Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear
Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear SummaryCultrex 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.
• 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.
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 7 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.
For research use only. Not for diagnostic use.
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).
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.
Citations for Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear
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Air-Liquid-Interface Differentiated Human Nose Epithelium: A Robust Primary Tissue Culture Model of SARS-CoV-2 Infection
Authors: BM Tran, SL Grimley, JL McAuley, A Hachani, L Earnest, SL Wong, L Caly, J Druce, DFJ Purcell, DC Jackson, M Catton, CJ Nowell, L Leonie, G Deliyannis, SA Waters, J Torresi, E Vincan
International Journal of Molecular Sciences, 2022;23(2):. 2022
Cell-intrinsic Aryl Hydrocarbon Receptor signalling is required for the resolution of injury-induced colonic stem cells
Authors: K Shah, MR Maradana, M Joaquina D, A Metidji, F Graelmann, M Llorian, P Chakravart, Y Li, M Tolaini, M Shapiro, G Kelly, C Cheshire, D Bhurta, SB Bharate, B Stockinger
Nature Communications, 2022;13(1):1827. 2022
Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage
Authors: Y Bollen, JH Hageman, P van Leenen, LLM Derks, B Ponsioen, JR Buissant d, I Verlaan-Kl, M van den Bo, LWMM Terstappen, R van Boxtel, HJG Snippert
PloS Biology, 2022;20(1):e3001527. 2022
Comprehensive metabolomics expands precision medicine for triple-negative breast cancer
Authors: Y Xiao, D Ma, YS Yang, F Yang, JH Ding, Y Gong, L Jiang, LP Ge, SY Wu, Q Yu, Q Zhang, F Bertucci, Q Sun, X Hu, DQ Li, ZM Shao, YZ Jiang
Cell Research, 2022;0(0):. 2022
Copy number amplification of ENSA promotes the progression of triple-negative breast cancer via cholesterol biosynthesis
Authors: YY Chen, JY Ge, SY Zhu, ZM Shao, KD Yu
Nature Communications, 2022;13(1):791. 2022
Lineage-specific silencing of PSAT1 induces serine auxotrophy and sensitivity to dietary serine starvation in luminal breast tumors
Authors: BH Choi, V Rawat, J Högström, PA Burns, KO Conger, ME Ozgurses, JM Patel, TS Mehta, A Warren, LM Selfors, T Muranen, JL Coloff
Cell Reports, 2022;38(3):110278. 2022
A Bioluminescent 3CLPro Activity Assay to Monitor SARS-CoV-2 Replication and Identify Inhibitors
Authors: C Mathieu, F Touret, C Jacquemin, YL Janin, A Nougairède, M Brailly, M Mazelier, D Décimo, V Vasseur, A Hans, JC Valle-Casu, X de Lamball, B Horvat, P André, M Si-Tahar, V Lotteau, PO Vidalain
Viruses, 2021;13(9):. 2021
Inhibition of mitochondrial function by metformin increases glucose uptake, glycolysis and GDF-15 release from intestinal cells
Authors: M Yang, T Darwish, P Larraufie, D Rimmington, I Cimino, DA Goldspink, B Jenkins, A Koulman, CA Brighton, M Ma, BYH Lam, AP Coll, S O'Rahilly, F Reimann, FM Gribble
Scientific Reports, 2021;11(1):2529. 2021
Sox9EGFP defines biliary epithelial heterogeneity downstream of Yap activity
Authors: DY Tulasi, DM Castaneda, K Wager, CB Hogan, KP Alcedo, JR Raab, AD Gracz
Cellular and Molecular Gastroenterology and Hepatology, 2021;0(0):. 2021
Culture and analysis of kidney tubuloids and perfused tubuloid cells-on-a-chip
Authors: L Gijzen, FA Yousef Yen, F Schutgens, MK Vormann, CME Ammerlaan, A Nicolas, D Kurek, P Vulto, MB Rookmaaker, HL Lanz, MC Verhaar, H Clevers
Nature Protocols, 2021;0(0):. 2021
Organoid-based drug screening reveals neddylation as therapeutic target for malignant rhabdoid tumors
Authors: C Calandrini, SR van Hooff, I Paassen, D Ayyildiz, S Derakhshan, MEM Dolman, KPS Langenberg, M van de Ven, C de Heus, N Liv, M Kool, RR de Krijger, GAM Tytgat, MM van den He, JJ Molenaar, J Drost
Cell Reports, 2021;36(8):109568. 2021
RAC1B modulates intestinal tumourigenesis via modulation of WNT and EGFR signalling pathways
Authors: V Gudiño, SÖ Pohl, CV Billard, P Cammareri, A Bolado, S Aitken, D Stevenson, AE Hall, M Agostino, J Cassidy, C Nixon, A von Kriegs, P Freile, L Popplewell, G Dickson, L Murphy, A Wheeler, M Dunlop, F Din, D Strathdee, OJ Sansom, KB Myant
Nature Communications, 2021;12(1):2335. 2021
Adult mouse and human organoids derived from thyroid follicular cells and modeling of Graves' hyperthyroidism
Authors: J van der Va, L Bosmans, SF Sijbesma, K Knoops, WJ van de Wet, HG Otten, H Begthel, IHM Borel Rink, J Korving, EGWM Lentjes, C Lopez-Igle, PJ Peters, HM van Santen, MR Vriens, H Clevers
Proceedings of the National Academy of Sciences of the United States of America, 2021;118(51):. 2021
