Cell-Vive™ T-NK Xeno-Free Serum Substitute, GMP

Product Details

Formulation

Xeno-free, serum-free formulation

Endotoxin Level

< 1 EU/mL

Storage & Handling

Store at -20°C to -5°C. Thaw overnight at 4°C. If necessary product can be kept at 4°C up to two weeks.

Application

Cell Culture

Recommended Usage

Recommended use at 5% v/v in basal media, combined with 200 IU/mL of human recombinant IL-2. Product should be titrated (2.5-10%) to determine optimal performance for your culture conditions.

Application Notes

Note: This product contains human-derived components.

Complete cell Culture Media Preparation:
Thaw Cell-Vive™ T-NK Xeno-Free Serum Substitute, GMP overnight at 4°C. The product can be stored at 4°C in the dark up to 2 weeks post-thaw. Do not refreeze after initial thaw. Presence of cloudiness in the product after thaw is normal, and relates to the high enrichment of the product. Make sure to mix product prior to use. Once the product is diluted into basal media, no cloudiness should be observed.

Dilute Cell-Vive™ T-NK Xeno-Free Serum Substitute, GMP to 5% v/v in IMDM and 200 IU/mL of recombinant human IL-2 (BioLegend, Cat. No. 791906 or equivalent).  Alternatively, the product can be used in combination with your basal media of choice and cytokines of interest. To determine optimal performance, a titration of the product under your desired cell culture setup may be required.

Cell Culture Platform:
Cell-Vive™ T-NK Xeno-Free Serum Substitute, GMP can be used with tissue culture plates, flasks and the G-REX® system. If using a bag format, testing is recommended with the specific bag being used.

Cell Activation methods:
Cell-Vive™ T-NK Xeno-Free Serum Substitute, GMP can be used in combination with anti-human CD3 and CD28 antibodies in solution, plate-coated or conjugated to magnetic beads. Optimal values should be determined if using a different culture setup.

For the protocols below, complete media refers to IMDM with 5% v/v Cell-Vive™ T-NK Xeno-Free Serum Substitute, GMP and 200 IU/mL recombinant human IL-2 (BioLegend, Cat. No. 791906 or equivalent) for T cell culture, and 1000 IU/mL for NK cell culture.

1. Coating plates with anti-human CD3 and CD28 antibodies
Add 1 µg/mL of anti-human CD3 (OKT3 clone, BioLegend, Cat. No. 317326 or equivalent) and 1 µg/mL of anti-CD28 antibodies (CD28.2 clone, BioLegend, Cat. No. 302934 or equivalent) to PBS. Add solution to the vessel, at recommended volume (table below). Incubate for 2 hrs at 37°C in a tissue culture incubator (or overnight at 4°C, preventing evaporation with Parafilm®). Aspirate solution and wash twice with 1 volume of PBS. Wells are ready to be used. Do not let wells dry.

TABLE 1: Recommended volumes of coating solution composed of anti-human CD3 and CD28 antibodies.

Vessel	Recommended volume
12- well plate	0.75 mL
6- well plate	2 mL
T25 flask	5 mL
T75 flask	15 mL

2. Soluble antibodies
Add 10 µg/mL of anti-human CD3 (OKT3 clone, BioLegend, Cat. No. 317326 or equivalent) and 5 µg/mL of anti-CD28 antibodies (CD28.2 clone, BioLegend, Cat. No. 302934 or equivalent) to complete media.

PBMC-Derived T cell culture protocol:

1. Equilibrate appropriate amount of complete media at 37°C for 10-15 min.
2. Plate PBMC (fresh or frozen) at 1 x 10⁶ cells/mL and incubate at 37°C and 5% CO₂.
3. Allow cells to get activated for 3-4 days.
4. At day 3-4, dissociate cell clumps by pipetting until a single cell solution is observed.
5. Determine cell density.
6. Dilute cell culture to 0.25 - 0.5 x 10⁶ cells/mL and incubate at 37°C and 5% CO₂.
7. Repeat steps 4-6 every 2 days.   
8. Monitor cell culture to make sure confluency is not reached. Pending donor-to-donor variability exponential expansion may require more frequent feeding. Complete media has been shown to support up to 4 x 10⁶ cells/mL without impact on cell viability and quality.
9. Follow with further processing and/or cell analysis.

PBMC-Derived NK cell culture protocol using K562 feeder cells:

1. Start by inactivating K562 cells with mitomycin-C (20 µg/mL in K562 growth media) for 45 min at 37°C and 5% CO₂.
2. Spin down inactivated K562 cells at 300xg for 5 min. Wash cell pellet twice with fresh media, to remove any leftover mitomycin-C.
3. Equilibrate appropriate amount of complete media at 37°C for 10-15 min.
4. Plate PBMC (fresh or frozen) at 0.5 x 10⁶ cells/mL and incubate at 37°C and 5% CO₂.
5. Add feeder cells (inactivated K562 cells (recommended value of 1 K562 cell to 2 PBMC cells; optimal value can be determined for desired culture setup).
6. Allow NK cells to get activated for 2-3 days.
7. Determine cell density.
8. Dilute cell culture to 0.25 - 0.5 x 10⁶ cells/mL and incubate at 37°C and 5% CO₂.
9. Repeat steps 4-6 every 2 days. 
10. Re-feed NK cell culture with freshly inactivated K562 cells at day 7.
11. Repeat steps 4-6 every 2 days up to day 14 (or appropriate culture end).
12. Follow with further processing and/or cell analysis.

G-REX® is a registered trademark of Wilson Wolf Corporation.
Parafilm is a registered trademark of Bemis Company, Inc.

Additional Product Notes

BioLegend has submitted a Device Master File (DMF) to the US Food & Drug Administration (FDA) for “Cell-Vive™ T-NK Xeno, Serum Substitute, GMP” product. Neither the FDA nor other outside party may have access to or release the DMF without the holder's consent as all information contained in a DMF is considered confidential to the holder.

 

As a BioLegend customer using our “Cell-Vive™ T-NK Xeno, Serum Substitute, GMP” product in a manufacturing process, we can provide a “Letter of Authorization (LOA) to you in support of a submission or filing that you have made to the FDA. To initiate the reference authorization of our DMF, please submit a request to BioLegend with the required information so we can provide a DMF Letter of Authorization to the respective FDA Center. Once we have your request, we will advise the FDA that a BioLegend DMF is relevant to its review and that the agency has our consent to support your submission. To submit a request please contact our Customer Service Department.

Disclaimer

BioLegend Cell-Vive™ GMP Cell Culture products are for research use only. Suitable for ex vivo cell processing. Not for injection or diagnostic or therapeutic use. Not for resale. BioLegend will not be held responsible for patent infringement or other violations that may occur with the use of our products.

Antigen Details

Gene ID

NA

Documentation

• Technical data sheet
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

Certificate of Analysis