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L-WRN

CRL-3276

Product category
Animal cells
Organism
Mus musculus, mouse
Morphology
fibroblast
Tissue
Subcutaneous connective tissue; Adipose; Areolar
Applications
3D cell culture
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

Biosafety Icon BSL 2

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

Cells contain SV40 sequences

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information

General

Specific 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.

Characteristics

Volume
1.0 mL
Growth properties
Adherent
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.
Age
100 days
Gender
Male
Strain
C3H/An
Genes expressed
wnt-3A; R-spondin; noggin
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).

Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete 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%
Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure
To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at 70°C. Storage at 70°C will result in loss of viability.
  1. Pre-warm growth medium (without G418 and without Hygromycin B) in flask at 37°C.
  2. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the Oring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
  3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
  4. Transfer the vial contents to the culture pre-warmed flask with medium. Because cells are very fragile after thawing, centrifugation and excess pipetting must be avoided.
  5. Incubate the culture at 37°C in a suitable incubator. A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet.
  6. After 24 hours, change medium with complete growth medium, which contains G418 and Hygromycin B.
Subculture when culture is subconfluent or proceed with protocol to prepare conditioned medium.
Subculturing procedure

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.
Reagents for cryopreservation
Complete growth medium supplemented with 20% (v/v) fetal bovine serum (ATCC 30-2020) and 10% (v/v) DMSO (ATCC 4-X)

Quality control specifications

STR profiling
CO1 species: mouse

History

Depositors
T Stappenbeck (Washington University)
Year of origin
2013

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

For-profit Research-use License

For-profit organizations
For every order of this item, you must have a research-use and/or commercial-use license from the contributor. Please refer to the details in the “Permits and Restrictions” section of the product page for licensing information. We cannot ship this item until we receive this license or authorization directly from the contributor in the form of an email.

If sending the license to us, email the license to [email protected] with a reference to both your account and sales order numbers. Once either the license or authorization is received, your order will be reviewed, and this item will be released for shipment if all requirements are met. If you need assistance with your order, please contact our Customer Care team or your applicable distributor.

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

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

  1. For-profit Recipients - must obtain a research use license prior to shipping.
  2. Commercial Use - all for-profit and non-profit Recipients must obtain a commercial use license prior to any Commercial Use.

Please contact Washington University in St. Louis at:

Washington University Office of Technology Management
Attn. Associate Director
Phone: 314-747-0920
Email:[email protected]

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

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

Frequently Asked Questions

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Telephone

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