In 3D cell culture models, cells are grown under conditions that allow the formation of multicellular spheroids or microtissues. Instead of growing in a monolayer on a plate surface, cells in 3D culture grow within a support matrix that allows them to interact with each other, forming cell:cell connections and creating an environment that mimics the situation in the body more closely than traditional 2D systems. Although 3D cultures are designed to offer a more physiologically accurate environment, the added complexity of that environment can also present challenges to experimental design when performing cell-based assays. For example, it can be a challenge for assay reagents to penetrate to the center of larger microtissues and for lytic assays to disrupt all cells within the 3D system.
Earlier this week Terry Riss, a Senior Product Specialist at Promega, presented a Webinar on the challenges of performing cell-based assays on microtissues in 3D cell culture. During the Webinar, Terry gave an overview of the different methods available for 3D cell culture, providing a description of the advantages of each. He then discussed considerations for designing and optimizing cell-based assays for use in 3D culture systems, providing several recommendations to keep in mind when performing cell viability assays on larger microtissue samples.
This blog is written by guest author, Maggie Bach, Sr. Product Manager, Promega Corporation.
Researchers are increasingly relying on cells grown in three-dimensional (3D) structures to help answer their research questions. Monolayer, or 2D cell culture, was the go-to cell culture method for the past century. Now, the need to better represent in vivo conditions is driving the adoption of 3D cell culture models. Cells grown in 3D structures better mimic tissue-like structures, better exhibit differentiated cellular functions, and better predict in vivo responses to drug treatment.
Switching to 3D cell culture models comes with challenges. Methods to interrogate these models need to be adaptable and reliable for the many types of 3D models. Some of the most popular 3D models include spheroids grown in ultra-low attachment plates, and cells grown in an extracellular matrix, such as Matrigel® from Corning. Even more complex models include medium flow over the cells in microfluidic or organ-on-a-chip devices. Will an assay originally developed for cells grown in monolayer perform consistently with various 3D models? How is measuring a cellular marker different when cells are grown in 3D models compared to monolayer growth?
Snakebite is a serious public health issue in many tropical countries. Every year, roughly 2 million cases of poisoning from snakebites occur, and more than 100,000 people die. Snake venom is extremely complex, containing a cocktail of chemicals, many of which are undefined. This complicates the development of new therapeutics for treating snakebite.
Antivenom is the most effective treatment for snakebites,
but its production is complex and dangerous. It involves manually milking the
venom from different species of live snakes, then injecting small doses of the
venom into animals (mostly horses) to stimulate an immune response. After a
period of time, antibodies form in the animal’s blood, which is purified for
use as antivenom.
But what if we could produce snake venom in the lab, instead
of using live snakes? Recently, a group from the Netherlands did just that by
growing organoids derived from snake venom glands.
Traditionally, scientists have relied on flat,
two-dimensional cell cultures grown on substrates such as tissue culture
polystyrene (TCPS) to study cellular physiology. These models are simple and
cost-effective to culture and process. Within the last decade, however, three-dimensional
(3D) cell cultures have become increasingly popular because they are more
physiologically relevant and better represent in vivo conditions.
Tissue culture using primary or cultured cell lines has long been a mainstay of testing compounds for inhibiting cell growth or promoting apoptosis during screening for cancer drugs. However, the standard culture conditions result in monolayers of cells, dividing and growing across the bottom of a well, plate or flask in a single layer. The drawback of this technique is that organisms do not come in monolayers; a three-dimensional (3D) spheroid is closer to the in vivo state, especially if the spheroids are made up of more than one cell type like tumors in multicellular organisms. Even more beneficial would be using 3D cultured cells in high-throughput screening to facilitate compound profiling for target effectiveness and cytotoxicity. In a recent PLOS ONE article, researchers used normal and breast cancer cells both in monoculture and coculture to test a set of compounds and found results differed between 2D and 3D cultured cells. Continue reading “Improving Cancer Drug Screening with 3D Cell Culture”
Based on the Illuminations article by Dr. Terry Riss, from our Cellular Analysis group.
Choosing the most appropriate cell health assay for your experiment can be difficult. There are several factors to consider when choosing an assay: the question you are asking, the nature of your sample, the number of samples being tested, the required sensitivity, the nature of the sample, the plates and plate readers and the reagent costs.
What question are you asking?
The first, and perhaps most important factor to consider, is the question you need answered. What do you want to know at the end of the experiment? There are cell health assays available that specifically detect the number of living cells, the number of dead cells, and for assessing stress response mechanisms or pathways that may lead to cell death. Matching the assay endpoint to the information you need is vital to choosing the appropriate cell health assay. Continue reading “Choosing the Right Cell Health Assay”
A quick search of the PubMed database for “dual luciferase” quickly returns over 1,000 papers. The Dual-Luciferase® Reporter Assay is a powerful tool that allows researchers to ask a multitude of questions about gene control and expression in a system that itself could be normalized and internally controlled. For more than 15 years, firefly and Renilla luciferases have formed the basis of a range of powerful assays and research tools for scientists who are asking questions about the deep and complex genetic and cellular story associated with cancer. Here we talk a bit of about bioluminescent chemistries, some of the newest bioluminescent tools available, and how some of these tools can be used to probe the deeper questions of cell biology, including cancer biology. Continue reading “Shining a Bright Light on Deep Questions in Biology with Bioluminescence”
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