ERBB3 overexpression due to miR-205 inactivation confers sensitivity to FGF, metabolic activation, and liability to ERBB3 targeting in glioblastoma
Authors: F De Bacco, F Orzan, J Erriquez, E Casanova, L Barault, R Albano, A D'Ambrosio, V Bigatto, G Reato, M Patanè, B Pollo, G Kuesters, C Dell'Aglio, L Casorzo, S Pellegatta, G Finocchiar, PM Comoglio, C Boccaccio
Cell Reports, 2021;36(4):109455. 2021
Ultrastructural analysis of breast cancer patient-derived organoids
Authors: L Signati, R Allevi, F Piccotti, S Albasini, L Villani, M Sevieri, A Bonizzi, F Corsi, S Mazzucchel
Cancer Cell International, 2021;21(1):423. 2021
Functional Radiogenetic Profiling Implicates ERCC6L2 in Non-homologous End Joining
Authors: P Francica, M Mutlu, VA Blomen, C Oliveira, Z Nowicka, A Trenner, NM Gerhards, P Bouwman, E Stickel, ML Hekkelman, L Lingg, I Klebic, M van de Ven, R de Korte-G, D Howald, J Jonkers, AA Sartori, W Fendler, JR Chapman, T Brummelkam, S Rottenberg
Cell Rep, 2020;32(8):108068. 2020
Slug-expressing mouse prostate epithelial cells have increased stem cell potential
Authors: Z Kahounová, J Remšík, R Fedr, J Bouchal, A Mi?ková, E Slabáková, L Binó, A Hampl, K Sou?ek
Stem Cell Res, 2020;46(0):101844. 2020
Single-cell derived tumor organoids display diversity in HLA class I peptide presentation
Authors: LC Demmers, K Kretzschma, A Van Hoeck, YE Bar-Epraïm, HWP van den To, M Koomen, G van Son, J van Gorp, A Pronk, N Smakman, E Cuppen, H Clevers, AJR Heck, W Wu
Nat Commun, 2020;11(1):5338. 2020
Loss of sphingosine 1-phosphate receptor 3 gene function impairs injury-induced stromal angiogenesis in mouse cornea
Authors: S Yasuda, T Sumioka, H Iwanishi, Y Okada, M Miyajima, K Ichikawa, PS Reinach, S Saika
Lab Invest, 2020;0(0):. 2020
Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells
Authors: C Donato, L Kunz, F Castro-Gin, A Paasinen-S, K Strittmatt, BM Szczerba, R Scherrer, N Di Maggio, W Heusermann, O Biehlmaier, C Beisel, M Vetter, C Rochlitz, WP Weber, A Banfi, T Schroeder, N Aceto
Cell Rep, 2020;32(10):108105. 2020
Preclinical evaluation of the anti-tumor activity of pralatrexate in high-risk neuroblastoma cells
Authors: RA Clark, S Lee, J Qiao, DH Chung
Oncotarget, 2020;11(32):3069-3077. 2020
An organoid biobank for childhood kidney cancers that captures disease and tissue heterogeneity
Authors: C Calandrini, F Schutgens, R Oka, T Margaritis, T Candelli, L Mathijsen, C Ammerlaan, RL van Inevel, S Derakhshan, S de Haan, E Dolman, P Lijnzaad, L Custers, H Begthel, HHD Kerstens, LL Visser, M Rookmaaker, M Verhaar, GAM Tytgat, P Kemmeren, RR de Krijger, R Al-Saadi, K Pritchard-, M Kool, AC Rios, MM van den He, JJ Molenaar, R van Boxtel, FCP Holstege, H Clevers, J Drost
Nat Commun, 2020;11(1):1310. 2020
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
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
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
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
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
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
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
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
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
A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity.
Authors: Sachs N, de Ligt J, Kopper O, Gogola E, Bounova G, Weeber F, Balgobind A, Wind K, Gracanin A, Begthel H, Korving J, van Boxtel R, Duarte A, Lelieveld D, van Hoeck A, Ernst R, Blokzijl F, Nijman I, Hoogstraat M, van de Ven M, Egan D, Zinzalla V, Moll J, Boj S, Voest E, Wessels L, van Diest P, Rottenberg S, Vries R, Cuppen E, Clevers H
Cell, 0;172(1):373-386.e10. 0
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.
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?
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.
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.
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.
Reviews for Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear
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dissolve it on ice, and don't put it outside for too not time
Aortic sprouting assay: mouse aortic ring cultured in a 48-well plate and imaged in an inverted microscope after 5 days.
Results are very consistent with this BME (used one layer under and another on top of the aortic ring).
We are using the BME at a final dilution of 1/30 for the maintenance of iPSC and differentiation into endoderm as well as mesoderm lineages. Attachment, growth, and differentiation of our iPSC is consistent, and we observed only minor variations between different lots.