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Permits and Restrictions	View Permits View Restrictions
Organism	Mus musculus, mouse
Tissue	subcutaneous connective tissue; areolar and adipose
Product Format	frozen 1.0 mL
Morphology	fibroblast
Culture Properties	adherent
Biosafety Level	2 [Cells contain SV40 viral DNA sequences] Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Age	100 days
Gender	male
Strain	C3H/An
Applications	This cell line is a source for producing Wnt-3A, R-spondin 3, and noggin conditioned medium which can be used for growing various mammalian tissue stem cells.
Storage Conditions	liquid nitrogen vapor phase

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Derivation	The L-WRN cells were derived by transfecting L-Wnt3A (ATCC^® CRL-2647) with an R-spondin 3 and noggin co-expressing vector and stable clones were selected in medium containing G418 and Hygromycin B.
Genes Expressed	Wnt-3A; R-spondin; noggin
Cellular Products	Wnt-3A protein; R-spondin; noggin protein
Comments	The L-WRN cells were derived by transfecting L-Wnt3A (ATCC® CRL-2647) with an R-spondin 3 and noggin co-expressing vector and stable clones were selected in medium containing G418 and Hygromycin B. The L-WRN cells secrete the factors Wnt3A, R-spondin 3 and noggin into the medium. Wnt3A binds the frizzled receptor family and activate β-catenin-dependent transcription. Members of the r-spondin protein family are potent co-activators of canonical Wnt signaling in the intestine and are essential for isolation of small intestinal stem cells. Noggin, a bone morphogenetic protein (BMP) signaling inhibitor, enables the maintenance and passge of small intestinal organoids in vitro. Although these three factors are commercially available, it is costly to maintain the large scale cultures that are required for standard assays currently with immortalized cell lines. Using the conditioned medium from CRL-3276 provides relatively intact and high titer proteins compared to medium made with reconstituted proteins and is a cost-effective alternative. Note: Since the conditioned medium contains other factors besides Wnt3A, R-spondin 3 and noggin proteins, it is necessary to control any experiments involving the Wnt3A conditioned medium with control conditioned medium from the grand-parental cell line (ATCC® CRL-2648).

Complete Growth Medium	The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: 0.5 mg/mL G-418 0.5 mg/mL hygromycin B fetal bovine serum to a final concentration of 10%
Subculturing	Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Remove and discard culture medium. Briefly rinse the cell layer with Ca⁺⁺/Mg⁺⁺ free Dulbecco's phosphate-buffered saline (D-PBS) (ATCC 30-2200) or 0.25% Trypsin – 0.53mM EDTA (ATCC 30-2101) solution to remove all traces of serum which contains trypsin inhibitor. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting. Add appropriate aliquots of the cell suspension to new culture vessels. Incubate cultures at 37°C. Protocol for Wnt-3A, R-spondin and Noggin Conditioned Medium: Split the cells 1:10 in culture medium (without G418 and Hygromycin B to prevent carryover of drugs in the conditioned medium) and seed 25 mL cell suspension into T-150 flasks Incubate the flasks for 3 or 4 days or until the cells become over-confluent and a number of cell aggregates come off. Remove medium and rinse flasks with 10 mL medium and discard rinse. Add 25 mL fresh medium and incubate flasks for 24 hours. Remove the medium to a centrifuge tube. Add new medium to the flasks. Centrifuge the conditioned medium at 2000 x g for 5 minutes and decant supernatant into a 1 L bottle. Store the conditioned medium at 4°C. This is the first batch of medium. Every 24 hours, collect 2^nd, 3^rd and 4^th conditioned medium. Centrifuge and add to same same bottle. After the 4^th collection, add an equal volume of fresh medium to the bottle (final concentration: 50%), mix well and aliquot media into 50 mL centrifuge tubes and store at -20°C. Collect 5^th to 8^th and 9^th to 12^th conditioned media, if desired. Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:10 is recommended.Medium Renewal: Every 2 to 3 days.
Cryopreservation	Freeze Medium: complete growth medium, 70%; fetal bovine serum, 20%; DMSO, 10%Storage Temperature: liquid nitrogen vapor phase
Culture Conditions	Temperature: 37°C; Atmosphere: air, 95%; carbon dioxide (CO₂), 5%

Volume	1.0 mL
STR Profile	CO1 species: mouse

Name of Depositor	T Stappenbeck (Washington University)
Year of Origin	2013
References	Miyoshi H, et al. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat. Protoc. 8(12): 2471-2482, 2013. PubMed: 24232249

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