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 Cultrex Basement Membrane Extract (BME) be stored and handled?

For best practices for handling our BME products, please refer to our helpful guide at the following link: https://www.bio-techne.com/reagents/cell-culture-reagents/cultrex-bme-and-ecm-proteins/cell-culture-matrix-for-your-research

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 is the Tube Formation Assay?

The Tube Formation Assay is based on the ability of endothelial cells to form three-dimensional capillary-like tubular structures when cultured on a hydrogel of reconstituted basement membrane, such as Cultrex Basement Membrane Extract (BME).

What are the advantages of the Tube Formation Assay?

The Tube Formation Assay is the most widely used in vitro angiogenesis assay. The assay is rapid, inexpensive and quantifiable. It can be used to identify potentially angiogenic and anti-angiogenic factors, to determine endothelial cell phenotype, and to study pathways and mechanisms involved in angiogenesis. It can be performed in a high throughput mode to screen for a large number of compounds.

What cell types can be used in the Tube Formation Assay?

The Tube Formation Assay is specific for endothelial cells, either primary cells or immortalized cell lines. Only endothelial cells form capillary-like structures with a lumen inside. Other endothelial cell types form other structures.

What are the variables associated with the Tube Formation Assay?

The major variables associated with tube formation are composition of the Cultrex Basement Membrane Extract (BME) hydrogel, thickness of the hydrogel, cell density, composition of angiogenic factors in the assay medium, and assay period.

Which Cultrex Basement Membrane Extract (BME) should I use for the Tube Formation Assay?

Cultrex Reduced Growth Factor BME (RGF BME) is generally used for testing compounds that promote angiogenesis because formation of capillary-like structures (tubes) is significantly less compared to non-growth factor reduced varieties of Cultrex BME. The Cultrex In Vitro Angiogeneis Assay (Tube Formation) includes a qualified production lot of Cultrex RGF BME that exhibits reduced background tube formation in the absence of angiogenic factors.

How do I reduce spontaneous formation of tubular structures on Cultrex BME in the absence of angiogenic factors?

Primary endothelial cells, such as Human Umbilical Vein Endothelial Cells (HUVECs) form capillary-like structures in the absence of added angiogenic factors less often than immortalized endothelial cells. Generally, reducing the number of cells per cm2 plated onto Cultrex BME will result in less background or spontaneous tube formation. Titrate the number of cells and find optimal conditions for your specific cell line. When endothelial cells fully form capillary structures in response to angiogenic activators, but not in their absence, you may proceed.

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 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 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 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 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.

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 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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Citations (21)

Product Datasheet / COA / SDS

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

Try it on your cultures! Request a Sample of Cultrex 3-D Culture Matrix RGF BME

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

Technical Data
Citations / Reviews (6) / FAQs

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

Species	Mouse
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.

Preparation & Storage

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.
Shipping Conditions	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.

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

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

Key Product Details

Summary

Why Use Cultrex 3-D Culture Matrix RGF BME ?

Product Specifications

Preparation & Storage

FAQs

Product Documents

Product Datasheets

COA

Certificate of Analysis

SDS

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