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The L-WRN is a cell culture medium that supports the growth and maintenance of Wnt-responsive cell lines. It contains the Wnt3a, R-spondin, and Noggin proteins necessary for maintaining stem cell potency and promoting cell proliferation.

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Lab products found in correlation

Glutamax, by Thermo Fisher Scientific (8 mentions) N acetylcysteine, by Merck Group (6 mentions) Advanced dmem f12, by Thermo Fisher Scientific (6 mentions) B27 supplement, by Thermo Fisher Scientific (6 mentions) Growth factor reduced matrigel, by Corning (5 mentions) Matrigel, by Corning (3 mentions) A83 01, by Bio-Techne (3 mentions) Nicotinamide, by Merck Group (3 mentions) Hepes, by Thermo Fisher Scientific (3 mentions) Human epidermal growth factor (hegf), by Thermo Fisher Scientific (2 mentions) Hfgf10, by Thermo Fisher Scientific (2 mentions) Hgastrin 1, by Bio-Techne (2 mentions) Y 27632, by Selleck Chemicals (2 mentions) Glutamax, by American Type Culture Collection (2 mentions) Advanced dmem f12, by American Type Culture Collection (2 mentions) B27 supplement, by American Type Culture Collection (2 mentions) Human epidermal growth factor (hegf), by American Type Culture Collection (2 mentions) Hfgf basic fgf2, by Merck Group (2 mentions) N2 supplement, by Thermo Fisher Scientific (2 mentions) Human epidermal growth factor (hegf), by Merck Group (2 mentions)

16 protocols using l wrn

1

Organoid Culture Conditions for hM1A and hT2

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Organoids (hM1A and hT2) were kindly provided by the Tuveson laboratory (Cold Spring Harbor Laboratory). Organoids were cultured at 37°C in 5% CO2. Cells were seeded in growth factor-reduced Matrigel (Corning; Cat# 356231) domes and fed with human complete feeding medium: advanced DMEM/F12-based WRN-conditioned medium (L-WRN (ATCC CRL-3276)), 1x B27 supplement, 10 mM HEPES, 0.01 μM GlutaMAX, 10 mM nicotinamide, 1.25 mM N-acetylcysteine, 50 ng/mL hEGF, 100 ng/mL hFGF10, 0.01 μM hGastrin I, 500 nM A83–01, 1 μM PGE2 and 10.5 μM Y27632 (44 (link)).
Hobbs G.A., Baker N.M., Miermont A.M., Thurman R.D., Pierobon M., Tran T.H., Anderson A.O., Waters A.M., Diehl J.N., Papke B., Hodge R.G., Klomp J.E., Goodwin C.M., DeLiberty J.M., Wang J., Ng R.W., Gautam P., Bryant K.L., Esposito D., Campbell S.L., Petricoin EF I.I.I., Simanshu D.K., Aguirre A.J., Wolpin B.M., Wennerberg K., Rudloff U., Cox A.D, & Der C.J. (2019). Atypical KRASG12R Mutant Is Impaired in PI3K Signaling and Macropinocytosis in Pancreatic Cancer. Cancer discovery, 10(1), 104-123.
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2

Culturing Patient-Derived Pancreatic Cancer Organoids

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The human pancreatic cancer organoids were provided by Dr. David Tuveson (Cold Spring Harbor Laboratory). The patient derived PDAC organoids were cultured at 37°C in 5% CO2. Cells were initially seeded in growth factor reduced Matrigel (Corning) domes and after reseeding were fed with complete human feeding medium: advanced DMEM/F12 (Thermo Fisher Scientific) based WRN conditioned medium (L-WRN (ATCC CRL-3276)), 1x B27 supplement (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), 0.01 μM GlutaMAX (Thermo Fisher Scientific), 10 mM nicotinamide (Sigma-Aldrich), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 50 ng/mL hEGF (Peprotech), 100 ng/mL hFGF10 (Peprotech), 0.01 μM hGastrin I (TOCRIS), 500 nM A83-01 (TOCRIS), and 10.5 μM Y27632 (Selleckchem) (Boj et al., 2015 (link)). Organoids were tested negative for mycoplasma.
Waters A.M., Khatib T.O., Papke B., Goodwin C.M., Hobbs G.A., Diehl J.N., Yang R., Edwards A.C., Walsh K.H., Sulahian R., McFarland J.M., Kapner K.S., Gilbert T.S., Stalnecker C.A., Javaid S., Barkovskaya A., Grover K.R., Hibshman P.S., Blake D.R., Schaefer A., Nowak K.M., Klomp J.E., Hayes T.K., Kassner M., Tang N., Tanaseichuk O., Chen K., Zhou Y., Kalkat M., Herring L.E., Graves L.M., Penn L.Z., Yin H.H., Aguirre A.J., Hahn W.C., Cox A.D, & Der C.J. (2021). Targeting p130Cas- and microtubule-dependent MYC regulation sensitizes pancreatic cancer to ERK MAPK inhibition. Cell reports, 35(13), 109291.
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3

