One option for 3D culture, or anchorage independent culture is to perform a soft agar assay. Here is one protocol: http://www.lbl.gov/LBL-Programs/lifesciences/BissellLab/labprotocols/softagar.htm

There are quite a few variations of this protocol which can be found with a quick internet search.

What kind of 3D cell culture you would like to do? To use the ready-to-use culture products or to produce 3D cell culture with conventional 2D technique?

More information please!

Techniques range from having singles cancer cells in 3-D environments to making 3-D spheroids with the cells themselves, to growing 3-D colonies from single cells... there are many, many 3-D culture methods.

One option for 3D culture, or anchorage independent culture is to perform a soft agar assay. Here is one protocol: http://www.lbl.gov/LBL-Programs/lifesciences/BissellLab/labprotocols/softagar.htm

There are quite a few variations of this protocol which can be found with a quick internet search.

we use spheroids made by the hanging drop method. If you google hanging drop and spheroids you will find lots of articles

We use BD Biosciences Matrigel for prostate cancer cell-lines. In addition to the plethora of information available in the literature regarding this method, our adapted protocol is outlined in our recently published article . In regards to "what methods of 3D culture is best" depends on what you are investigating. More info is needed...

Hela cells grow extremely well in softagar and can be used in xenograft assays. Actually we use Hela as positive controls for those assays. Sphere assays are usually used for either breast or prostate cancer cells, and rarely used for cervical cancer lines. Air-liquid interphase 3D assay works for normal cervical and other epithelial cells, but not Hela.

Thank you every one for your kind suggestion

Here's a protocol for the hanging droplet technique which works very well for most cell lines:

Hanging Drop (procedure Kelm et al (2002)). Cells were harvested in medium at a concentration of 1 X 106 cells/ml. From this 10 – 15 drops, each containing a volume of 20µl, was placed on the lid of a petri dish, which was then inverted over a dish containing 10ml of sterile water. The dish was then incubated at 37°C. Within 24-48hrs (depending on the cell line used), the cells will have formed aggregates. Using a pipette, aggregates were gently transferred to a fresh plate coated with agar (prevents attachment) and 10ml media. This dish was incubated at 37°C. After a period of 24-48hrs, the aggregates, unable to adhere to the dish, will have formed dense spheroids.

Another option is to seed cells into either a spinner or shake flask at low speed. When they become confluent spheroids will usually form.

Perhaps, another method that is perhaps less laborious because it can be automated at least in part, is to prepare a stack of layered cells from single layers, thus having a tessellation, or grid that allows the sequential construction of one layer at a time, and then joining the grid layers by pressing them gently together. This would also allow one to check for reproducibility and the interactions between the individual cell layers. As grids one might be able to use coated electron microscope grids, or a plastic mesh that can be easily sterilized and kept sterile while an automatic sorter/pipette can add the cells one at a time to the grid. Awhile ago we were thinking of using this method to produce a model of an artery from smooth muscle cells so that we could follow the atherosclerosis process in a controlled cell culture environment. To summarize, the basic idea is to build up the 3D from many 2D layers that have been well-studied and are reported in the literature on cell cultures on supports.

If it is about Hela cells, a cervical epithelial cells, it is important to have a 3D culture which can mimic the epithelial cell differentiation process. I am highly encourage everybody take a look at the air-liquid interphase system. One of the example is from a Swiss company called CellnTec.