Cultrex^® Basement Membrane Extract, Type 2, PathClear^® Protocol

Product Description

Basement membranes are continuous sheets of the specialized extracellular matrix that form an interface between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma. Basement membranes are degraded and regenerated during development and wound healing. They not only support cells and cell layers, but they also play an essential role in tissue organization that affects cell adhesion, migration, proliferation, and differentiation. Basement membranes provide major barriers to invasion by metastatic tumor cells.

Cultrex^® Basement Membrane Extract (BME) is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor. The extract gels at 37 ⁰C to form a reconstituted basement membrane. The major components of BME include laminin, collagen IV, entactin, and heparin sulfate proteoglycan.

BME can be used in multiple applications, under a variety of cell culture conditions, for maintaining growth or promoting differentiation of primary endothelial, epithelial, smooth muscle, and stem cells. BME can also be utilized in cell attachment, neurite outgrowth, angiogenesis, in vitro cell invasion, and in vivo tumorigenicity assays. Recently we have developed two additional formulations of Cultrex^® BME known as Cultrex^® BME Type 2 and Cultrex^® BME Type 3. Cultrex^® BME Type 2 provides a proprietary formulation that is higher in tensile strength when compared to our original BME, while Cultrex^® BME Type 3 is physiologically aligned with the in vivo solid tumors environment and is recommended for xenografts and other in vivo applications.

Specifications

1. Concentration: 12 - 18 mg/mL
2. Source: Murine Engelbreth-Holm-Swarm (EHS) tumor
3. Storage buffer: Dulbecco’s Modified Eagle’s medium without phenol red (Product No. D5030), with 10 μg/mL gentamicin sulfate (Product No. G1264)

Precautions and Limitations

1. For Research Use Only. Not for use in diagnostic procedures.
2. The physical, chemical and toxicological properties of these products may not yet have been fully investigated; therefore, we recommend the use of gloves, lab coats, and eye protection while using these chemical reagents.

Material Qualification

1. Functional Assays
   Tube formation assay - BME promotes the formation of capillary-like structures by a human (HBMVEC; HUVEC) or mouse (SVEC4-10) endothelial cells.
2. Sterility Testing
   1. PathClear^® - Negative by PCR test for mycoplasma; 17 bacterial and virus strains typically included in mouse antibody production (MAP) testing, plus 13 additional murine infectious agents including LDEV, for a total of 31 organisms and viruses
   2. No bacterial or fungal growth detected after incubation at 37 ⁰C for 14 days following USP sterility testing guidelines
   3. Endotoxin concentration ≤8 EU/mL by LAL assay
3. Gelling
   BME gels in less than 30 minutes at 37 ⁰C and maintains the gelled form in culture medium for a minimum of 14 days at 37 ⁰C.

Name	Buffer	Tensile Strength	Concentration	Applications
Cultrex® BME, PathClear	DMEM	medium	12 – 18 mg/mL	xenograft/tumorgraft, 2D cell culture, 3D culture spheroids/organoids, stem cell
Cultrex® BME, Type 2, PathClear®	DMEM	high	12 – 18 mg/mL	xenograft/tumorgraft, 2D cell culture, 3D culture spheroids/organoids, stem cell
Cultrex® BME, Type 3, PathClear®	RPMI1640	high	12 – 18 mg/mL	xenograft/tumorgraft

Storage and Stability

The product is stable for a minimum of 3 months from date of shipment when stored at –20 ⁰C in a manual defrost the freezer. For optimal stability, store at –80 ⁰C. Avoid freeze-thaw cycles.

Coating Protocol

Thaw Cultrex^® BME overnight at 2-8 ⁰C. Refrigerator temperatures may vary; therefore it is recommended to keep BME on ice in a refrigerator during the thawing process. Thawed BME solidifies quickly at the temperatures above 15 ⁰C; when working with extract, keep it on ice to prevent untimely gelling.

There are many applications for Cultrex^® BME, which require different thicknesses and concentrations. In general, BME at a protein concentration ≥ 10 mg/mL is used for differentiation studies of primary cells. For applications such as endothelial cell formation of capillary-like structures (Tube Formation Assay), the differentiation of rat aorta tissue into capillary-like structures (Aortic Ring Assay), epithelial organoid formation, or tumor organoid formation, a thick gel is needed. Some applications, such as propagation of primary cells, require a thin layer coating and not a thick gel; therefore, the thin layer method should be used.

