FAQs – 3D Cell Culture

No need to use cell stripping reagents such as trypsin. As spheroids do not adhere strongly to the culture surface, it can be recovered by pipetting with medium into centrifuge tube. Precipitate the cells by centrifuging about 5 minutes at 200xg. Please avoid strong pipetting when it is needed to keep spheroid morphology.

Please keep the plate on ice while collecting the spheroids. Use cold medium to recover spheroids and put into centrifuge tubes. Centrifuge at 200xg for 5 minutes at 4oC. Please extract RNA or protein from the cell pellet by using commercial kits according to your experiments.

The most basic and accurate way is to disperse spheroids into single cells and count the living cell number. We recommend CellTiter-Glo assay kit by Promega for the cell growth assay by measuring ATP contents.

*Reference: Application notes No.11

Those can be used by dispersing spheroids into single cells as they do not penetrate inside the spheroids.

NCP is compatible with SBS standard, and be used with various plate readers.

Spheroids formed on NCP can be observed by differential interference contrast microscope.

By using microscope, colony counter, or high-content screening equipment, which can clearly observe and take picture of the whole well, can analyze spheroid number or size from the image.

In case of evaluating the compounds that is effective during the period when single cells migrate and adhere into cell cluster (spheroid), add compounds at the same time when seeding. Meantime, in case of evaluating the effect after spheroid formation, add compounds 3-5 days after spheroid formation. We recommend adding small amount of high concentration solution, when adding compounds or factors after spheroid formation. As a guideline, please prepare 10 times concentration compounds, and add 1/10 volume of the culture volume. Please let the compounds disperse naturally, because pipetting after adding of compounds may cause spheroids to fall apart or to cause uneven distribution.

Yes, it is possible. Dispersing spheroids into single cells and seeding again into new well will form spheroids again. Also, it can be subcultured just by changing medium in spheroid form and not dispersing, or can also be subcultured into new well after recovering in spheroid form into centrifuge tube and changing medium.

Yes, it can be immunostained either on NCP or after moving to culture slide. In case of spheroid detachment during cell washing process, it can be stained inside the centrifuge tube. However, please be careful in case of spheroids with weak cell-to-cell adhesion, which may fall apart during cell washing process. Further, in case of spheroids with strong cell-to-cell adhesion, antibody may not penetrate into the center part of the spheroids.

*Reference: Application notes No.12

Yes, it is possible to transfect gene or siRNA into cells before seeding or at the same time of seeding. Though we have not evaluate gene transfection after spheroid formation, it should be difficult to transfect gene into cells in the center part of the spheroids.

Yes, it is possible to analyze cells with flow cytometry by labeled antibody. Please use our spheroid dispersion solution for dispersing spheroid into single cells.

Yes, it is possible to make a slice of spheroids by freeze-sectioning using CMC or OTC compound, or a paraffin embedding method.

Yes, it is possible. You can co-culture and observe morphology at the same time, by labeling cell types into different colors with fluorescent dye.

There is ultrafine structural difference between those plates, square one is the Micro-square (MS pattern), and hexagonal one is the Micro-honeycomb (MH pattern). They are suitable with different cell types, you may choose the suited pattern depending on your cell types.

H plate is a grade with enhanced adhesiveness of the culture surface.

We recommend you to confirm spheroid forming condition with the L-type plate first. In case of, the spheroids detach from culture surface and condense in the well center, or easily detach when changing medium or washing cells, please try H-type plate. However, in case of H-type plate, cell-to-plate adhesion become stronger than cell-to-cell adhesion, which result in a monolayer cell culture and may not form spheroids with some cell types.

This is an example of procedure to determine culture condition, for your reference.

• Prepare plates (NCP-LH-96, NCP-LS-96) and medium
  (ATCC recommended growth medium, NCM-M, NCM-R).
• After collecting monolayer cultured cells, dilute it with culture medium and seed 5,000, 10,000, 20,000 and 50,000 cells/well.
• Make microscopic observation and ATP assay, on every 1, 3, 5, 7 day. (Change medium if necessary).
• Find optimum condition from spheroid morphology and ATP levels over time. (Also can fix optimum condition by using expression level of the targeted molecules).

It is recommended to compare your own medium and NCM during the optimizing of 3D culture condition, because spheroid formability may differ greatly by the culture medium. NCM is a medium used for benchmarking NCP, which can be used when you lose reproducibility with your own medium.

NCM-M is a medium with minimum recipe (10%FBS, antibiotic added basal medium). NCM-R has spheroid forming factor added to NCM-M.

Yes, NCP is capable to form spheroid with your standard medium. Please try.

Yes, it is inactivated.

Due to micro-processing of culture surface, it is easy to form bubble on the culture surface. We recommend to remove the bubbles by pre-incubation which improve compatibility of medium with culture surface, because remaining bubbles may inhibit spheroid formation.

*Reference: Handbook-NCP96

If it is before cell seeding, tap plate lightly to bring the bubbles to the surface.

If the bubbles do not float by tapping, it can be removed by centrifuging at 300-500xg for 1-3 minutes with microplate centrifuge (if you cannot use microplate centrifuge, pipette medium with micropipettor and remove the bubbles. Make sure not to damage the culture surface when pipetting.)
If it is after cell seeding, please remove by pipetting in as same manner. If some time has passed after seeding and the cells had already adhered to the culture surface, we recommend you to continue culturing as it is, because it is difficult to remove the bubbles without disturbing spheroids.

It should be all right in case of just a slight poke by the micropipettor, however, if it made a scratch, the damaged part will not be suitable for 3D culture. Please do not use that well if you feel uncomfortable.

