Cancer Research

Assuming you are looking to culture a cancer organoid in the apical chamber of Corning® FluoroBlok™ Cell Culture Inserts and measure immune function, then the answer is most likely.  Although it does not appear much published work has been done in this area, FluoroBlok inserts are a great choice when one is looking to exclude fluorescent signal generated by cells on the apical surface of the membrane.  Even though they are typically used for migration and invasion assays, they may find some use here. It is also possible to combine Corning 96HTS inserts with the Corning spheroid microplate in order to look at immune cell migration/invasion towards the cancer organoid. Corning scientists have done a similar assay with NK92 cells and a lung cancer cell line.

Please find some useful links below:

Video: An In vitro FluoroBlok Tumor Invasion Assay

Webinar: Applications of Corning® FluoroBlok™ Permeable Supports

Review Article: Culture and Assay Systems Used for 3D Cell Culture

Poster: A Novel Spheroid-based Three-dimensional Invasion Model for Evaluating Potentially Anti-tumor Compounds

Application Note: A Novel Three Dimensional Immune Oncology Model for High Throughput Testing of Tumoricidal Capability

Product Information:

FluoroBlok Cell Culture Inserts

Corning® FluoroBlok™ Inserts Frequently Asked Questions

Although Corning does not have any DU145 specific transfection protocols, we do know that DU145 as well as many other prostate lines behave differently in 3D as opposed to 2D. 3D transfection is possible in Corning® Matrigel® matrix, as well as in Corning Ultra Low Attachment vessels and Corning spheroid microplates. There are several protocols for 3D transfection that can be found in the literature as well as some commercially available 3D transfection kits. We’ve included some helpful links below:

Influence of Matrices on 3D-Cultured Prostate Cancer Cells' Drug Response and Expression of Drug-Action Associated Proteins

Transfection of human prostate cancer cells (DU145) with the human short prolactin receptor sensitizes the cells to cell cycle regulation by S179D prolactin

DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: Implications for the androgen receptor functions and regulation

Gene transfection to spheroid culture system on micropatterned culture plate by polyplex nanomicelle: a novel platform of genetically-modified cell transplantation

Protein Transfection Study Using Multicellular Tumor Spheroids of Human Hepatoma Huh-7 Cells

There are numerous options for high-throughput screening spheroids. The right option will be dependent on what you want to measure and the instrument available to you. A good homogenous method for assessing viability is using Promega’s CellTiter-Glo 3D reagent to measure ATP. It’s important to use the 3D version of the reagent since spheroids typically require stronger lytic reagents than what is required for a 2D monolayer assay. It’s also possible to use high throughput imagers via phase or fluorescent microscopy and image analysis software to quantify changes in spheroid size, metabolic activity and fluorescent staining.

Please find some useful links below:

Drug screening in 3D in vitro tumor models: overcoming current pitfalls of efficacy read-outs

High-Throughput Confocal Imaging of 3D Spheroids

CAR-T Cell Screening in Tumor Spheroids Using Corning Spheroid Microplates

Spheroid Formation Protocol

There are many exciting things happening in the cancer research space, but two areas that we feel will have the largest impact on cancer research in the next few years are through improved modeling and superior imaging. An interesting trend we have noticed, is that the technologies are not necessarily all new, but have been reimagined by researchers to be used in new and innovative applications. With tools such as Ultra-low Attachment microplates and ECM’s like Corning® Matrigel® matrix researchers can create 3D cancer models that are more complex, and what’s more is that they are easier to develop and are more consistent than ever. These 3D models do a much better job capturing the complexity of the cancer environment compared to more traditionally used 2D models. Imagers that have confocal capabilities are also likely to play an important role in cancer research and personalized medicine as they can deliver so much more information than instrumentation of the past.

There are several different formats of laminin available. Here is an overview of what Corning offers, whether vialed (murine), coated, or recombinant (human).

1. Vialed products (mouse)

Corning® Laminin (mouse) is used as a thin coating on tissue-culture surfaces or as a soluble additive to culture medium. Laminin is a major component of basement membranes. It has numerous biological activities including promotion of cell adhesion, migration, chemotaxis, growth, and differentiation, including neurite outgrowth.