Thick Gel Method

1. Thaw BME as stated above.
2. Mix BME by slowly pipetting solution up and down; be careful not to introduce air bubbles.
3. Pipette 200-300 μL per cm² onto the growth surface.
4. Place the coated object at 37 ⁰C for 30 minutes.
5. Coated objects are ready for use.

Thin Layer Method (non-gelling)

1. Thaw BME as stated above.
2. Mix BME by slowly pipetting solution up and down; be careful not to introduce air bubbles.
3. Dilute BME to desired concentration in cold serum-free medium. Empirical determination of the optimal coating concentration for your application may be required. A protein concentration of 150 μg/mL is a recommended starting concentration for the propagation of primary cells.
4. Add a sufficient amount of solution to cover the entire area onto the growth surface. A volume of 300 μL per cm² is recommended.
5. Incubate the coated object at room temperature for an hour.
6. Aspirate coating solution and immediately plate cells. Do not allow the coated surface to dry out.

Legal Information

Cultrex and PathClear are registered trademarks of Trevigen, Inc.

References

1. Albini A, Iwamoto Y, Kleinman H, Martin G, Aaronson S, Kozlowski J, McEwan R. 1987. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res.. 473239-3245.

2. Fridman R, Giaccone G, Kanemoto T, Martin GR, Gazdar AF, Mulshine JL. 1990. Reconstituted basement membrane (matrigel) and laminin can enhance the tumorigenicity and the drug resistance of small cell lung cancer cell lines.. Proceedings of the National Academy of Sciences. 87(17):6698-6702. https://doi.org/10.1073/pnas.87.17.6698

3. Fridman R, Kibbey MC, Royce LS, Zain M, Sweeney TM, Jicha DL, Yannelli JR, Martin GR, Kleinman HK. 1991. Enhanced Tumor Growth of Both Primary and Established Human and Murine Tumor Cells in Athymic Mice After Coinjection With Matrigel. JNCI Journal of the National Cancer Institute. 83(11):769-774. https://doi.org/10.1093/jnci/83.11.769

4. Fridman R, Sweeney TM, Zain M, Martin GR, Kleinman HK. 1992. Malignant transformation of NIH-3T3 cells after subcutaneous co-injection with a reconstituted basement membrane (matrigel). Int. J. Cancer. 51(5):740-744. https://doi.org/10.1002/ijc.2910510513

5. Kubota Y, Kleinman HK, Martin GR, Lawley TJ. 1988. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.. 107(4):1589-1598. https://doi.org/10.1083/jcb.107.4.1589

6. Ponce ML, Nomizu M, Delgado MC, Kuratomi Y, Hoffman MP, Powell S, Yamada Y, Kleinman HK, Malinda KM. 1999. Identification of Endothelial Cell Binding Sites on the Laminin ?1 Chain. Circulation Research. 84(6):688-694. https://doi.org/10.1161/01.res.84.6.688

7. Eisenstein M. 2006. Thinking outside the dish. Nat Methods. 3(12):1035-1043. https://doi.org/10.1038/nmeth1206-1035

8. Benton G, George J, Kleinman H, Arnaoutova I. 2009. Advancing science and technology via 3D culture on basement membrane matrix. J. Cell. Physiol.. 221(1):18-25. https://doi.org/10.1002/jcp.21832

9. Arnaoutova I, George J, Kleinman HK, Benton G. 2009. The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogenesis. 12(3):267-274. https://doi.org/10.1007/s10456-009-9146-4

10. Arnaoutova I, Kleinman HK. 2010. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc. 5(4):628-635. https://doi.org/10.1038/nprot.2010.6

11. Benton G, Kleinman HK, George J, Arnaoutova I. 2011. Multiple uses of basement membrane-like matrix (BME/Matrigel) in vitro and in vivo with cancer cells. Int. J. Cancer. 128(8):1751-1757. https://doi.org/10.1002/ijc.25781

12. Arnaoutova I, George J, Kleinman HK, Benton G. 2012. Basement Membrane Matrix (BME) has Multiple Uses with Stem Cells. Stem Cell Rev and Rep. 8(1):163-169. https://doi.org/10.1007/s12015-011-9278-y

13. Fridman R, Benton G, Aranoutova I, Kleinman HK, Bonfil RD. 2012. Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection. Nat Protoc. 7(6):1138-1144. https://doi.org/10.1038/nprot.2012.053