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FAQs – 3D Cell Culture

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What is the use of NanoCulture Plate?

NanoCulture Plate (NCP) is a cell culture plate used for three dimensional cell culture. Cells grown in NCP can form multi-cellular clusters (spheroid) after seeding cells like conventional monolayer cell culture plates. No specialist equipment is required. Microscopic observation can be made with clear well-bottom film, while culturing.

Also, black upper structure allow fluorescence imaging.

It is used for various applications, such as 1) evaluation of intracellular changes during 3D cell culture, 2) anticancer drug screening (compatible for HTS), 3) evaluation of knockdown effect with siRNA, 4) hepatotoxicity screening, 5) differentiation of stem cells, etc.

What is three-dimensional (3D) cell culture? What is the advantage?

Three-dimensional (3D) cell culture is a method to form three-dimensional multi-cellular cell cluster (spheroid). Cell morphology is different compared to conventional culture method using polystyrene culture ware (two-dimensional (2D) culture, monolayer culture). Multi-cellular spheroid is known to have closer characteristics to in vivo compared to monolayer cultured cells. 

How do cells on NCP form spheroids?

Cells seeded on NCP attach to the ultrafine processed mesh structure on the culture surface, grown in a spherical morphology. Cells migrate on the microstructure, and continuously contact/adhere to each other and form multi-cellular clusters (spheroids). Spheroids grow larger with proliferation at the same time.

*Reference: Technology

What is the difference between other three-dimensional cell culture methods?

Two of the most popular 3D cultures are, by forming spheroids 1) on matrix or gel, or 2) suspended with Ultra-low attachment microplates. NCP is a ready-to-use microplate, which can form spheroids with a standard culture medium, and can be easily observed by standard microscope through the transparent bottom film, and has following advantages.

*No foreign unknown materials
*Uniformly grown spheroids
*Compatible with High-throughput
*Easy to observe through transparent bottom film
*High cell viability (compared to Ultra-low attachment microplates)
*Easy to collect cells
*Easy to change medium (spheroids attach to the plate, compatible with fluid medium) 

What material is NCP made of?

NCP is made of synthetic polymer, with low attachment surface. By imprinting micro-structure patterning, cell adhesion is controlled to provide suitable environment for cells to form spheroids.

Is the culture surface coated?

No, it is not coated with any chemical substances, protein, gel, etc.

What types of NCP are available?

SBS format micro-titer plates (384-, 96- and 24-well) and 35 mm dish are available. Also, two types of adhesion (high/low) and different imprint pattern (honeycomb/square) can be chosen.

*High-binding grades are produced on order. 

Are the spheroids attached to the culture surface (bottom film)?

Spheroids are attached to the bottom film, however, its adhesion is not strong. Cells contacting the culture surface and grab the micro-structure as a scaffold.

How can the spheroid formation be confirmed?

It can be confirmed by microscopic observation.

Does 3D cell culture by NCP require special kind of equipment?

No, it does not require any special equipment. It can be used with same experimental equipment or device as the standard monolayer culture. We recommend to pre-incubate the plates before culturing, and it is useful if you have a microplate centrifuge. Also, it is useful to apply microplate compatible colony counter, which can measure spheroid size and its distribution.

Can NCP be reused?

No, you cannot reuse the used NCP wells. The unused wells within the plate can be reused for another experiment, by making aseptic handling (such as by covering the wells with plastic seal).

Can NCP be sterilized by autoclaving?

No, please do not autoclave NCP as it may cause deformation. NCP is sold in sterilized package. To maintain sterility, please make sure not to open the lid of NCP outside the clean bench.

Is NCM a must-use medium for 3D cell culture using NCP?

No, it is not a must for NCP spheroid culture. Spheroids can be formed on NCP by using standard culture medium with 10% FBS content.

What kind of medium is NCM?

Two types of NCM are available, 1) M Type, and 2) R Type with enhanced spheroid formation. Both medium are sold in a set of basal medium added with antibiotic and FBS. FBS is a lot-checked by spheroid formation on NCP. Normally, 10% FBS content is recommended, however, can be changed to a suitable concentration by monitoring the spheroid morphology.

What is the difference between Spheroid Dispersion Solution and Spheroid Lysis Buffer? And, when should it be used?

Spheroid Dispersion Solution is a solution which will disperse spheroids into single cells enzymatically (not trypsin). Single cells obtained by the dispersion of spheroids can be used for successive subculture, or Flow Cytometry analysis, etc.

Meantime, Spheroid Lysis Buffer lyse spheroids. This solution completely destroy the cells, which cannot be used for successive subculture. Further, as it is a very strong detergent, it cannot be used for experiments where protein conformation reservation required. It can be used for PicoGreen Assay (method to measure viability by DNA content).

What cell types can form spheroids on NCP?

