So with the hanging drop method, the plates are designed to fit over the top of a 96 well plate.  This is so that you can place the plate on top of a 96-well and then use media or PBS added from the top of the hanging drop apparatus to push the culture containing drops out of the bottom of the hanging drop into your 96 well plate.  

Another method that you can use is encapsulation of cells within sodium alginate beads.  Its pretty simple and works well for adherent cells.  You merely combine a given number of cells in about 250 uL of media (I use about a million but you can get cell densities very high with this method) with 750uL culture grade sodium alginate, then using a syringe drop that mixture into a well of a 6 well plate containing Ca2+ enriched (100 mM) media (you add culture grade Ca to normal culture media yourself).  The Ca polymerizes the alginate forming an easily manipulated bead (you can actually use a small scoop to move the beads from well to well for your experiments.  Then you wait five minutes and wash 3x for five min each with normal media and you have your 3D cultures.

Finally, I have used matrigel, but this is pretty expensive so I really only use it for more advanced cultures such as making organoids.  For this you can simply let the matrigell thaw on ice for about two to three hours combine it with the cell types and densities you desire and put it in a prewarmed plate.  Unlike with other applications of matrigel (such as invasion assays), making 3D cultures with matrigel is best done with matrigel that has not been diluted with the media prior to solidification.  We have found that U bottom 96 well plates work well (especially if your reagents are expensive).  The cell densities I am using are ranging from 5K to 50K cells per well depending on the needs of the experiment and the number of cell types I am including in the culture (typically I am limiting the number of cancer cells to be at or under 15K).  Also note that using this method, a few of the cells will stick to the bottom of the well, but we have not found that this is a big problem.  

I hope this helps 

Best, 

Andrew 

I will start by saying that I have no experience with A549 cells, but that I have worked with a wide variety of 3D culture systems in a variety of settings mainly with jurkat cells, and pancreatic cancer cell lines.  I think there are several options that you can consider, but before I go on and list a bunch of methods, some of which may or may not work for you, I think it would be very helpful for both of us if you give a little bit more information as to why the 3D culture methods you have tried so far have not worked, and how you know this to be the case. 

Andrew

Hi matrigel working good so you can try either 6,12 and 24 well plates, use martrigel from corning company and follow the instructioncarefully. see the link from Harvard

http://brugge.med.harvard.edu/protocols

Recently Lonza company also have 3D reageants try . good luck

You can use rotating wall vessel.

http://www.nature.com/nrmicro/journal/v8/n11/abs/nrmicro2423.html

Dear Andrew,

The culture in the 96 well plate did not work because they cells stuck to the bottom even though the wells are U-shaped and despite using methylcellulose (I tried different concentrations from .25 to 2%) as well as agarose, but we could not figure out why.

The hanging drop method failed because they spehroids kept breaking when i tried to pipette them to transfer them to the 96 well-plate.

I would like to know the seeding density you normally use, that might be helpful.

thank you a lot Mr. Panchanathan and Pourashghary

So with the hanging drop method, the plates are designed to fit over the top of a 96 well plate.  This is so that you can place the plate on top of a 96-well and then use media or PBS added from the top of the hanging drop apparatus to push the culture containing drops out of the bottom of the hanging drop into your 96 well plate.  

Another method that you can use is encapsulation of cells within sodium alginate beads.  Its pretty simple and works well for adherent cells.  You merely combine a given number of cells in about 250 uL of media (I use about a million but you can get cell densities very high with this method) with 750uL culture grade sodium alginate, then using a syringe drop that mixture into a well of a 6 well plate containing Ca2+ enriched (100 mM) media (you add culture grade Ca to normal culture media yourself).  The Ca polymerizes the alginate forming an easily manipulated bead (you can actually use a small scoop to move the beads from well to well for your experiments.  Then you wait five minutes and wash 3x for five min each with normal media and you have your 3D cultures.

Finally, I have used matrigel, but this is pretty expensive so I really only use it for more advanced cultures such as making organoids.  For this you can simply let the matrigell thaw on ice for about two to three hours combine it with the cell types and densities you desire and put it in a prewarmed plate.  Unlike with other applications of matrigel (such as invasion assays), making 3D cultures with matrigel is best done with matrigel that has not been diluted with the media prior to solidification.  We have found that U bottom 96 well plates work well (especially if your reagents are expensive).  The cell densities I am using are ranging from 5K to 50K cells per well depending on the needs of the experiment and the number of cell types I am including in the culture (typically I am limiting the number of cancer cells to be at or under 15K).  Also note that using this method, a few of the cells will stick to the bottom of the well, but we have not found that this is a big problem.  

I hope this helps 

Best, 

Andrew 

Also, sorry I omitted this from my first post, but the amount of matrigel I suspend my cells in is 25 uL per well in U bottom, 96-well plate.

Have you tried preventing adhesion to the bottom of the plates by pre-coating the plates with polyHEMA?

I'm also interested in finding out how to transfer spheroids without destroying them with the pipetting action, so will be watching this thread avidly!

Thanks

Mhairi

We have not mainly because this has not been a big problem (when we use the U bottom plates we are really only focusing on the development of architecture within the droplet of matrigel).  Once we start developing functional assays for these advanced cultures we might care a little more or If in these U-bottom plates we were trying to develop tumor spheroids (rather than organoids) this adhesion would be a bigger problem as adhesion to the bottom would really detract from the formation of the spheroid itself (your spheroid would essentially lack at least one of the cell components you are interested in.  I have dealt with this type of problem before using stromal cell line cultured with malignant T-cells.  In this case the main way we overcame adhesion to the plate was using the hanging drop method.  Finally, to move the organoids in matrigel we plate them on top of cover slips and move the cover slip rather than pipetting.  We are still trying to develop methods to use the 96 well plates for experiments were the cultures must be moved prior without destroying tissue architecture.  One thing is for sure though, pipetting is not the solution to this.