Patient-derived Pancreatic and Colorectal Organoids

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The human pancreatic cancer organoids were provided by Dr. David Tuveson (Cold Spring Harbor Laboratory). The patient-derived PDAC organoids hM1A KRASG12D and hT2 KRASG12R were cultured at 37°C in 5% CO2. Cells were seeded in growth factor reduced Matrigel (Corning) domes and fed with complete human feeding medium: advanced DMEM/F12 based WRN condition media (L-WRN (ATCC CRL-3276)), 1x B27 supplement, 10 mM HEPES, 0.01 μM GlutaMAX, 10 mM nicotinamide, 1.25 mM N-acetylcysteine, 50 ng/mL hEGF, 100 ng/mL hFGF10, 0.01 μM hGastrin I, 500 nM A83-01, 1 μM PGE2 and additionally 10.5 μM Y27632 (Boj et al., 2015 (link)). Organoids were tested for mycoplasma. The patient-derived colorectal organoids OT227 KRASG13D, OT238 KRASG12D and OT302 KRASG12D were previously fully characterized in terms of genomic alterations by Schütte et al. (2017) (link). CRC organoids were cultured in crypt culture medium (CCM) containing advanced DMEM/F12 (GIBCO) supplemented with 1x GlutaMAX (GIBCO), 10 mM HEPES buffer (GIBCO), Penicillin/Streptomycin (100 U/ml/100 μg/ml), 1 mM N-acetylcysteine (Sigma), 1x N2 Supplement (GIBCO), 1x B27 supplement (GIBCO) and prepared with freshly added hFGF basic/FGF2 (20 ng/ml) (Sigma) and hEGF (50 ng/ml) (Sigma).
Ozkan-Dagliyan I., Diehl J.N., George S.D., Schaefer A., Papke B., Klotz-Noack K., Waters A.M., Goodwin C.M., Gautam P., Pierobon M., Peng S., Gilbert T.S., Lin K.H., Dagliyan O., Wennerberg K., Petricoin EF I.I.I., Tran N.L., Bhagwat S.V., Tiu R.V., Peng S.B., Herring L.E., Graves L.M., Sers C., Wood K.C., Cox A.D, & Der C.J. (2020). Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers. Cell reports, 31(11), 107764.
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4

Generating Wnt-3A, R-spondin3 and Noggin

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Wnt-3A, R-spondin3 and noggin expressing cell line L-WRN was purchased from ATCC and was used to produce WNT3A-RSPO3-NOGGIN conditioned medium (Miyoshi and Stappenbeck, 2013 (link)).
Ghosh A., Syed S.M., Kumar M., Carpenter T.J., Teixeira J.M., Houairia N., Negi S, & Tanwar P.S. (2020). In Vivo Cell Fate Tracing Provides No Evidence for Mesenchymal to Epithelial Transition in Adult Fallopian Tube and Uterus. Cell reports, 31(6), 107631.
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5

