HCM-BROD-0783-C71

ICD-10-CM code: C71, malignant neoplasm of cerebrum; Recurrent glioblastoma

NeuroCult NS-A Basal Medium (StemCell Technologies #05750) with NS-A Proliferation Supplement (StemCell Technologies #05754) + 20 ng/mL EGF (StemCell Technologies #78003.1) + 20 ng/mL bFGF (Peprotech #AF-100-15) + 2 µg/mL Heparin (StemCell Technologies #07980).

Prepare media according to the manufacturer’s instructions: Stem Cell Technologies Catalog #5751

Culture vessels must be pre-coated with laminin prior to seeding with cells.

Coating procedure (perform all steps in a biosafety cabinet)

1. Thaw (Corning #354232) laminin on ice.
2. Transfer 10 mL of D-PBS (ATCC 30-2200) to a 15 mL conical tube.
3. Add 100 uL stock laminin to the 15 mL conical tube with D-PBS and mix thoroughly.
4. Added diluted laminin to vessels at a volume of approximately 1 mL per 10 cm2. For example, use 7.5 mL diluted laminin for a 75cm2 flask.
5. Tilt plate to ensure entire surface is coated.
6. Place in a humid cell culture incubator at 37C for 1-24 hours.

Immediately prior to seeding cells, aspirate off the laminin coating solution and discard. Do not allow the plates to dry out.

To ensure 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. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
2. 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.
3. Transfer the vial contents to a centrifuge tube containing  9.0 mL complete culture medium. and spin at approximately 200 x g for 5 minutes.
4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6). pH (7.0 to 7.6).
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.

Culture vessels must be pre-coated with laminin prior to seeding with cells.

Coating procedure (perform all steps in a biosafety cabinet)

1. Thaw (Corning cat#354232) laminin on ice.
2. Transfer 10 mL of D-PBS (ATCC 30-2200) to a 15 mL conical tube.
3. Add 100 µL stock laminin to the 15 mL conical tube with D-PBS and mix thoroughly.
4. Added diluted laminin to vessels at a volume of approximately 1 mL per 10 cm². For example, use 7.5 mL diluted laminin for a 75 cm² flask.
5. Tilt plate to ensure entire surface is coated.
6. Place in a humid cell culture incubator at 37°C for 1-24 hours.

Immediately prior to seeding cells, aspirate off the laminin coating solution and discard. Do not allow the plates to dry out.

1. Passage cells when the culture has reached approximately 70% to 80% confluence.
2. Pre-coat new culture vessels with laminin, if necessary.
3. Warm Accutase (StemCell Technologies  cat# 07920) and complete growth media to room temperature.
4. For each flask, carefully aspirate the spent media without disturbing the monolayer.
5. Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.
6. Add room temperature Accutase (1 to 2 mL for every 25 cm²) to each flask.
7. Gently rock each flask to ensure complete coverage of the Accutase solution over the cells, and then aspirate the excess fluid off of the monolayer.
8. Observe the cells under the microscope.
9. When the majority of cells appear to have detached (typically 2-5 minutes), quickly add an equal volume of the complete growth medium to each flask.
10. Transfer the dissociated cells to a sterile centrifuge tube.
11. Centrifuge the cells at 200 x g for 5 minutes.
12. Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, pre-warmed, complete growth medium.
13. Count the cells and seed new culture flasks at a density of 5 x 10⁴ viable cells per cm2. Prior to seeding, aspirate the coating and discard the coating laminin solution from the vessel.
14. Place newly seeded flasks in a 37°C, 5% CO₂ incubator for at least 24 to 48 hours before processing the cells further.
15. Perform a complete medium change every 3-4 days.

Note: This model has exhibited significant instability in culture. Refer to the lot # CoA for the STR profile.

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.

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.