3D analysis using z-stack imaging.

      Z-stack image acquisition and analysis

      In addition to developing advanced algorithms for detection of organoids in the single focal plane 2D scans, a new software tool was designed to acquire and analyze z-stack images of CellRafts containing organoids of interest. Data acquired from the z-stack images are used to calculate more accurate organoid diameter measurements of large organoids than from the full array scans since the 2D, single focal plane scans tend to sample the organoid near the surface of the CellRaft. Diameters at each slice can be further integrated to estimate total organoid volume, and while not yet available, analysis of Hoechst-stained images will ultimately provide aggregate cell counts. A viewer was developed in the software that allows for navigation through composite brightfield and fluorescence z-stack images and that provides richer phenotypic and morphologic information from the three-dimensional imaging data.

      Image-driven cellraft isolation

      After an automated full array scan is performed with the AIR System, CellRafts containing cells of interest can be designated for isolation using software-guided or manual selection tools. Once selected, a CellRaft is dislodged from its microwell by a motorized release needle that penetrates the elastomeric bed of the CytoSort Array (Fig. 2A) and – by dint of its paramagnetism from doping with iron nanoparticles – collected on the tip of a magnetic wand. The instrument then aligns and inserts the wand into the designated well of a 96-well tissue culture or PCR plate – while retracting its internal magnet – to deposit the CellRaft in the collection plate (Fig. 2B). The process is repeated for each CellRaft selected by the user for isolation, one CellRaft (with its attached cells) per collection well.
      Fig 2
      Fig. 2The CellRaft AIR System enables automated isolation and transfer of organoid-containing CellRafts from the 3D CytoSort Array. (A) First, the release needle punctures the elastomeric floor of the CytoSort Array to dislodge the CellRaft from its microwell. The collection wand, which houses a retractable magnet, is lowered into the CytoSort Array to collect the released CellRaft, which is doped with paramagnetic iron nanoparticles. (B) The collection wand is then inserted into the designated well of a 96-well plate or PCR tube while the internal magnet is retracted, allowing the CellRaft to fall into the well. (C) The original “off axis” needle design relies on factory calibration to conduct prescribed “pokes” for CellRaft release. (D) The concentric needle design aligns the release needle with the objective, enabling dynamic image-based guidance of the release needle (and magnetic wand), which increases the accuracy and speed of dislodging (and collecting) organoid-containing CellRafts in extracellular matrix (E).
      The hardware and software were designed to isolate CellRafts from arrays filled with standard cell culture media. The process has been validated (>95% success rate) to release 100μm and 200μm CellRafts from their microwells using a regimented 2-poke pattern and then collect them with the magnetic wand positioned up to 5mm away. The prescribed poke locations and large attraction distance have allowed isolations to date to be conducted “off axis” from the microscope imaging path (Fig. 2C), relying on system calibration between the microscope, needle, and wand to align the CellRaft of interest for release and collection.
      Release of the larger 500µm format and collection through an extracellular matrix are significantly more challenging than in liquid cell culture. They require more targeted needle pokes to release corners that remain engaged with the PDMS microwell and much closer proximity (0-1mm) of the magnetic wand tip to the CellRaft to overcome the ECM viscosity. To address these challenges, a “concentric” release needle design (Fig. 2D) was developed and validated to align the release needle and collection wand with the microscope imaging axis, which facilitates real-time imaging of release and three-dimensional alignment of the collection wand tip to the target CellRaft. Imaging data is instantly analyzed to achieve and detect CellRaft release (Fig. 2E) after 1-4 targeted needle pokes and to dynamically control the height, lateral position, and dwell time of the magnetic wand tip to achieve collection. A video demonstrating the release and collection of an organoid-containing CellRaft from the 3D CytoSort Array can be found in the supplementary material (Supplementary Video 1). The new concentric design, paired with software that performs image-based decisions, yields faster organoid isolation with a higher success rate than the previous “off axis” system configuration.
      Collectively, these engineering advancements in our consumable, hardware, and software, as described in the data above, uniquely position this technology to facilitate organoid workflows from initial seeding to isolation of intact 3D structures that are incredibly challenging, if not impossible, with standard culture and imaging.

      Organoid workflows enabled by the 3D CytoSort Array and CellRaft AIR system

      To demonstrate the functionality of the engineering improvements for 3D cell biology, we performed proof-of-concept experiments to demonstrate the utility of the automated workflow for 3 key aspects of organoid biology: 1) clonal organoid track and traceability, 2) on-array real-time phenotypic assessment of organoids, and 3) isolation of intact organoids for downstream clonal propagation or endpoint assays.

