Hanging Drop Spheroid Culture

• Use the appropriate complete culture medium for the selected cell type;
• Reported examples include α-MEM, DMEM, or RPMI-1640 supplemented with fetal bovine serum and penicillin-streptomycin.

• Cell suspension is the starting material for each hanging drop, and published studies have used seeding densities ranging from very low cell numbers, including 10 ovarian cancer cells per drop in a 384-well hanging-drop array, to hundreds of cells per 1 µL PDMS-HDA drop or larger conventional drops, depending on cell type and platform.

• Type I collagen can be included when the experimental goal is to improve spheroid formation or incorporate matrix components;
• One PDMS-HDA study tested 0, 50, 500, and 1000 µg/mL collagen and reported improved compact spheroid formation with 500 µg/mL collagen in MCF7 cells, while MDA-MB-231 cells required a higher collagen condition in that system.

• Methylcellulose can be used as a medium additive when compactness and circularity are experimental endpoints, because it improved spheroid circularity and compactness in hanging-drop cultures.

• Calcein AM and ethidium homodimer-1 can be used for live/dead fluorescence staining;
• One PDMS-HDA study used 2 µM calcein AM and 4 µM ethidium homodimer-1 with 1 h incubation at 37 °C and 5% CO₂ before imaging.

• CellTiter-Blue or comparable fluorescence/colorimetric viability assays can be used when drug-response or viability readouts are required in hanging-drop spheroids.

• A humidified 37 °C, 5% CO₂ incubator is used to maintain hanging-drop cultures under physiological culture conditions.

• Hanging drops may be generated on an inverted culture-dish lid, a 384-well hanging-drop array plate, a superhydrophobic hanging spherical drop substrate, or a PDMS-based hanging-drop array, depending on throughput and imaging needs.

• A pipette or liquid-handling instrument is used to dispense cell-containing droplets;
• Reported implementations include manual droplet placement and automated dispensing of 1 µL drops on PDMS-HDA devices.

• Bright-field and fluorescence microscopy are used for spheroid morphology and viability imaging, and Fiji/ImageJ can be used to measure orthogonal spheroid diameters and calculate spheroid volume.

• Prepare a single-cell suspension in complete culture medium and adjust the cell concentration to deliver the desired number of cells per drop; the chosen seeding density should be optimized for the cell type because spheroid size and formation depend on initial cell number.

• For PDMS-HDA collagen-containing drops, prepare neutralized type I collagen near pH 7.4 and mix cells into the selected collagen concentration before dispensing.

• Prepare the hanging-drop platform before seeding: for conventional hanging drops, droplets are placed on the inner surface of a culture-dish lid and the lid is inverted;
• For PDMS-HDA, the chip can be UV-sterilized for 30 min before use, and the device is placed in a culture dish containing medium to reduce evaporation.

• Dispense cell suspension as discrete droplets onto the selected hanging-drop surface, then invert the lid or device so the droplets hang downward;
• Published protocols include conventional hanging drops and array formats, while PDMS-HDA used 1 µL droplets dispensed onto PDMS and then flipped into a 6-cm dish containing 750 µL medium sealed with parafilm.

• Incubate the inverted droplets at 37 °C and 5% CO₂ until spheroids form;
• Reported formation times include overnight to 24 h in PDMS-HDA and longer culture windows in conventional or 384-array formats depending on cell type and assay endpoint.

• For viability staining in PDMS-HDA, add or expose spheroids to 2 µM calcein AM and 4 µM ethidium homodimer-1, incubate for 1 h at 37 °C and 5% CO₂, and acquire bright-field and fluorescence images.

• For drug-response assays, form spheroids first, expose them to compounds for the reported assay period, and measure viability by fluorescence or colorimetric assay;
• Examples include 5-FU and tirapazamine testing in 384 hanging-drop spheroids and paclitaxel/cisplatin testing in PDMS-HDA spheroids.

• Acquire bright-field images to document spheroid formation, morphology, and size, and acquire fluorescence images when live/dead or viability dyes are used.

• For spheroid volume analysis, measure two orthogonal diameters, calculate mean diameter as the square root of their product, and estimate spheroid volume as 4π(l/2)³/3, as reported for PDMS-HDA spheroids.

• For drug-response studies, include untreated spheroids as negative controls and treated spheroids as experimental groups;
• 2D monolayer cultures may be included as a comparator when the aim is to test whether 3D culture changes drug sensitivity.

• For high-throughput assay quality, Z-factor analysis has been used to evaluate fluorescence- and colorimetric-based assays in 384 hanging-drop arrays.

Problem: Drops evaporate or become unstable during culture.

• Possible Cause: Small hanging-drop volumes are vulnerable to evaporation and handling disturbance.
• Literature-supported Solution: Place the hanging-drop device in a humidified culture dish containing medium and seal the dish, as reported for PDMS-HDA culture.

Problem: Spheroids are loose, irregular, or poorly compacted.

• Possible Cause: Cell type, seeding density, matrix condition, and medium additives influence aggregation and morphology.
• Literature-supported Solution: Optimize starting cell number and consider literature-supported additives such as methylcellulose or collagen when compactness and circularity are required.

Problem: Imaging is blurred by spheroid movement inside hanging drops.

• Possible Cause: Motion of spheroids within the droplet can reduce image quality.
• Literature-supported Solution: Methylcellulose-containing media reduced motion-induced image blur and improved imaging in hanging-drop spheroid cultures.