Here are links with more information on the offerings from Corning:

Laminin

Laminin/Entactin High Concentration

2. BioCoat™ surfaces

Use Corning® Laminin surfaces as a thin coating on tissue culture surfaces or as a soluble additive to culture medium to promote cell adhesion, migration, chemotaxis, growth, and differentiation. Laminin/Entactin Complex High Concentration (HC) is a special formulation that Corning has developed to enhance three-dimensional (3D) cell culture applications. Culturing cells in or on gels such as Laminin/Entactin Complex enable the study of specific mechanisms that dictate cell differentiation and functionality.

Applications include:

• Promotion of neurite outgrowth
• Reduction of fibroblast migration in neuronal cell cultures
• Neural crest cell attachment
• Gene expression in rat cortical neurons
• Maintenance of undifferentiated pluripotent stem cells

To learn more about these Corning surfaces, please use the links below:

Corning Laminin Coating Surfaces

Corning BioCoat Surfaces

Specifications and ordering information:

Corning BioCoat Surfaces

Corning Laminin Coating Surfaces

3. rLaminin-521 (human)  

Corning rLaminin-521 (Human) is a heterotrimer composed of α5, β2, and γ1 chains expressed in a mammalian cell culture system. rLaminin-521 (Human) supports long-term self-renewal of human pluripotent stem cells (hPSC), including embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) in defined and xeno-free environments. rLaminin-521 provides additional benefits, including ROCK inhibitor independent single cell expansion of PSCs, which improves hPSC culture ease and efficiency.  Corning rlaminin-521 is available in vialed formats as well as pre-coated 6-well plates.

Related Application Notes:

Corning® PureCoat™ rLaminin-521 Cultureware: A Ready-to-use, Animal-free Vessel Platform for the Culture and Single Cell Passaging of Human Pluripotent Stem Cells

Corning® PureCoat™ rLaminin-521 (Human) for Expansion and Differentiation of Human Neural Stem Cells

To learn more about this Corning surface, please use the link below:

Corning rLaminin-521, Human

Specifications and ordering information:
Corning® rLaminin-521, Human

A great option for this type of work would be ultra-low attachment plates like the Corning® spheroid microplate as an optimal cell culture vessel. They are easy to use, promote formation of one organoid per well of uniform size and allow for fluorescent, luminescent and imaging assays to be conducted right in the culture plate. As far as ECMs, Corning Matrigel® matrix has always been considered the gold standard. It is the most cited ECM and is routinely used for 3D cell culture applications. Download the Ultimate Guide to Matrigel matrix or visit Corning’s website to learn more.

Hoechst is great for counting all cells since it stains all nuclei (live and dead cells). To enumerate dead cells we routinely use Propidium iodide (PI). PI is a membrane impermeable dye that is excluded form viable cell membranes. If your cells are positive for PI they are most likely dead or apoptotic. Both stains are incorporated very quickly into cells at low concentrations.

The first step in B cell isolation is to isolate mononuclear cells from whole blood through centrifugation with a Ficoll gradient. Here is a protocol for reference that uses Corning Lymphocyte Separation Medium as part of this process. There are many different B cell isolation kits that are commercially available, however, you will find it difficult to find one that does not use magnetic beads as part of the isolation process.

One of the simplest methods to create co-culture spheroids containing multiple cell types is to seed the different cell types together into a low attachment round bottom plate such as the Corning® spheroid microplate. The cells can be seeded at the same time or added at different time points. Here is an application note as an example that uses tumor cells from a lung cancer cell line and primary human lung fibroblasts for reference. In this example, PBMCs were also included as a treatment in the wells, but were not shown to be incorporated into the spheroid. When working with multiple cell types some optimization may be required. For example, different media conditions may need to be tested and some models may require the addition of extracellular matrices such as Corning Matrigel® matrix. The cells may also have different doubling rates so cell seeding densities for each cell type may need optimization as well. You can also access this guidelines for use document on considerations for 3D cell culture as another useful resource.