How does Cultrex® Basement Membrane Extract (BME) promote cell differentiation?

All epithelial and endothelial cells are in contact with a basement membrane matrix on at least one of their surfaces. By providing them with their natural matrix in vitro as a substrate for the cells that provides biological cues, the cells can assume a more physiological morphology (i.e. correct shape) and begin expression of cell-lineage specific proteins. Two-dimensional plastic surfaces, in combination with serum-containing media, cause cells to flatten, proliferate and de-differentiate.

How should cells be cultured prior to setting up the 3-D culture?

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.

What are 3-D cultures?

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.

What are the different types of 3-D 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.

What are the variables associated with 3-D culture?

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.

What is the advantage of 3-D culture over traditional 2-D culture?

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.

What is the difference between Cultrex® Basement Membrane Extract (BME) and Cultrex® 3D Culture Matrix?

Cultrex® 3D Culture Matrix was developed to provide the most standardized basement membrane extract for use in 3D Cultures. A special process is employed to reduce growth factors. This material is then incorporated in a 3D culture to validate efficacy. 3D Culture Basement Membrane Extract promotes differentiation of a human epithelial cell line derived from mammary gland (MCF-10A) and human prostate (PC-3) into acinar structures. The Cultrex® 3D Culture Matrix is essentially the same as our standard Cultrex® BME, but has been additionally qualified with the functional 3D assay as described above.

What type of analysis is typically applied for organoid or 3-D cell cultures?

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.

What type of plates are recommended?

In order for the Cultrex 3-D Culture Matrix RGF BME to stick to the plate, it must be tissue-culture treated. However, if the application is spheroids or when using BME diluted in media as a slurry to form floating 3-D structures, then low attachment plates are recommended.

Cultrex 3-D Culture Matrix Reduced Growth Factor Basement Membrane Extract, Pathclear

Name: Cultrex 3-D Culture Matrix Reduced Growth Factor Basement Membrane Extract, Pathclear
Brand: R&D Systems
SKU: 3445-010-01
Rating: 5 (6 reviews)

R&D Systems, part of Bio-Techne | Catalog # 3445-010-01

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Product Datasheet / COA / SDS

Reduced Growth Factor Basement Membrane Extract for 3-D Cell Culture

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Product Summary
Product Specifications
Preparation & Storage
Background
Product Documents

Key Product Details

Features

Cultrex 3-D Culture Matrix RGF BME is an extracellular matrix hydrogel that directs cells to grow in three dimensions and assemble into organotypic structures in vitro.

Key Benefits

Qualified to support 3-D cell culture applications
Reduced growth factor formulation provides a more defined culture system
Quality controlled for performance consistency

Species

Mouse

Product Summary

Why Use Cultrex 3-D Culture Matrix RGF BME ?

Cultrex 3-D Culture Matrix Reduced Factor Basement Membrane Extract (RGF BME) is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor. This extract provides a natural extracellular matrix hydrogel that polymerizes at 37°C to form a reconstituted basement membrane. Cultrex 3-D Culture Matrix RGF BME is produced and qualified specifically for use in 3-D culture studies. It provides the foundation for cells to grow in three dimensions allowing for the formation of complex in vitro structures. To provide the most standardized basement membrane extract for use in 3-D cultures, a special process is employed to reduce growth factors and manufacture matrix at a standard and consistent concentration. This product is then evaluated in 3-D culture assays to validate efficacy. Basement membranes are continuous sheets of specialized extracellular matrix that form an interface between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma and that play an essential role in tissue organization by influencing cell adhesion, migration, proliferation, and differentiation. The major components of BME include laminin, collagen IV, entactin, and heparin sulfate proteoglycans.

Product Specifications

Source

Murine Engelbreth-Holm-Swarm (EHS) tumor

Protein Concentration

8-12 mg/mL as determined by Lowry assay

Endotoxin Level

≤ 8 EU/mL by Limulus Amoebocyte Lysate (LAL) assay

Sterility Testing

No bacterial or fungal growth detected following 14 days in culture

Cell Culture Testing

3-D Culture - Cultrex 3-D Culture Matrix RGF BME promotes differentiation of a human epithelial cell line derived from mammary gland (MCF-10A) or human prostate (PC-3) into acinar structures.

Tube Formation Assay - Cultrex 3-D Culture Matrix RGF BME Supports formation of capillary-like structures by human (HBMVEC; HUVEC) or mouse (SVEC4-10) endothelial cells.

Gelling Assay - Cultrex 3-D Culture Matrix RGF BME gels in less than 30 minutes at 37 °C, and maintains the gelled form in culture medium for a minimum of 7 days at 37 °C.

Viral Testing

Tested negative by PCR test for a total of 31 organisms and viruses, including: mycoplasma, 17 bacterial and virus strains typically included in mouse antibody production (MAP) testing, and 13 additional murine infectious agents including LDEV.

Formulation, Preparation, and Storage

Shipping

The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended on the product label.

Storage

Store the unopened product at -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles.

Stability

Product is stable for at least two years from date of manufacture when stored at ≤ -70 °C. See lot specific Certificate of Analysis for expiration date.

Background: Basement Membrane Extracts

Basement membrane extract (BME) is a type of extracellular matrix (ECM) derived from Engelbreth-Holm-Swarm mouse sarcomas. BME is commonly used in cell culture and tissue engineering to provide a 2-D or 3-D scaffold for cells, allowing them to grow, differentiate, and interact in an environment that closely mimics the native ECM. This makes BME invaluable for modeling the behavior of cells in vivo. All Cultrex BME formulations are rigorously tested to ensure performance, quality, and lot-to-lot consistency.