Cultrex 3-D Culture Matrix Laminin I

Within the organoid, spheroid, or 3-D culture, cells may be assessed for morphology, apical/basal polarity, protein localization, and relative proliferation. In addition, cells may be isolated from the 3-D culture and evaluated for levels of RNA and protein expression, as well as modifications to DNA.

The recommended working concentration for thin coating is 0.05-10 µg/cm² . However, conditions must be optimized for each cell line or model. Cultrex Laminin I should be used at 6 mg/mL for 3-D culture applications.

Laminin I is a major component of extracellular matrix. Cultrex Laminin I is purified from murine EHS sarcoma. It is composed of α1β1γ1 chains with a total MW of 800,000 Da. Cultrex Laminin I increases cell adhesion, migration, growth, differentiation, neurite outgrowth, protease production, and malignancy. The response is dependent on cell type.

3-D cultures are in vitro cultures where immortalized cell lines, primary cell lines, stem cells, or tissue explants are placed within hydrogel matrices, such as Cultrex Basement Membrane Extract, that mimic in vivo cell environments and allow cells to proliferate in three dimensions.

While 2-D culture has been used for studying many aspects of cell function and behavior, the tissue-culture treated plastic environment is unlike anything found within living organisms. As a result, cells in 2-D culture exhibit altered morphology, function, proliferation, and gene expression when compared to their emanating tissues. By placing these cells in a 3-D environment, they assume biological and biochemical characteristics similar to what is observed in vivo.

The major variables associated with 3-D culture are cell type, cell seeding density, composition of hydrogel, thickness of hydrogel, stiffness of hydrogel, composition of cell culture medium, and time of culture.

The two principal methods for performing 3-D culture are the top assay and embedded assay. For the top assay, cells are seeded on a thick gel of Cultrex Basement Membrane Extract (BME) or Extracellular Matrix Protein. A thin overlay of cell culture medium is then applied to the cells. For the embedded assay, cells are resuspended within a thick gel of Cultrex BME or ECM and the culture media is applied on top. The top assay is easier to setup, to control seeding densities, and to keep cells within one focal plane for analysis.

Choice of matrix should correspond to the environment that you wish to recapitulate. Cultrex Basement Membrane Extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Cultrex Collagen I is the major constituent of connective tissue, and is commonly inhabited by stationary cells, such as fibrocytes and adipose cells, as well as migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.

Cells need to be healthy and actively dividing in 2-D culture. Cells should be passaged two or three times after resuspension from cryopreservation, and they should never surpass 80% confluency during each passage. Cells should also be assessed for viability using trypan blue, and they should exhibit less than 5% staining.