Choose ECM concentration, cell density, dome volume, culture duration, and endpoints.

Keep Matrigel, tubes, and tips cold; lift cells, count them, and prepare cell-gel solution to target final concentrations.

Dispense the hydrogel-cell suspension into the inner ring.

Allow gelation before gently adding warm complete medium pipetted against the wall.

Perform media exchange and brightfield monitoring.

Fix, stain, and image intact domes, or perform other endpoint assays.

Physical support, shape mold, and central positioning of Matrigel domes. The inserts also enable lifting and transfer of whole intact domes with minimal deformation.

Use the plate format supplied or specified by VivoTex. Matched control wells should use the same plate type where possible.

Prepare final target concentration after accounting for dilution by cells, medium, and additives. Keep cold before dispensing.

Use healthy cultures and model-specific passage, viability, and dissociation requirements.

Replace with model-specific complete medium for organoids or primary cells.

Minimize premature Matrigel gelation and reduce material loss during mixing. model-specific complete medium for organoids or primary cells.

Measures relative metabolic activity; does not uniquely distinguish proliferation, cell number, apoptosis, necrosis, or metabolic state.

Optional fixed-cell imaging workflow used in the application note.

1. Bring the GelControl™ plate or insert-containing well plate into the BSC while maintaining sterility.
2. Keep the plate closed until immediately before use.
3. Prepare matched control wells in the same plate type where possible. Label plate maps clearly to distinguish GelControl™-supported domes from conventional control domes.
4. Assemble equipment and materials for Matrigel.
   
   • Pre-chill tubes, pipette tips, and reservoirs that will contact Matrigel.
   • Keep Matrigel and Matrigel-containing mixtures cold until the moment of dome dispensing.
   • Note: Matrigel will start polymerization if any part of it reaches 10ºC.

1. Harvest cells or organoids according to the model-specific protocol.
2. Count cells or organoid fragments and determine viability.
3. Prepare a concentrated suspension that will yield the desired final cell density after mixing with Matrigel.
4. Keep the suspension cold (if compatible with the cell model).

1. Prepare volumes recordings of stock Matrigel, cells, diluent, and additives required to reach the desired final matrix concentration and cell density.
2. Mix stock Matrigel with diluent gently by pipetting with pre-chilled tips and avoid bubbles.
3. Combine cells and Matrigel gently in a chilled tube. Mix until homogeneous without over-pipetting.
   
   • Note: Matrigel viscosity and gelation behavior change as the mixture warms. This may make large experiments with many domes more difficult to seed. Calibrate your containers and cooling equipment to best suit your workflow.

1. Using a pre-chilled pipette tip, aspirate the required dome volume. For the 24-well use 60 µL per dome; for 48-well use 40 µL per dome.
2. Seed the same Matrigel-cell suspension and the same dome volume for supported domes with GelControl™ inserts and conventional, unsupported, Matrigel domes.
3. For accurate evaluation of the effects of the inserts, interlace the seeding order and well position within the same plate to form matched controls.
4. Inspect each dome immediately for bubbles, overflow, incomplete filling, or off-center loading. Record any seeding defects.
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‍GelControl™ Inserts‍

1. Position the pipette tip above the inner ring of the GelControl™ insert. Dispense solution slowly into the ring. The solution should fill the defined inner ring.

• To improve pipetting control the pipette tip can be leaned against the top of the wall edge. Watch demonstration video below.
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• Avoid touching the microfiber grid with the pipette tip. Do not scrape or push against the grid. Watch demonstration video below.
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Conventional Matrigel Domes‍

1. Dispense Matrigel domes directly into matched wells.
2. Use consistent pipetting angle, dispense speed, and well location across controls.

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1. Transfer the plate carefully to a humidified 37 °C incubator.
2. Allow domes to gel undisturbed for 20 minutes before media addition.
   
   • Some protocols advise turning Matrigel dome upside down for gelation to discourage cell attachment to the tissue culture plastic. GelControl™ inserts are compatible with this optional step, and reliably remain on the bottom of wells.
3. Do not add medium until domes are visibly gelled.

1. Warm complete medium to 37°C (or appropriate temperature for the cell model).
2. Gently add medium along the wall of each well, away from the dome. For the 24-well, add 1000 µL per well; for 48-well add 400 µL.
   
   • Handle plates with domes carefully between an incubator and a BSC. The domes are highly fragile and can easily detach. Avoid abrupt plate movement.
   • Avoid dispensing directly onto the gel surface.
3. Return the plate to the incubator.

1. Monitor cultures by brightfield imaging or visual inspection according to the experimental schedule.
2. During media exchange, aspirate and dispense along the well wall. Keep the tip away from the gel construct and microfiber grid.
   • GelControl™ inserts allow near 100% media exchange without disturbing the domes. Tip plates and position aspirator next to the wall. Aspirate the desired amount of liquid with while reducing the risk of aspirating or damaging the cultures.
   • For media exchange of unsupported domes, be extra vigilant. Aspirate media slowly, and ensure the domes are disturbed as little as possible.
3. Document dome position, deformation, detachment, floating, contraction, and any observations during each exchange.

1. Monitor your cells during culture using brightfield images.
2. Acquire low-magnification images to document construct position and shape, and higher-magnification images to monitor cellular phenotype.
3. Image supported and conventional cultures with similar settings. The GelControl™ microfiber grid will be visible in the images.

Watch demonstration video below.

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1. Use autoclaved forceps to grasp the GelControl™ handle, not the gel or the microfiber grid.
2. Lift slowly to allow excess medium to drain back into the well. Avoid rapid acceleration or contact with the well wall.
3. Transfer the insert-supported construct into a fresh well, staining vessel, imaging dish, or processing container containing appropriate medium or buffer.
4. After transfer, confirm that the construct remains attached to the insert and centrally positioned. Record any deformation or gel loss.

1. Prepare alamarBlue working solution according to the reagent manufacturer’s protocol and the validated plate-reader settings in your laboratory.
2. Remove or dilute culture medium as required by the assay plan, then add assay solution gently along the well wall. Use 300 µL alamarBlue solution per well in a 24-well format and 1 hour incubation.
3. Include controls containing a clear medium and alamarBlue solution that was incubated without cells.
4. Read fluorescence or absorbance within the linear range of the assay. Normalize within the experiment using appropriate controls.

1. Fix constructs using 4% paraformaldehyde for 30 minutes.
2. Wash gently with PBS. Add and remove liquids along the wall of the well or transfer vessel.
3. Permeabilize constructs using 0.1% Triton X-100 for 30 minutes at room temperature.
4. Stain with antibodies according to the manufacturer’s recommendations.
5. Counterstain with Hoechst (2000X) or DAPI and phalloidin for 1 hour at room temperature.
6. Image domes using consistent objective, and processing settings. Note that the microfiber grid may appear in transmitted-light or fluorescence images. When using laser scanning confocal microscope, the grid can be avoided with the desired focal area.