Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear

For Robust Organoid Cultures - Reduced Growth Factor Basement Membrane Extract
Catalog # Availability Size / Price Qty
3533-001-02
3533-005-02
3533-010-02
Mouse Intestinal Organoids Cultured in Cultrex RGF BME Type 2_3533-005-02
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Product Details
Citations (9)
FAQs
Reviews (1)

Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear Summary

Cultrex Reduced Growth Factor Basement Membrane Extract (RGF BME ),Type 2 is specifically qualified to support the establishment and expansion of robust organoid cultures. It's composition mimics the in vivo microenvironment to improve take rate and growth of organoids.

Key Benefits

• Qualified for use in organoid cell culture
• Commonly used robust and established organoid systems
• Reduced growth factor formulation provides a more defined culture system
• Quality controlled for performance consistency

Why Use Cultrex RGF BME, Type 2?

Cultrex Reduced Growth Factor Basement Membrane Extract (RGF BME), Type 2 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. 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 heparan sulfate proteoglycans.

Cultrex RGF BME, Type 2 provides a proprietary formulation that is high in tensile strength and is designed for use in robust tissue organoid culture as well as other applications requiring an extracellular matrix scaffold.

Protocols Utilizing Cultrex RGF BME, Type 2 for Organoid Cell Culture.

Cultrex RGF BME, Type 2 is ideal for use as a scaffold for organoid and 3D cell culture. Listed below are protocols designed by our research and development groups for organoid culture featuring Cultrex RGF BME, Type 2 as well as growth factors, media supplements, and small molecules from Bio-Techne.

Protocol for Mouse Enteric Organoid Culture.
Protocol for Human Intestinal Organoid Culture.
Protocol for Human Liver Organoid Culture.
Protocol for Human Lung Organoid Culture.
Protocol for Harvesting Organoids for Biochemical Analysis.

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
Tested following USP <71> sterility guidelines.
Testing Cell Culture
Organoid Culture - Cultrex RGF BME, Type 2 supports growth and expansion of mouse small intestine organoid progenitor cells.

Gelling Assay - Cultrex RGF BME, Type 2 gels in less than 30 minutes at 37 °C, and maintains the gelled form in culture medium for a minimum of 14 days at 37 °C.

Dome Assay Cultrex RGF BME, Type 2 forms and maintains stable 3-D dome structures on cell culture plates.

Tube Formation Assay - Cultrex RGF BME, Type 2 supports formation of capillary-like structures by human (HBMVEC; HUVEC) or mouse (SVEC4-10) endothelial cells.
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.
Stability
Product is stable for two years from date of manufacture. 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.
Species
Mouse

Limitations

For research use only. Not for diagnositic use.

Product Datasheets

Data Example

Mouse Intestinal Organoids Cultured in Cultrex RGF BME, Type 2. Mouse intestinal organoids cultured in Cultrex RGF BME, Type 2 were immersion fixed and processed for whole mount staining or paraffin embedding and sectioning for immunocytochemistry. A) Whole mount mouse intestinal organoids were stained using Goat Anti-Human/Mouse E-Cadherin Antigen Affinity-purified Polyclonal Antibody (green; Catalog # AF748) at 10 µg/mL for 3 hours at room temperature. Cells were counterstained with DAPI (blue). B) Paraffin-embedded mouse intestinal tissue stained for Human Cadherin‑17 Antibody (green; Catalog # MAB1032), Human Ki67/MKI67 Antibody (red; Catalog # AF7617), and counterstained with DAPI (blue). View our protocol for the

Human Lung Organoids Cultured in Cultrex RGF BME, Type 2. A) Representative brightfield image of human lung organoids cultured using Cultrex RGF BME, Type 2. B) Expression of Sox2 (green; Catalog # AF2018) and Acetylated Tubulin (red; Novus Biologicals, Catalog # NB600-567). C) Expression of p63/TP73L (green; Catalog # AF1916) and Cytokeratin 10 (red; Novus Biologicals, Catalog # NBP2-61736). D) Expression of Podoplanin (green; Catalog # AF3670) as a marker of type 1 alveolar cells.

Liver Organoid Formation and Differentiation in Cultrex RGF BME, Type 2. Human Liver organoids were derived from human biopsy tissue. Undifferentiated organoids were formed by embedding dissociated tissue in Cultrex RGF BME, Type 2 and culturing in specialized media. The organoids were differentiated using media containing Recombinant Human FGF-19 (Catalog # 969-FG), DAPT (Catalog # 2634), and Dexamethasone (Catalog # 1126). A) Undifferentiated liver organoids. B) Liver organoids shrink as they differentiate. C) Representative images of differentiated liver organoids expression hepatocyte markers, Albumin and HNF3 beta, as well as E-Cadherin.

Human Intestinal Organoids Cultured using Cultrex RGF BME, Type 2. Human transverse colon organoids (A,B) and human ileum organoids (C, D) were grown using cells isolated from transverse colon and ileum biopsy tissue, respectively. Organoids were embedded in Cultrex RGF BME, Type 2 as a scaffold matrix. A) Brightfield image of human transverse organoids. B) Human transverse organoid stained using Goat Anti-Human/Mouse E-Cadherin Antigen Affinity-purified Polyclonal Antibody (green; Catalog # AF748), a MUC2 Antibody (red; Catalog # NBP2-44431), and DAPI (blue). C) Brightfield image of human ileum organoids. D) Human ileum organoid stained using a Aldolase B Antibody (red; Catalog # NBP2-15345), a Human Cadherin-17 Antibody (green; Catalog # MAB1032, and DAPI (blue).

