L-WRN (ATCC® CRL-3276)

Organism: Mus musculus, mouse  /  Tissue: subcutaneous connective tissue; areolar and adipose  / 

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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
Images Cell Micrograph of L-WRN Mouse Transfected Fibroblast Cells
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.

  1. 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.
  2. 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).
  3.      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.

  4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
  5. 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:
  1. 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
  2. Incubate the flasks for 3 or 4 days or until the cells become over-confluent and a number of cell aggregates come off.
  3. Remove medium and rinse flasks with 10 mL medium and discard rinse.
  4. Add 25 mL fresh medium and incubate flasks for 24 hours.
  5. 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.
  6. Every 24 hours, collect 2nd, 3rd and 4th conditioned medium. Centrifuge and add to same same bottle.
  7. After the 4th 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.
  8. Collect 5th to 8th and 9th to 12th 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

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • For-profit customers must obtain a research use license agreement from the Contributor prior to shipment.
  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
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Other Documentation
Restrictions

This material is subject to the following restrictions in addition to those outlined in the ATCC Material Transfer Agreement:

  1. Prior to purchase, any for-profit Purchaser must obtain a research use license from Washington University; and
  2. When a Commercial Use is contemplated, all for-profit and non-profit Purchasers must obtain a Commercial Use license from the contributor.  For information on executing a license to use this product for research (for-profit entities) or Commercial Use (for-profit and non-profit entities), contact:
Washington University Office of Technology Management
Attn. Associate Director
E-mail: otm@dom.wustl.edu
Phone: 314.747.0920.

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