Generating Wnt-3A, R-spondin3 and Noggin

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Wnt-3A, R-spondin3 and noggin expressing cell line L-WRN was purchased from ATCC and was used to produce WNT3A-RSPO3-NOGGIN conditioned medium (Miyoshi and Stappenbeck, 2013 (link)).
Ghosh A., Syed S.M., Kumar M., Carpenter T.J., Teixeira J.M., Houairia N., Negi S, & Tanwar P.S. (2020). In Vivo Cell Fate Tracing Provides No Evidence for Mesenchymal to Epithelial Transition in Adult Fallopian Tube and Uterus. Cell reports, 31(6), 107631.
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6

Patient-derived Pancreatic and Colorectal Organoids

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The human pancreatic cancer organoids were provided by Dr. David Tuveson (Cold Spring Harbor Laboratory). The patient-derived PDAC organoids hM1A KRASG12D and hT2 KRASG12R were cultured at 37°C in 5% CO2. Cells were seeded in growth factor reduced Matrigel (Corning) domes and fed with complete human feeding medium: advanced DMEM/F12 based WRN condition media (L-WRN (ATCC CRL-3276)), 1x B27 supplement, 10 mM HEPES, 0.01 μM GlutaMAX, 10 mM nicotinamide, 1.25 mM N-acetylcysteine, 50 ng/mL hEGF, 100 ng/mL hFGF10, 0.01 μM hGastrin I, 500 nM A83-01, 1 μM PGE2 and additionally 10.5 μM Y27632 (Boj et al., 2015 (link)). Organoids were tested for mycoplasma. The patient-derived colorectal organoids OT227 KRASG13D, OT238 KRASG12D and OT302 KRASG12D were previously fully characterized in terms of genomic alterations by Schütte et al. (2017) (link). CRC organoids were cultured in crypt culture medium (CCM) containing advanced DMEM/F12 (GIBCO) supplemented with 1x GlutaMAX (GIBCO), 10 mM HEPES buffer (GIBCO), Penicillin/Streptomycin (100 U/ml/100 μg/ml), 1 mM N-acetylcysteine (Sigma), 1x N2 Supplement (GIBCO), 1x B27 supplement (GIBCO) and prepared with freshly added hFGF basic/FGF2 (20 ng/ml) (Sigma) and hEGF (50 ng/ml) (Sigma).
Ozkan-Dagliyan I., Diehl J.N., George S.D., Schaefer A., Papke B., Klotz-Noack K., Waters A.M., Goodwin C.M., Gautam P., Pierobon M., Peng S., Gilbert T.S., Lin K.H., Dagliyan O., Wennerberg K., Petricoin EF I.I.I., Tran N.L., Bhagwat S.V., Tiu R.V., Peng S.B., Herring L.E., Graves L.M., Sers C., Wood K.C., Cox A.D, & Der C.J. (2020). Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers. Cell reports, 31(11), 107764.
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7

Establishing Pancreatic and Colorectal Cancer Organoids

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Pa01C, Pa02C, Pa03C, Pa04C, Pa14C and Pa16C cell lines were provided by A. Maitra (MD Anderson Cancer Center). The remaining PDAC cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in recommended media (DMEM or RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin). Previously established organoids derived from pancreatic cancer were provided by David Tuveson (Cold Spring Harbor Laboratory). No patient samples were used in this study. All cell lines were maintained in a humidified chamber with 5% CO2 at 37°C. Pancreatic organoid cells were seeded in growth factor–reduced Matrigel (Corning) domes and fed with complete human feeding medium: advanced DMEM/F12-based WRN condition medium (L-WRN, ATCC CRL-3276), 1× B27 supplement, 10 mM HEPES, 0.01 μM GlutaMAX, 10 mM nicotinamide, 1.25 mM N-acetylcysteine, 50 ng ml–1 hEGF, 100 ng ml–1 hFGF10, 0.01 μM hGastrin I, 500 nM A83-01, 1 μM PGE2 and 10.5 μM Y27632. Colorectal cancer organoids were maintained as described (16 (link)). All cell lines were validated using short-tandem-repeat (STR) profiling and confirmed regularly as mycoplasma-negative.
Goodwin C.M., Waters A.M., Klomp J.E., Javaid S., Bryant K.L., Stalnecker C.A., Drizyte-Miller K., Papke B., Yang R., Amparo A.M., Ozkan-Dagliyan I., Baldelli E., Calvert V., Pierobon M., Sorrentino J.A., Beelen A.P., Bublitz N., Lüthen M., Wood K.C., Petricoin EF I.I.I., Sers C., McRee A.J., Cox A.D, & Der C.J. (2023). Combination Therapies with CDK4/6 Inhibitors to Treat KRAS-mutant Pancreatic Cancer. Cancer research, 83(1), 141-157.
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8

