Hi, I have done spheroids in spinoflast, hanging drop and 96 well plate. Both worked but 96 well plate is the one that give a higher number of spheroids being them more similar in size, they are more homogenous than with spniflast method and you can have more spheroids than with hanging drop. I usually coated my 96 well plate first with 1% agarose (with 100mL of 1% agarose and pipette it in and out to leave a thin film). It is not difficult and works well.

One important issue is the media. In DMEM media I achieve HCT116 and MCF7 cell line spheroids, but with other medias they did not form spheroids. Change the media every 48h to keep them growing properly.

Also, you may want to consider which size of spheroids you want to and which day may be the best to analyse them. Usually spheroids less than 400um do not have necrotic core.

You may have to seed different cell concentrations per well first and see which one is best for you. I would recommend to wait until day 4 at least, to have time for the cells to form good spheroids (see paper below).

I found useful this paper:

Spheroid-based drug screen: considerations and practical approach.

Friedrich J1, Seidel C, Ebner R, Kunz-Schughart LA.

Nat Protoc. 2009;4(3):309-24

You may try this

https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis

but, the yield for Western blot will be lower. You will have to scale-up.

Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...

A common way is to use bacteria petri dishes. It may be complemented with coating bottom with common agar made in the medium of choice as a base. This should be enough for a start!

One of the easiest way to make suspension culture (generating spheroids) is culturing them on hydrophobic surface. Ultra lLow attachment plate can be an ideal.

Another cheap and simple way is to coat plates with poly-HEMA (Poly (2-hydroxyethyl methacrylate). The following is a simple protocol.

1) Make 20 mg~50 mg/ml solution in ethanol. (Dissolve 2~5 g Poly-HEMA in 100 ml 95% ethanol overnight at 37 degree Celcius.

2) Clarify solution by centrifugation (2,000 x g, 20 min).

3) Apply 100 ul per 1 cm2 of poly-HEMA solution to culture plates. Let them dry very slowly. (Quick evaporation of alcohol leaves glassbead-like spots).  You can keep them for a few days.

4) Rinse the plate with PBS.

Try non-treated culture plates (suspension culture plates) or 2% agar coated plate and increase seeding density.

God Bless...

When doing organoid culture in the past I have used low attachment nunc plates - however this was with matrigel and I am not sure what your culture is like, but they worked well. Hope this helps!

Matrigel is a good choice.

This is very cell line dependant some gorw well with simple seeding in 96 well low attachment plates some require spinning in a spheroid spinner flask. What cell lines are you wanting to use?

Hi all,

Many thanks for all your generous replies! I am using HepG2 cell line at the moment.

I found a protocol using agarose/poly-HEMA to coat the well plates but my here's my other concerns (apart from having enough spheroids) in case I am using the coating protocol:

• Will the coating affect the microscopy? Can we get a good resolution picture of them?
• How do I harvest the cells? Can I just aspirate the whole thing and spin them down to get cell pellet, then Trizol the spheroid pellets?
• and how do I get the cells lysate if I am using RIPA buffer?

HI,

You can use agarose coating in your  well plate, make sure you make a very thin coating so that you can further do the microscopy as well. I would rather suggest to perform your experiments with 2nd or 3rd generation spheroids. So that you would rule out all the possibilities of having cellular clusters which are not really spheroids. Then You can spin down and the cells and use it for further analysis.

To get cell lysate you have to amplify the cultures in multiple plates.

Hope this helps.

Good Luck

poly HEMA coating is OK for microscopy. Yes, the cell resolution is OK. Even if coating generates a strange patterns (like glass bead-like image  in the bottom of the plates), simply you can transferyour spheres to a new vessels (plate) for taking pictures.

Cells are in suspension and you can simply harvest them with pipets.

After collecting spheres, wash (centrifuge) them to make pellets for subsequent study (trizol treatment for RNA or lysis for Western analysis)

Here's an alternative way of generating spheres from cultured cells.

Spinner culture flask. Buy one with glass (not single use, disposable). Flasks with different culture volumes (50, 100, 250, 500 ml and even liters) are available. Because it is glass, continuous stirring (100~250 revolution per min) with magnetic stirrer makes cells suspend and spontaneously aggregate to form spheres.

Thank you all, these are very helpful!!

There are also ultra-low attachment plates available, e.g. Costar/Corning and they come in different formats, e.g. 6-well, 96-well. They are not exactly cheap but look like a normal 6-well plate and you won't have any problems in imaging those on a microscope

Hi, I have done spheroids in spinoflast, hanging drop and 96 well plate. Both worked but 96 well plate is the one that give a higher number of spheroids being them more similar in size, they are more homogenous than with spniflast method and you can have more spheroids than with hanging drop. I usually coated my 96 well plate first with 1% agarose (with 100mL of 1% agarose and pipette it in and out to leave a thin film). It is not difficult and works well.

One important issue is the media. In DMEM media I achieve HCT116 and MCF7 cell line spheroids, but with other medias they did not form spheroids. Change the media every 48h to keep them growing properly.

Also, you may want to consider which size of spheroids you want to and which day may be the best to analyse them. Usually spheroids less than 400um do not have necrotic core.

You may have to seed different cell concentrations per well first and see which one is best for you. I would recommend to wait until day 4 at least, to have time for the cells to form good spheroids (see paper below).

I found useful this paper:

Spheroid-based drug screen: considerations and practical approach.

Friedrich J1, Seidel C, Ebner R, Kunz-Schughart LA.

Nat Protoc. 2009;4(3):309-24

Hi for anyone reading this post, I would like tom suggest another option: This is Happy Cell ASM http://valelifesciences.com/. This technology has been designed to allow the formation of Spheroids in a few days (depending on the cell type). Its very easy to use ....just add cells to the media and the spheroids will form spontaneously. This work can be conducted in a variety of lab ware (although if you are needing to use multi well plates I do recommend low attachment or non-tc treated micro plates). Also using Happy Cell ASM can result in improved cell viability and growth in primary cell cultures Article Short term ex-vivo expansion of circulating head and neck tumour cells

Anthony

Use non adherent plate and add high density of cell to form quick cluster or spheroids formation.

Best regards,

soosai

I used ultra low attachment (round bottom 96 well plates) but after several days when I wanna change their medium I see that cells are like a sheet and attached to the bottom of the well plate. Can anyone tell me weather they are still spheroids? should I keep them or they are just mass of the cells not spheroids? Although my well plates have not any attachment factors but they attach and make a cells sheet.

Hello Elmira. In the process of spheroid formation, cells have to change the expression of cadherins and integrins, and this allow the formation and compactation of the spheroid. If your cells stay as a sheet after several days in culture I would try first to coat my 1% agarose and if not you can use the hanging drop culture method, maybe the microgravity happening in the drop helps in the formation of the spheroid. Good Luck!!!

Elmira Keramatfar