Comparison of 2D and 3D cell culturing:

Cell cultures are a widely used in vitro tool for understanding cell biology, tissue morphology and disease mechanisms, as well as drug effects, protein production and tissue engineering development. Most cell research is based on experiments using two-dimensional (2D) in vitro cultures. However, 2D cultures have many limitations, such as disrupting the interaction between the cellular and extracellular environment, as well as changes in cell morphology, polarity and partitioning. These disadvantages have led to the creation of new models that mimic physiological conditions much better. One of these new methods is the development of three-dimensional (3D) cultures using a hydrogel with a structure which resembles the extracellular matrix. Cells grown on hydrogels are in many respects very similar to those grown in vivo, e.g. in terms of morphology, the gene expression and protein profile, and cell communication. Optimizing such conditions in culturing allows a better understanding of the biology of cells, such as cancer cells, and can also facilitate the study of biomarkers and targeted therapies.

Comparison of the cultivation of cells in 2D and 3D conditions: This service consists of plating cells into two 48-well plates, one with and one without LifeGel. The most commonly used tiles are panels from different LifeGels, which means that clients can get a comparison between gels with different density and hardness parameters. The cells are cultivated for a fixed number of days (as required by the client) and the experiment ends with imaging of the cells with a bright-field microscope. The photos are then analyzed and sent to the client in the form of a report. It is possible to carry out additional tests and analyses by prior arrangement. This service applies to both cell lines and primary cells provided by the client and is particularly important for those people who are just starting their experience with 3D cultures and want to compare the two types of cell cultivation.

Sample results from the comparison of 2D and 3D cell culture:

Pancreatic tumor cultured in 2D conditions

Pancreatic tumor cultured in 3D conditions

Conducting long-term cell cultures:

The long-term cell culture test measures the behavior of cells after stimulation with external factors at a given time. Many toxic substances or medicines such as anti-cancer drugs are administered to the body at intervals. Cells exposed to external stimulation in this way may alter their morphology, function or behavior relative to each other depending on the duration of the therapy/exposure and its frequency. Standard 2D cultures allow only one-time administration of a dose of the test substance, but with 3D cultures it is possible to administer several doses of the test substance at specified intervals.
This service is based on detailed arrangements and the client’s specific requirements. For example, the cells are seeded into a 48-well plate with the appropriate type of LifeGel (as required by the client). Then, at specified time intervals, the test substance is administered to the culture and the cells are imaged using a bright-field microscope after a given exposure time. This activity is repeated until the culturing process is terminated (at a time determined by the client, also dependent on the type of cells being tested). The photos are then analyzed and sent to the client in the form of a report. Other tests and analyses can be carried out by prior arrangement. This service applies to both cell lines and primary cells provided by the client, and it is designed for people who want to examine, for example, the impact on a cell culture of a given substance administered two or three times at various intervals. Such culturing can also be used to achieve a much larger number of cells in one culture than when carrying out standard 2D culturing.

Breast cancer cultures using LifeGel; Days of breeding: A- 14 days, B- 17 days; C- 20 days

Angiogenesis test:

Angiogenesis, or the formation of new blood vessels, is a key process in certain physiological conditions, such as wound healing and the growth and function of the female reproductive organs. In addition, disorders of the mechanisms of physiological angiogenesis in the form of the excessive spread of blood vessels can play an important role in the pathogenesis of certain diseases, such as cancer, psoriasis, arthritis, obesity, asthma and atherosclerosis. Impairment of angiogenesis is also observed during heart or brain ischemia, hypertension, osteoporosis, as well as neurodegenerative diseases. Research into angiogenesis provides an opportunity to treat these above-mentioned diseases and is a very important element when testing new drugs.

The angiogenesis test is used to determine the ability of vascular endothelial cells to form capillary-like structures. In this study, cells (e.g. HUVEC) are plated into a 48-well plate with the appropriate LifeGel in a dedicated culture medium for this assay, and then, kept in the culture for about 24 hours, they form capillary structures. The experiment ends with the imaging of the capillaries with a bright-field microscope, with the photos able to be analyzed using dedicated image analysis software. The photos are then analyzed and sent to the client in the form of a report. Other tests and analyses can be carried out by prior arrangement. This test is particularly important for people testing anti-cancer drugs designed to inhibit angiogenesis.

Forming blood vessels with LifeGel (HUVEC)

Aortic ring test:

Angiogenesis, or the growth of blood vessels from the pre-existing vascular system, is associated with both natural and pathological processes. Different angiogenesis assays involve the study of individual endothelial cells under culture conditions. An aortic ring examination is a model of angiogenesis based on organ culture. In this test, angiogenic vessels grow from the aortic segment. An aortic ring test is a useful tool for assessing angiogenic, as well as anti-angiogenic, factors.

This test is similar to the angiogenesis test but it uses a sample of aortic tissue instead. In this test, the aortic ring is placed on the appropriate LifeGel, after which the culture medium for the test is added. After a few days, cells of the blood vessels migrating from the aortic ring are observed. Carried out on a 48-well plate, the experiment ends with the imaging of the capillaries with a bright-field microscope. The photos are then analyzed and sent to the client in the form of a report. Other tests and analyses can be carried out by prior arrangement. This test is particularly important for people testing anti-cancer drugs designed to inhibit angiogenesis.

Aortic ring on LifeGel

Intestinal permeability test:

The intestinal permeability test allows you to assess the level of absorption and bioavailability of a given substance in the intestine. The small and large intestines function in a unique way as a digestive/absorption organ for nutrients, as well as a strong immunological and mechanical barrier preventing the excessive absorption of bacteria, food antigens and other macromolecules. Both malabsorption and increased intestinal permeability are associated with chronic gastrointestinal disturbances, as well as many systemic disorders. Human cell cultures of in vitro enterocytes allow the use of cell lines as models in studies on transmembrane transport. This test assesses the extent to which the substances are transported by the intestinal cells. The most commonly used cell line is Caco-2, which are cells derived from human colon adenoma (large intestine) and characterized by adherent growth (meaning they form permanent connections to the ground). Caco-2 cells display many morphological and biochemical similarities to intestinal cells – enterocytes. A distinctive feature of Caco-2 cells compared to other cell lines is their ability to create a brush border, i.e. a microvilli-covered cell surface.

This test is performed on Transwell inserts that are coated with LifeGel and provide a very good base for the growth and differentiation of Caco-2 cells. After approximately 21 days of cultivation, a test is carried out using selected molecules. The use of the Transwell inserts allows samples to be taken from the apical portion (above the shed cells, where the test molecule is administered) and from the base part (under the seeded cells, the molecule under investigation must be transported by the cells). The collected samples can be analyzed for the concentration of the tested molecule.

Intercellular migration test:

Migration is a key property of living cells and is very important for proper development and immune response, as well as in disease processes such as tumor metastasis. Methods for testing cell migration are very useful in a wide range of biomedical research areas, such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Studying cell migration in cancer research is particularly important because metastasis is one of the leading causes of death in cancer patients. When the cancer spreads throughout the body, the cancer cells migrate and attack the extracellular matrix (ECM).

This test is performed on 48-well plates covered with the appropriate type of LifeGel which provides cells with the ability to migrate and communicate. The duration of the test is selected individually according to the client's needs and specific experience, and the experiment ends with imaging of the spheroids formed in the gel, showing the degree of movement between neighboring spheroids or towards the chemoattractant.

Other services:

3D cell cultures are an increasingly used tool in modern research. Our goal is to enable research using 3D culturing on a large scale. Due to our many years of experience, we can help to create individual research models and develop research techniques using 3D cultures for our clients’ needs.