      Clonal identification and temporal phenotypic assessment of organoids on the 3D CytoSort Array

      Traditional culture methods of organoids in semi-solid basement membrane extract (BME) dome, such as Matrigel, result in random arrangement in the x, y, and z dimensions, making imaging challenging due to overlapping 3D structures and multi-focal imaging requirements. In addition, the random arrangement of cells in a BME dome does not permit clonal organoid growth or temporal growth assessment of individual organoids (Fig. 3A). The new 3D CytoSort Array, which provides segregated microwell positions that have unique IDs, and our revised seeding protocol, which facilitates alignment of organoids onto the predictable z-plane of the microwells, overcome this bottleneck of traditional organoid culture methods.
      Fig 3
      Fig. 3Traditional dome culture methods (A) present challenges in imaging and clonal propagation due to random arrangement of organoids in the x, y, and z dimensions, whereas organoids are organized in segregated microwells in a single focal plane on the 3D CytoSort Array (B). The CellRaft AIR System and 3D CytoSort Array enable clonal propagation and temporal monitoring of clonal organoid development. With serial imaging, beginning 4 hours after cell seeding (Day 0) on the array (B), CellRafts with single cells (green box), or small clusters of cells (red boxes), can be easily identified using the CellRaft AIR System software and tracked over time (C) allowing for a complete data record that verifies clonality.
      Using both mouse pancreatic (Supplemental Fig. 2) and mouse hepatic organoids, we have demonstrated a robust and reliable method for obtaining high-quality, time-course images of developing organoids on the 3D CytoSort Array to maintain a dynamic growth record of each organoid during the entire development process from single cell to isolation. By performing an array scan shortly after cell seeding (4 hours), CellRafts containing single or multiple cells can be identified, either manually or using the Raft Cytometry tool (Fig. 3B). After single-cell identification, serial scans were acquired every 24 hours to capture temporal images of organoid development (Figs. 3C, 4). The ability to reliably image CellRafts in every field of view on the array enables clonal organoid workflows that are not currently possible using standard culture methods and imaging tools.
      Fig 4
      Fig. 4Temporal imaging of the development of a mouse hepatic organoid from a small fragment of cells for 10 days on the 3D CytoSort Array. On day 10, the organoids on the array were stained with a FITC-conjugated primary antibody for EpCAM and imaged for phenotypic assessment.
      In addition, the AIR System can perform advanced fluorescence-based phenotypic assessment for a variety of applications, including live cell staining, CRISPR editing, and on-array viability assays (Supplemental Fig. 3). Importantly, the imaging is incredibly rapid; a scan in brightfield and three fluorescent channels takes approximately 15 minutes to image the entire array (under 9 minutes for brightfield only). Such temporal brightfield and fluorescence imaging for rapid multiparameter phenotypic and morphologic screening of hundreds of individual organoids on a single consumable represents a significant advancement over current methods that require dozens of standard cell culture consumables, expensive imaging platforms, and extensive manual upstream and downstream effort by the user.
      To demonstrate the ability of the consumable and instrument to enable real-time imaging and analysis of intact live organoids, mouse hepatic organoids grown on the array were stained in situ with EpCAM FITC-conjugated antibody (Figs. 4, 5) and Hoechst to label nuclei. Two CellRafts containing 200-300µm mouse hepatic organoids were selected for z-stack imaging. Using the AIR System and software, brightfield, blue fluorescence (exposure = 50, 50ms), and green fluorescence (exposure = 200, 100ms) images were acquired every 10µm across focal ranges that encompassed the full height of the two organoids (24 and 30 images, respectively). The phenotypic content in the z-stack images was explored using a new interface within the software for users to step through composite brightfield and fluorescence images within the stack (Supplemental Fig. 4). Composite slices were exported using the z-stack viewer, and the images were compiled, using external software, to create videos for z-stack display (Supplementary Videos 2 and 3). Such a suite of software capabilities is not available using standard organoid culture methods and imaging platforms.
      Fig 5
      Fig. 5The proprietary CellRaft AIR System software includes automated z-stack acquisition in brightfield and 3-color fluorescence for organoids of interest. Mouse hepatic organoids were co-stained with a FITC-conjugated antibody for EpCAM and Hoechst 33342 to highlight cell membranes (green) and nuclei (blue). Using the CellRaft AIR System, z-stack images were taken every 10µm through the full height of the organoid.

