We use cookies to provide visitors of our website with the best possible experience. To learn more how we use cookies or how to block cookies, please visit our cookie policy.

Biocompare Bench Tip - 3D Cell Culture

Here we describe how to create a custom protocol incorporating optimized parameters for best performance with specific assay workflows.

 

Script:

Over the last decade a central focus for improving drug efficacy in clinical trials has been to increase the biological relevance of assays performed early in the drug discovery process. However it remains difficult to simulate a drug in vivo response using in in vitro assay.

Today we're going to focus on the importance of 3D cell culture and the impact it has had on advancing drug discovery research.

In vitro assays typically use cells grown on two-dimensional hard plastic or glass substrates which are not representative of the true in vivo cell environment. In tissue cells interact with neighboring cells and with the extracellular matrix or ECM to form a communication network that affects many cellular processes including proliferation, migration and apoptosis.

In a simplified in vitro 2D environment most of the tissue specific architecture and cell-cell communication cues are lost, therefore there is a need for advanced culture methods that better mimic cellular function within living tissue.

Three-dimensional cell culture methods provide a matrix that encourages  cells to organize into structures that resemble the in vivo environment thereby  developing normal cell to cell and cell ECM interactions in an in vitro environment.

What options do researchers have to aid in the formation of 3D cultured cells? One, there are polymeric hard scaffolds typically composed of a cross-linked plastic such as polystyrene. Two, there are biological scaffolds where the cells are combined with and then  suspended within a matrix of extracellular matrix proteins typically  found in vivo which provide structure and support to the cells, and three the spheroid model which is also very popular as it can mimic both tissues and tumors depending on the included cell types pre-made spheroids or micro tissues can be provided to you in microtiter plates to simplify or even  eliminate the cell culturing process.

If costs are a hurdle for you or you want to have more control over your culturing process there are two more options to create your own spheroid models. First there is the hanging drop where cells are suspended in a 30 to 50 micro liter drop which literally hangs below the bottom of the well. Cell aggregation then occurs in the drop over time. Another popular method is to use microplates coated with an ultra low attachment or ULA surface. These plates look and behave much like other round bottom plates. However when the cells are added the ULA coating prevents attachment to the bottom of the well which promotes the cells to aggregate together into a spheroid structure, and as the spheroids are already in the bottom of the micro plate well addition of 3D overlays such as soft auger or Matrigel Matrix is easily accomplished.

Once you have selected the culturing format you'll need to think about how to collect the information that will answer your questions. Are you looking to monitor a cellular response to an experimental treatment or are you focused on learning about detailed changes in the cellular morphology. Or it could be a combination of both of these. Here are two methods that can be used to collect information on your cells. They provide different results so it really depends on the level of detail you are looking for to achieve and answering your questions. Microplate readers which typically employ a PMT based detector provide a rapid means to make Holwell measurements. Experiments performed on 3D cultured cells and commonly run on micro plate readers include cytotoxicity and signal  transduction assays. On the other hand, microscopes and cell imagers increase the amount of information attained from each experiment through the use of widefield microscopy by incorporating magnification through the use of appropriate objectives. Unnecessary areas of the well are excluded from examination increasing the detail of the image being viewed as well as the data generated.

High content imagers provide the greatest quantity and breath of imaging based data. Automation of the imaging process in addition to the inclusion of high content analysis software allows image acquisition and analysis to be performed in a high throughput manner. If you need to combine multiple functionalities to obtain your results there are options available to  integrate microscopy with multimode microplate reading can provide both phenotypic and quantitative results. From a single precious sample multiple pieces of information can be captured but quickly and easily.  Depending on your research needs a multifunctional instrument may be the right choice for you.

3D cell culture is an exciting and important experimental tool that has enabled researchers to better simulate living tissue in an in vitro environment. You have many options to choose from in regards to 3D culturing methods and the level of detail required in the data collected while it can be difficult to navigate through the available choices. Once you make your selection the results you can get from using this technique will be truly invaluable.

 

link