It is for adherent cells. SCIVAX confirmed spheroid formation using various cell types, mostly cancer cell lines. Please contact us for updated information as we are continuing to evaluate various cell types.Floating cells, such as blood corpuscle cells may be difficult to form spheroids, however, if it has an adhesive property, it may be worth trying.

*Reference: Spheroid List.

Is it possible to request spheroid culture on contract basis?

Yes, it is possible. SCIVAX is providing contract spheroid culture service. We also offer on-sight training.

How long is the shelf life of NCP?

Date of expiry is printed outside of package. It is recommended to check the date before using the plate.

How should I store NCP?

Please avoid the light and store at room temperature. Please do not store inside the clean bench, because NCP degrade quickly by UV light and will lose spheroid formability.

There is a scratch on the culture surface. Can I use it?

Culture surface may be damaged by scratching with the tip of micropipettor, etc. This scratched part cannot be used for 3D culture, so please do not use the well when the culture surface is damaged.
In case of scratch on the plate bottom (the other side of culture surface), it will not affect 3D cell culture, however it may affect the observation.

Can I use the plate stored inside a CO2 incubator?

Yes, it can be used, however, sterility cannot be guaranteed.

Can NCP be kept in a freezer?

Though there is no problem with NCP quality, keeping inside a freezer will not extend its expiry date.

Are there any reagents that cannot be used with NCP?

Please avoid long time contact with 100% ethanol or 100% methanol. However, there is no problem when contacting with methanol for a short time (around 30 minutes).

What is the shelf life of NCM?

Shelf life of NCM after thawing is 1 year with storage condition of 4-8℃. After adding FBS, it is 1 months.

How should I preserve NCM?

Please preserve both basal medium and FBS below -20℃. Please preserve at 4℃ after thawing, and please avoid freeze-thaw cycle.

The received NCM is yellowish than usual. Can I use it?

NCM is delivered refrigerated with dry ice. In some cases, frozen basal medium may become yellowish due to the dry ice, however it will become normal after thawing and has no affect with culturing. Please move to the freezer immediately after receive. If it remains yellowish even after thawing, please contact us.

Is spheroid dispersion solution stable for freeze-thaw cycle?

Please avoid freezing/thawing cycle. We recommend to aliquot when using first time, and store inside freezer. Please store the thawed dispersion solution in a refrigerator, and use as soon as possible. Please do not preserve the dispersion solution when diluted.

Do cells proliferate on NCP?

Most cancer cell lines proliferate at a rate equivalent to monolayer culture or slightly less. Meantime, in case of normal cells, most of which form spheroids, but does not proliferate much.

How can I harvest the spheroids?

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.

How to prepare RNA or protein from collected spheroids?

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.

What type of cell growth assay is recommended?

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

Can I use MTT or WST-8 for cell growth assay?

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

Is NCP compatible with microplate reader?

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

Can NCP be observed with differential interference contrast microscope?

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

How can I count the spheroid number?

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.

I want to test drug sensitivity screening with NCP. When is the suitable timing to add compounds or factors to the wells? Also, let me know if I need any precaution.

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.

Can NCP be used for subculture?

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.

Can spheroids be immunostained?

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

Can I transfect gene or siRNA?

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.

Can I analyze cells by flow cytometry assay after dispersing?

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.

Can I make a slice of spheroids?

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

Can I co-culture on NCP?

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.

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.

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.

How should I 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).

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.

Can I use my standard medium?

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

Is the FBS of NCM-M inactivated?

Yes, it is inactivated.

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-NCP96

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.

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.

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.

What is the recommended volume of medium?

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

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.

How long 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

What solvent should be used for the reagent?

DMSO is the recommended solvent.

Why the reagent sometimes doesn’t dissolve properly in old DMSO?

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.

What is the storing condition or length of dissolved LOX-1 solution in DMSO?

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

What is the recommended final concentration of LOX-1 for Hypoxia detecting assay?

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

How can I detect/observe the signal/region of LOX-1?

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

How long time is needed for hypoxia detection after LOX-1 treatment?

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

What type of filter is needed for detecting LOX-1 signal with fluorescent microscopy?

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.

Can I measure the LOX-1 signal with microplate-readers?

We don’t recommend that. 

Is it possible to detect the LOX-1 signal with cell imagers?

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.

Can I measure oxygen concentration accurately?

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

Can I conduct time-lapse observation of the LOX-1 signal inside spheroid?

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

Can I use the reagent for cell culture under 2D condition?

Yes, you can.

Can LOX-1 visualize the hypoxic region of sliced spheroid or tissue?

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

Can I use the reagent for in vivo imaging?

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

Are there any reference paper for detecting hypoxia of spheroids by LOX-1?

Yes, there are. Please see the paper list.

How should ExoCapTM be stored?

ExoCapTM should be stored at 4°C.

Do I need to let ExoCapTM come to room temperature before use?

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.

Can ExoCapTM be stored in a freezer?

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