Suspension Spheroid Formation (Low-Adhesion/Forced Aggregation)
Materials Required
Principle
Suspension spheroid formation by low-adhesion or forced aggregation is a scaffold-free 3D culture method in which cells are prevented from attaching to plastic and are guided to interact with each other, forming compact multicellular aggregates through cell-cell adhesion, gravity-driven settling, microwell confinement, or centrifugation-assisted aggregation. The method detects the capacity of a cell population to self-assemble into spheroids, and the main readouts are spheroid formation efficiency, morphology, compactness, projected area or diameter, circularity, viability, proliferation, and experimental responses such as drug sensitivity. Classic implementations include hanging drops, agarose or hydrogel microwells, ultra-low-attachment round-bottom wells, and centrifugation-assisted aggregation in non-adherent wells. Low-adhesion culture shifts the system away from cell-substrate adhesion and toward cell-cell adhesion, while round-bottom or microwell geometry concentrates cells into a defined location and improves spheroid uniformity.
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use the culture medium appropriate for the selected cell type, such as DMEM supplemented with fetal bovine serum and antibiotics when this medium is reported for that cell line, and prepare cells as a single-cell suspension before seeding.
• PBS is used for washing, trypsin-EDTA is used to detach adherent cells before spheroid seeding, and agarose can be used to fabricate non-adherent microwell scaffolds or liquid-overlay surfaces for spheroid formation.
• Methylcellulose may be included when the published protocol uses it to increase medium viscosity and support forced cell aggregation in suspension.
• For spheroid characterization, published studies used live/dead viability staining, phalloidin for F-actin, DAPI for nuclei, and Ki-67 immunostaining to assess proliferative cells within spheroids.
• Drug-response studies may use viability assays or image-based morphology readouts when the assay has been validated for the selected spheroid model.
• Required equipment may include sterile low-adhesion round-bottom or U-bottom plates, agarose microwell arrays, a humidified 37 °C incubator with 5% CO2, a centrifuge for forced aggregation when used, a pipette system for gentle medium exchange, an inverted light microscope for routine monitoring, and fluorescence or confocal microscopy for stained spheroid analysis.
• PBS is used for washing, trypsin-EDTA is used to detach adherent cells before spheroid seeding, and agarose can be used to fabricate non-adherent microwell scaffolds or liquid-overlay surfaces for spheroid formation.
• Methylcellulose may be included when the published protocol uses it to increase medium viscosity and support forced cell aggregation in suspension.
• For spheroid characterization, published studies used live/dead viability staining, phalloidin for F-actin, DAPI for nuclei, and Ki-67 immunostaining to assess proliferative cells within spheroids.
• Drug-response studies may use viability assays or image-based morphology readouts when the assay has been validated for the selected spheroid model.
• Required equipment may include sterile low-adhesion round-bottom or U-bottom plates, agarose microwell arrays, a humidified 37 °C incubator with 5% CO2, a centrifuge for forced aggregation when used, a pipette system for gentle medium exchange, an inverted light microscope for routine monitoring, and fluorescence or confocal microscopy for stained spheroid analysis.
Experimental Procedure
• Maintain cells under the culture conditions reported for the selected cell type, detach adherent cultures into a single-cell suspension, count viable cells, and adjust the suspension to the seeding density used in the selected literature model.
• Reported spheroid protocols vary by platform and cell type, so seeding density should be selected from the relevant paper rather than generalized across cell lines.
• For agarose or microwell-based culture, prepare the non-adherent microwell surface before seeding and equilibrate it with culture medium when reported.
• For ultra-low-attachment round-bottom culture, seed cells directly into non-adherent wells so that one spheroid can form per well when the cell line is spheroid-forming.
• Seed the prepared single-cell suspension into the low-adhesion or microwell culture format, using the reported cell number or concentration for the selected cell line and platform.
• If forced aggregation is used, centrifuge the plate after seeding only under the speed and duration reported by the selected protocol, because centrifugation is a protocol-specific step rather than a universal requirement.
• Incubate seeded cultures at 37 °C and 5% CO2, then monitor spheroid formation by bright-field microscopy over the time course reported for the model.
• Published examples show that some cells form compact spheroids within days, whereas other models show progressive aggregation, compaction, and growth over longer culture periods.
• Exchange medium gently to avoid spheroid loss or displacement, especially in low-adhesion and microwell formats where aggregates are not attached to the plate.
• If staining is performed, published workflows include PBS washing, paraformaldehyde fixation, permeabilization with Triton X-100, blocking, primary antibody incubation, fluorescent secondary antibody incubation, phalloidin staining, DAPI staining, and fluorescence imaging.
• Acquire bright-field images at defined time points and quantify spheroid morphology using projected area, diameter, circularity, roundness, major axis, minor axis, perimeter, or compactness when these measurements are appropriate for the biological question.
• Image-based monitoring can distinguish compact spheroids from loose cell assemblages, and automated segmentation has been used to follow aggregation dynamics at high throughput.