      Isolation of individual organoids for downstream assays, growth and subcloning, and -omics applications

      In addition to the issue of multifocal imaging requirements, traditional organoid culture methods are susceptible to producing large variations in organoid size, shape, viability, and growth rate due to inconsistent starting material. Standard Matrigel dome culture methods used to evaluate organoid development, viability, or molecular-based changes in response to toxicants or therapies homogenize the response of many organoids, ignoring phenotypic and genetic heterogeneity. The 3D CytoSort Array and CellRaft AIR System present a novel solution for investigating heterogenous organoid populations, as well as generating clonally derived organoid populations. In addition to image-based phenotypic characterization, the presented technology permits isolation and transfer of individual organoids of interest for downstream applications and expansion.
      Using organoid models to understand the dynamics and evolution of intra- and inter-tumor heterogeneity on the molecular level is becoming widely implemented to better predict drug efficacy [
      • Li X.
      • Francies H.E.
      • Secrier M.
      • et al.
      Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics.
      ,
      • Bolck H.A.
      • Corrò C.
      • Kahraman A.
      • et al.
      Tracing clonal dynamics reveals that two- and three-dimensional patient-derived cell models capture tumor heterogeneity of clear cell renal cell carcinoma.
      ]. While studies have been performed using standard culture methods, largely focused on populations of organoids, we have used our technology to demonstrate the reliability and efficiency of our system for enabling such applications for individual organoids. Using both mouse pancreatic and mouse hepatic organoids, we have evaluated the utility of the technology for downstream organoid growth and subcloning and nucleic acid isolation from individual organoids isolated from the 3D CytoSort Array. After isolation from the array into 96-well collection plates, organoids remain viable for downstream assays and continue to grow in dilute Matrigel growth media (Fig. 6A). We have also demonstrated the ability to create clonal organoid populations by identifying organoids derived from single cells, as verified by temporal imaging, and isolating them into 96-well collection plates. After 5 days of growth off-array, each “parent” organoid was enzymatically dissociated in the 96-well plate into small fragments of cells, then re-seeded onto a new array to propagate hundreds of second-generation “child” organoids for further expansion or evaluation of lineage-based phenotypes (Fig. 6B).
      Fig 6
      Fig. 6Organoids isolated from the 3D CytoSort Array continue to grow post-isolation in dilute Matrigel media (A), and can be used for downstream applications, including organoid sub-cloning. (B) Mouse hepatic organoids were dissociated into single cells and seeded onto the 3D CytoSort Array and imaged every 24 hours for 8 days to monitor clonal organoid development. Clonal “parent” organoids were isolated into 96-well collection plates, containing dilute Matrigel media, using the CellRaft AIR System and allowed to grow for 5 additional days off-array. In the 96-well collection plate, individual “parent” organoids were enzymatically dissociated and the second-generation “child” cells were seeded onto a second 3D CytoSort Array for clonal organoid propagation.
      In addition to isolations for downstream growth, assays, and subcloning, the system can deposit single organoids into PCR strip tubes or 96-well PCR plates for nucleic acid isolation, a commonly investigated endpoint for drug discovery and toxicology. Mouse hepatic organoids, ranging in size from 200 to 700 µm, were isolated directly into lysis buffer in standard PCR strip tubes for RNA purification to determine a size threshold for RNA quality and concentration. We obtained high-quality RNA (RIN > 9.4) suitable for use in downstream -omics applications (n=8), and the amount of RNA obtained was directly correlated with organoid size (Fig. 7). While there are other platforms that aim to perform single organoid isolation, the data presented demonstrate the flexibility and utility of the technology to provide an all-in-one platform that facilitates efficient, user-friendly workflows for temporal phenotypic assessment of individual organoids upstream of single organoid applications.
      Fig 7
      Fig. 7Using the CellRaft AIR System, single organoids can be isolated from the 3D CytoSort Array for downstream -omics applications. Mouse hepatic organoids, grown to various sizes (200-700µm) on the 3D CytoSort Array, were isolated into PCR strip tubes containing lysis buffer for RNA isolation. High quality RNA was purified from all organoids (RIN > 9.4) and RNA concentration was correlated to organoid diameter.

      Ongoing work for optimizing the CellRaft Technology for 3D workflows

      Although the substantial advancements in the consumable and instrument have been successful in automating organoid assays, we are continuing to improve and validate our technology for single organoid workflows. We are performing high-throughput, quantitative validation studies to ensure our products maintain a rigorous level of accuracy when performing isolation and transfer of large sample sizes. We are also determining the maximum concentration of ECM that permits our threshold of 95% isolation efficiency, as we anticipate there may be some organoid cell types or workflows that require a larger concentration of ECM for optimal growth or assay conditions. The data presented herein represent a huge step for our technology in enabling 3D culture. Continuing to robustly test the ability of the CellRaft technology to image, identify, and isolate organoids will provide assurance that the combined power of the consumable, hardware, software, and protocols presented here can be leveraged to enable cost-efficient, user-friendly, automated organoid workflows.

      Declaration of Competing Interest

      The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: All authors are employed by Cell Microsystems, Inc., and their research and authorship of this article were completed within the scope of their employment with Cell Microsystems, Inc.
      This article is being reproduced in print post-publication in a sponsored print collection for distribution. The company sponsoring the print collection was not involved in the editorial selection or review of this article.

      Acknowledgments

      The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by NIH SBIR Grant 5R44ES032782-03

      Appendix. Supplementary materials

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