The ultrafine mesh structure of NCP culture surface might be infilled by overlaid collagen. In that case, it may not form the spheroids.

We recommend 100-200µL/well for 96well plate, and 600-1200 µL/well for 24well plate.

It is recommended to study suitable condition at around 1×104 cell/well for 96well plate, and 6×104 cell/well for 24well plate. In case of small spheroid forming cell types, increasing the cell number result in larger spheroids.

Spheroid forming rate and growth curve will be affected by the number of cell seeded, thus it is recommended to optimize the culture condition in the beginning.

Most cells start migration and cell-to-cell adhesion within 24 hours, and clear spheroids are formed after 3 to 4 days. Spheroids keep growing until day 7, and after that it is possible to continue culturing for at least 1 month depending on changing medium and supplying nutrition.

Unlike monolayer culture, there is no such phenomenon which can be applicable to so called “confluent”. However, in some cell types, growth of the spheroids cease and saturate in 7 to 10 days after seeding.

Though spheroids size differ by cell types, spheroids grow up to 50µm average even for the small spheroid forming cell types, and average of 200µm with the large spheroid forming cell types. Also, the size is affected by the cell numbers, increasing the seedling cell numbers would result in larger spheroids.

Spheroid morphology differ by cell characteristics, such as cells which form tight spherical spheroids with strong cell-to-cell adhesion, or a grapevine-like spheroid with slack cell-to-cell adhesion.

NCP has very small well-to-well, plate-to-plate and day-to-day variation, and all the wells form similar spheroids.

In case of insufficient cell-to-cell adhesion, please try NCM-R medium. Also, by adding small amount of extracellular matrix in medium may promote spheroid formation. Our data showed that 0.5-1% BD Matrigel TM addition is effective.
In case of monolayer cell formation owing to strong adhesion with the plate bottom, please try either by changing FBS content of NCM-M between 1 to 20%, or using NCM-R.

Continuous subculturing may cause change in cell characteristics, which will effect spheroid formability. Also, seeding cells cultured at 50 to 80% confluency may form spheroids consistently.

The medium inside the well which is closer to the outer of the well is heated faster, causing convection current of medium inside the well. This convection current cause uneven distribution of the spheroids. In some cell types, this phenomenon occurs very easily. In such cases, it can be prevented by heating the plate and medium to 37℃ before seeding cells.

Spheroid adhesion to the culture surface of NCP is not very strong compared to monolayer. Please be cautious not to shake the plate while observation by microscope after seeding. In case of light tapping or continuous mechanical vibration may detach spheroids from the culture surface and float.

As the spheroid adhesion to the culture surface is not very strong, we never recommend changing whole medium inside the well. Please remove half the medium gently, and add another half gently. Please be careful when pipetting as spheroids easily breakdown or become unevenly distributed.

Larger culture surface per well increase the pitching of medium, which makes spheroids detach and float easily. Floating spheroids agglomerate with each other and form larger spheroids with uneven shape and size, which result in a well-to-well variation. Please try not move the plate as much as possible for 10-15 minutes after seeding. Also, by adding larger volume of medium (2mL/well) than usual may prevent floating of spheroids in some cases. Further, using H plate may prevent floating of the cells. Please contact us for further information.

For HT-29 cells, up to 0.5% of DMSO concentration does not affect spheroid formation. The concentration may vary depend on cell types.

Cell number can be counted by dispersing spheroids into single cells using Spheroid Dispersion Solution, and counting with a hemocytometer. Also, living cells can be counted by ATP assay using CellTiter-Glo Promega.

Basically, we do not recommend using used plate for different day experiment. However, by covering the unused wells with aseptic sealing tape before using, the unused wells can be used on next culture occasion. However, we cannot guarantee the sterility with such use.

We confirmed that the cells in the center part of spheroids are alive until 7-10 days culture period, by staining with EthD-1 or Calcein-AM.

DMSO is the recommended solvent.

Old DMSO maybe contain moisture due to the absorbency in bad condition. We recommend using new one or the one stored in good condition, because it will be difficult for LOX-1 to dissolve into moisturized DMSO.

LOX-1 solution is stable for couple of months under light protection in -20℃.

We recommend adding LOX-1 into the culture medium at 2 µmol/L.

One can detect the signal with microscope or cell imager that has fluorescence detector feature.

In monolayer culture, 2 hours is recommended. And in 3D culture, 24 hours is needed.

We have confirmed the adequateness of G-2A filter set (Nikon) and filter set 14 (Zeiss) for the observation. You can use the other filter set matching with the spectrum (Ex: 483, Em: 616) of LOX-1.

We don’t recommend that. 

Yes it is. We have confirmed that some cell imager equipment i.e. Celigo (Nexcelom), InCell series (GE Healthcare) and BioStation CT (Nikon) are capable to detect the signal.

Measuring oxygen concentration can be possible by measuring phosphorescence lifespan. Because the phosphorescence lifespan is correlated with oxygen concentration.

Yes, you can. But superoxide which is produced by excitation light maybe affect cytotoxicity to cells.

Yes, you can.

No, it cannot. This is the reagent for only living cells.

Yes, you can. Please contact us for detailed information.

Yes, there are. Please see the paper list.

ExoCapTM should be stored at 4°C.

We recommend that the buffers are allowed to come to room temperature prior to performing the enrichment. To avoid unnecessary changes in temperature, the elution buffer can remain stored at 4°C if intact exosomes are not required in the downstream process.

No. Freeze-thaw cycles will cause denaturation of the antibodies.