• Use negative or comparative controls that are reported in the literature, such as non-spheroid-forming sublines, adherent 2D cultures, or untreated spheroids in drug-response assays.
• Interpret poor compaction as a biological or technical outcome rather than assuming protocol failure, because loss of essential cell-cell adhesion molecules can prevent spheroid formation in some cancer-cell sublines.
• Reported spheroid protocols vary by platform and cell type, so seeding density should be selected from the relevant paper rather than generalized across cell lines.
• For agarose or microwell-based culture, prepare the non-adherent microwell surface before seeding and equilibrate it with culture medium when reported.
• For ultra-low-attachment round-bottom culture, seed cells directly into non-adherent wells so that one spheroid can form per well when the cell line is spheroid-forming.
• Seed the prepared single-cell suspension into the low-adhesion or microwell culture format, using the reported cell number or concentration for the selected cell line and platform.
• If forced aggregation is used, centrifuge the plate after seeding only under the speed and duration reported by the selected protocol, because centrifugation is a protocol-specific step rather than a universal requirement.
• Incubate seeded cultures at 37 °C and 5% CO2, then monitor spheroid formation by bright-field microscopy over the time course reported for the model.
• Published examples show that some cells form compact spheroids within days, whereas other models show progressive aggregation, compaction, and growth over longer culture periods.
• Exchange medium gently to avoid spheroid loss or displacement, especially in low-adhesion and microwell formats where aggregates are not attached to the plate.
• If staining is performed, published workflows include PBS washing, paraformaldehyde fixation, permeabilization with Triton X-100, blocking, primary antibody incubation, fluorescent secondary antibody incubation, phalloidin staining, DAPI staining, and fluorescence imaging.
• Acquire bright-field images at defined time points and quantify spheroid morphology using projected area, diameter, circularity, roundness, major axis, minor axis, perimeter, or compactness when these measurements are appropriate for the biological question.
• Image-based monitoring can distinguish compact spheroids from loose cell assemblages, and automated segmentation has been used to follow aggregation dynamics at high throughput.
• Use negative or comparative controls that are reported in the literature, such as non-spheroid-forming sublines, adherent 2D cultures, or untreated spheroids in drug-response assays.
• Interpret poor compaction as a biological or technical outcome rather than assuming protocol failure, because loss of essential cell-cell adhesion molecules can prevent spheroid formation in some cancer-cell sublines.
Troubleshooting
Problem: Cells remain as loose aggregates instead of forming compact spheroids.
• Possible cause: The cell population may have weak cell-cell adhesion or loss of adhesion-related molecules.• Literature-supported solution: Compare spheroid morphology with a known spheroid-forming control or analyze adhesion-related molecules, because loss of E-cadherin, P-cadherin, or α-catenin was linked to impaired spheroid formation in colon cancer sublines.
Problem: Spheroids vary substantially in size or shape.
• Possible cause: Cell distribution is not sufficiently confined or synchronized during aggregation.• Literature-supported solution: Use round-bottom low-adhesion wells, microwell arrays, or centrifugation-assisted aggregation when supported for the selected model, because these formats concentrate cells and improve uniform spheroid formation.
Problem: Spheroids are lost or disturbed during medium exchange.
• Possible cause: Suspension spheroids are not attached to the culture surface.• Literature-supported solution: Use gentle aspiration and medium replacement, or microwell systems designed to retain aggregates during handling, because microwell geometry has been reported to improve handling and medium exchange compared with some droplet-based approaches.
References:
- [1]. Maritan SM, et al. An efficient and flexible cell aggregation method for 3D spheroid production. J Vis Exp. 2017;(121):55544. [Content Brief]
- [2]. Stadler M, Scherzer M, Walter S, Holzner S, Pudelko K, Riedl A, et al. Exclusion from spheroid formation identifies loss of essential cell-cell adhesion molecules in colon cancer cells. Sci Rep. 2018;8(1):1151. [Content Brief]
- [3]. Foty R. A simple hanging drop cell culture protocol for generation of 3D spheroids. J Vis Exp. 2011;(51):2720. [Content Brief]
- [4]. Wang Y, Kim MH, Tabaei SR, Park JH, Na K, Chung S, et al. Spheroid formation of hepatocarcinoma cells in microwells: experiments and Monte Carlo simulations. PLoS One. 2016;11(8):e0161915. [Content Brief]
- [5]. Deckers T, Lambrechts T, Viazzi S, Hall GN, Papantoniou I, Bloemen V, et al. High-throughput image-based monitoring of cell aggregation and microspheroid formation. PLoS One. 2018;13(6):e0199092. [Content Brief]
- [6]. LaBarbera DV, et al. The multicellular tumor spheroid model for high-throughput cancer drug discovery. Expert Opin Drug Discov. 2012;7(9):819-830. [Content Brief]
- [7]. Thomsen AR, Aldrian C, Bronsert P, Thomann Y, Nanko N, Melin N, et al. A deep conical agarose microwell array for adhesion independent three-dimensional cell culture and dynamic volume measurement. Lab Chip. 2018;18(1):179-189. [Content Brief]
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