Cryopreserved Intestinal Organoid Culture

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Intestinal stem cells were harvested from the duodenum of adult C57BL/6 (Charles River, MA) mice as previously described25 (link). Cells were grown as organoids in 25 uL droplets of GFR Matrigel® (Corning) in 24 well plates in Advanced DMEM/F12 (Gibco) containing 50% L-WRN (ATCC) conditioned media25 (link), B27 and N2 supplements (Gibco), Glutamax (Gibco), N-acetyl cysteine (Sigma), HEPES (Sigma), primocin (InvivoGen), and murine epidermal growth factor (EGF, Peprotech) under standard growth conditions (37 °C, 5% CO2). Cells were fed every other day and were passaged every 3–5 days, or when organoids began to contact one another or darken. Organoids used in this study had previously been cryopreserved for up to 6 months in liquid nitrogen.
Puzan M., Hosic S., Ghio C, & Koppes A. (2018). Enteric Nervous System Regulation of Intestinal Stem Cell Differentiation and Epithelial Monolayer Function. Scientific Reports, 8, 6313.
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9

Culturing Patient-Derived Pancreatic Cancer Organoids

Cited 1 time
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The human pancreatic cancer organoids were provided by Dr. David Tuveson (Cold Spring Harbor Laboratory). The patient derived PDAC organoids were cultured at 37°C in 5% CO2. Cells were initially seeded in growth factor reduced Matrigel (Corning) domes and after reseeding were fed with complete human feeding medium: advanced DMEM/F12 (Thermo Fisher Scientific) based WRN conditioned medium (L-WRN (ATCC CRL-3276)), 1x B27 supplement (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), 0.01 μM GlutaMAX (Thermo Fisher Scientific), 10 mM nicotinamide (Sigma-Aldrich), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 50 ng/mL hEGF (Peprotech), 100 ng/mL hFGF10 (Peprotech), 0.01 μM hGastrin I (TOCRIS), 500 nM A83-01 (TOCRIS), and 10.5 μM Y27632 (Selleckchem) (Boj et al., 2015 (link)). Organoids were tested negative for mycoplasma.
Waters A.M., Khatib T.O., Papke B., Goodwin C.M., Hobbs G.A., Diehl J.N., Yang R., Edwards A.C., Walsh K.H., Sulahian R., McFarland J.M., Kapner K.S., Gilbert T.S., Stalnecker C.A., Javaid S., Barkovskaya A., Grover K.R., Hibshman P.S., Blake D.R., Schaefer A., Nowak K.M., Klomp J.E., Hayes T.K., Kassner M., Tang N., Tanaseichuk O., Chen K., Zhou Y., Kalkat M., Herring L.E., Graves L.M., Penn L.Z., Yin H.H., Aguirre A.J., Hahn W.C., Cox A.D, & Der C.J. (2021). Targeting p130Cas- and microtubule-dependent MYC regulation sensitizes pancreatic cancer to ERK MAPK inhibition. Cell reports, 35(13), 109291.
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10

Generating Organoid Culture from L‐WRN Cells

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The L‐WRN (CRL‐3276) cell line30 was purchased from ATCC on November 6, 2015. The third and fourth passages were used to generate conditioned media for organoid culture. The mycoplasma detection test (ATCC) was performed on all cultures before experiments (last check on May 18, 2018).
Razumilava N., Shiota J., Mohamad Zaki N.H., Ocadiz‐Ruiz R., Cieslak C.M., Zakharia K., Allen B.L., Gores G.J., Samuelson L.C, & Merchant J.L. (2018). Hedgehog Signaling Modulates Interleukin‐33‐Dependent Extrahepatic Bile Duct Cell Proliferation in Mice. Hepatology Communications, 3(2), 277-292.
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