Formation of HepG2 Spheroids in Cultrex RGF BME, Type 2. Hepatocyte spheroids were formed by plating HepG2 liver hepatocellular carcinoma cells (10,000 cells per well) in a 24-well plate coated with 5 mg/mL of Cultrex RGF BME, Type 2. Spheroids were cultured for 21 days prior imaging by brightfield microscopy.

Cultrex RGF Basement Membrane Extract, Type 2

Reagents Provided

Citations for Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

9 Citations: Showing 1 - 9
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  1. Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages
    Authors: JM Rosenbluth, RCJ Schackmann, GK Gray, LM Selfors, CM Li, M Boedicker, HJ Kuiken, A Richardson, J Brock, J Garber, D Dillon, N Sachs, H Clevers, JS Brugge
    Nat Commun, 2020;11(1):1711.  2020
  2. Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia
    Authors: E Driehuis, N Oosterom, SG Heil, IB Muller, M Lin, S Kolders, G Jansen, R de Jonge, R Pieters, H Clevers, MM van den He
    PLoS ONE, 2020;15(5):e0231588.  2020
  3. Release of transcriptional repression via ErbB2-induced, SUMO-directed phosphorylation of myeloid zinc finger-1 serine 27 activates lysosome redistribution and invasion
    Authors: DM Brix, SA Tvingsholm, MB Hansen, KB Clemmensen, T Ohman, V Siino, M Lambrughi, K Hansen, P Puustinen, I Gromova, P James, E Papaleo, M Varjosalo, J Moreira, M Jäättelä, T Kallunki
    Oncogene, 2019;0(0):.  2019
  4. Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses
    Authors: J Fakhiri, MA Schneider, J Puschhof, M Stanifer, V Schildgen, S Holderbach, Y Voss, J El Andari, O Schildgen, S Boulant, M Meister, H Clevers, Z Yan, J Qiu, D Grimm
    Mol Ther Methods Clin Dev, 2019;12(0):202-222.  2019
  5. Neutrophils escort circulating tumour cells to enable cell cycle progression
    Authors: BM Szczerba, F Castro-Gin, M Vetter, I Krol, S Gkountela, J Landin, MC Scheidmann, C Donato, R Scherrer, J Singer, C Beisel, C Kurzeder, V Heinzelman, C Rochlitz, WP Weber, N Beerenwink, N Aceto
    Nature, 2019;0(0):.  2019
  6. SH3BP4 Regulates Intestinal Stem Cells and Tumorigenesis by Modulating ?-Catenin Nuclear Localization
    Authors: P Antas, L Novellasde, A Kucharska, I Massie, J Carvalho, D Oukrif, E Nye, M Novelli, VSW Li
    Cell Rep, 2019;26(9):2266-2273.e4.  2019
  7. Oral mucosal organoids as a potential platform for personalized cancer therapy
    Authors: E Driehuis, S Kolders, S Spelier, K Lohmussaar, SM Willems, LA Devriese, R de Bree, EJ de Ruiter, J Korving, H Begthel, JH Van Es, V Geurts, GW He, RH van Jaarsv, R Oka, MJ Muraro, J Vivie, MMJM Zandvliet, APA Hendrickx, N Iakobachvi, P Sridevi, O Kranenburg, R van Boxtel, GJPL Kops, DA Tuveson, PJ Peters, A van Oudena, H Clevers
    Cancer Discov, 2019;0(0):.  2019
  8. Pancreatic cancer organoids recapitulate disease and allow personalized drug screening
    Authors: E Driehuis, A van Hoeck, K Moore, S Kolders, HE Francies, MC Gulersonme, ECA Stigter, B Burgering, V Geurts, A Gracanin, G Bounova, FH Morsink, R Vries, S Boj, J van Es, GJA Offerhaus, O Kranenburg, MJ Garnett, L Wessels, E Cuppen, LAA Brosens, H Clevers
    Proc. Natl. Acad. Sci. U.S.A., 2019;0(0):.  2019
  9. AGE-RAGE interaction in the TGF?2-mediated epithelial to mesenchymal transition of human lens epithelial cells.
    Authors: Cibin T Raghavan, Ram H Nagaraj
    Glycoconjugate Journal, 2016;0(0):1573-4986.  2016

FAQs

  1. What kinds of tumor cells or biopsy specimens grow in vivo with Cultrex® BME?

    • Many cell lines and tumor biopsy specimens (usually cut into small fragments) have been found to grow in vivo when implanted with Cultrex® BME. These include melanoma, intestinal, prostate, breast, lung, renal, and liver cancers as well as the 3T3 mouse embryonic fibroblast cell line.

  2. 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.

  3. How should Cultrex Basement Membrane Extract (BME) be stored and handled?

    • Cultrex BME should be stored at or below -20°C for optimal stability. Preparation of working aliquots is recommended. Cultrex BME should be thawed overnight on ice at 4°C, however long term storage at 4°C is not recommended. Freeze/thaw cycles and gel-liquid phase transitions should be avoided, since they can compromise product integrity.

  4. 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).

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. Does Cultrex BME, Catalog # 3533-005-02, affect fluorescence readings when Alamar Blue is used for final readout of assay? 

    • BME is known to have autofluorescence, but if appropriate controls are evaluated, background can be successfully subtracted. A BME only control well with no cells should be used to subtract the background fluorescence.

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Cultrex PathClear Reduced Growth Factor BME (2 x 5 mL)
By Anonymous on 06/01/2018
Application: Cell migration/motility
Reason for Rating: it can form a very nice tube in our control group within 6-8 hour.