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  3. FAQ NCP – Start-up




Q.201 – What is the difference between Micro-square and Micro-honeycomb pattern?
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.
Q.202 – What is the difference between Low-attachment type (L-type plate) and High-attachment type (H-type plate)?
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.
Q.203 – How should l determine the 3D cell culture condition?
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.)
Q.204 – When should I use NCM?
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.
Q.205 – Can I use my standard medium?
Yes, NCP is capable to form spheroid with your standard medium. Please try.
Q.206 – Is the FBS of NCM-M inactivated?
Yes, it is inactivated.
Q.207 – Why the pre-incubation of NCP is necessary?
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-NCP
Q.208 – There are Bubbles on the well bottom culture surface. What should I do?
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.
Q.209 – Damaged culture surface with the tip of a pipette. What should I do?
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.
Q.210 – Is it possible to coat NCP surface with collagen?
The ultrafine mesh structure of NCP culture surface might be infilled by overlaid collagen. In that case, it may not form the spheroids.
Q.211 – What is the recommended volume of medium?
We recommend 100-200µL/well for 96well plate, and 600-1200 µL/well for 24well plate.
Q.212 – How many cells should I seed?
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
Q.213 – How long time will it take to form spheroids? And how many days can the spheroids be cultured at the longest?
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.
Q.214 – Will it become confluent?
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.
Q.215 – What is the size of the spheroids?
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.
Q.216 – Do spheroids become spherical?
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.
Q.217 – Does it form similar spheroids with every well?
NCP has very small well-to-well, plate-to-plate and day-to-day variation, and all the wells form similar spheroids.
Q.218 – Cells do not form spheroids, what should I do?
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.
Q.219 – Cells were able to form culture spheroids formerly, however, become unable to reproduce. What should I do?
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.
Q.220 – Spheroids are formed unevenly distributed inside the well. What should I do?
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.
Q.221 – Cells float and gather around the center of the well. What can I do to prevent it?
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.
Q.222 – How can I change medium while culturing?
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.
Q.223 – Spheroids formed nicely and evenly in case of 96well plate, while with 24well plate spheroids float and form large aggregates. What can I do to prevent it?
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.
Q.224 – What concentration of DMSO will affect spheroid formation?
For HT-29 cells, up to 0.5% of DMSO concentration does not affect spheroid formation. The concentration may vary depend on cell types.
Q.225 – How do you count cell number?
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.
Q.226 – Can I use the unused well on a different cell culture occasion?
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.
Q.227 – Are the cells in the center part of spheroids alive?
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.