SARTORIUS 4Cell NutriVero Flex 10 500mL Bottle Instructions

SARTORIUS 4Cell NutriVero Flex 10 500mL Bottle

A chemically defined (CD), animal component-free (ACF) medium, designed to support the expansion of Vero Cells and viral vaccine production

Product Description

4Cell®
NutriVero™ Flex 10 is a chemically defined, animal component-free medium developed to support Vero cell growth in both microcarrier suspensions and monolayer cultures in the areas of virology, virus production, and biotechnology . There are certain risks associated with the use of animal sera, e .g ., fear of contamination with adventitious agents such as BSE, Hepatitis, and HIV . Culturing cells in a serum-free and animal component-free medium eliminates these risks. Furthermore, it allows cells to be grown under a defined set of conditions . 4Cell® NutriVero™ Flex 10 is appropriate for both microcarrier suspensions and monolayer cultures of Vero cells. 4Cell® NutriVero™ Flex 10 is suitable for large-scale culturing and for virus production as well as other cell culture applications.

Features

• Suitable for 2D and 3D cultures
• Chemically defined – no animal or plant components
• Very low protein concentration
• Lot-to-lot consistency
• Optimal for the production of various viruses (incl . measles, enterovirus, and polio)
• Suitable for direct adaptation
• Easy downstream product purification

Precaution and Disclaimer

• Do not use if a visible precipitate is observed in the medium.
• Do not use 4Cell® NutriVero™ Flex 10 Medium beyond the expiration date indicated on the product label

Storage and Stability

• 4Cell® NutriVero™ Flex 10 Medium should be stored at 2-8°C, and protected from light.
• Shelf Life: refer to the product label for the expiration date

Instructions for Use

Procedure
A successful transition from serum-containing media to serum-free media often requires the use of techniques that were specifically developed for this purpose . Special techniques for trypsinization, neutralization of trypsin, cryopreservation of cells, as well as the use of an effective serum-free growth medium are all essential . Careful attention to the details of procedures outlined here are therefore essential in order to guarantee the successful use of 4Cell® NutriVero™ Flex 10. 4Cell® NutriVero™ Flex 10 is a ready-to-use serum-free medium, which does not require the addition of L-glutamine or Alanyl glutamine. Antibiotics are not specifically recommended, but if required, use a lower concentration than usual.

Adaptation of the cells to 4Cell® NutriVero™ Flex 10
In most cases, it is possible to seed cells that have been removed from the freezing medium directly into 4Cell® NutriVero™ Flex 10, when the cell concentration is approximately 8 × 103 cells per cm2. The cells will begin to grow in 4Cell® NutriVero™ Flex 10, and after a few passages, the adaptation will be complete . Cells should be passaged for a minimum of three passages in 4Cell® NutriVero™ Flex 10 prior to testing in a growth promotion assay . After successful adaptation, it is recommended to cryopreserve the cells in NutriFreez™D10 Cryopreservation Medium (05-713-1) in order to avoid the need for any further adaptation in the future. For cell dissociation, we recommend using Recombinant Trypsin EDTA Solution (Cat . No . 03-079-1), and for trypsin neutralization Soybean Trypsin Inhibitor (Cat . No . 03-048-1), which are both chemically defined reagents and contain no products of animal origin

Adaptation from serum-containing media:
If adopting directly from a serum-supplemented medium to 4Cell® NutriVero™ Flex 10, the initial cell plating density should be greater than that used with serum-containing media for the first 2 – 3 passages . In cases where the cells do not adapt successfully after the direct transfer, it will be necessary to perform gradual adaptation (weaning). The cells should be seeded with 4Cell® NutriVero™ Flex 10 containing 5% serum, and the serum concentration is then gradually reduced with each passage. The stage at which serum is completely removed is determined in the course of the weaning for each specific case . In order to save time, we recommend parallel experiments with direct adaptation and with weaning. Generally, after the first or second passage, it will become evident if direct adaptation has been successful, and if not, only the weaning experiments are continued.

Culturing Vero cells in 4Cell® NutriVero™ Flex 10

Recovery of cryopreserved Vero cells
Note: for the recovery of cryopreserved and subculturing of Vero cells always pre-warm 4Cell® NutriVero™ Flex 10 to room temperature

• Pre-warm to room temperature 5 – 10 mL of 4Cell® NutriVero™ Flex 10 in a 50 mL conical tissue culture tube.
• Rapidly thaw frozen vial of Vero cells in a 37°C water bath, with gentle agitation
• Slowly add the cells into the pre-warmed 4Cell® NutriVero™ Flex 10 until no ice remains
• Centrifuge cells at 400 g for 4 .5 minutes at room temperature.
• Remove supernatant and re-suspend the cell pellet in 0 .5 – 1 mL of pre-warmed 4Cell® NutriVero™ Flex 10
• Perform a viable cell count (e .g ., using Trypan Blue Exclusion Assay)
• Add the desired volume of pre-warmed 4Cell® NutriVero™ Flex 10
• Transfer the cells into desired culture are . Seeding densities should be calculated (see Table 1)
• Incubate in a humidified CO2 incubator (37°C).

Table 1: Recommended seeding densities
(approximately 8000 cells/cm2)

Cultureware| Surface area cm2| Volume of

4Cell® NutriVero™ Flex 10 (mL/well)

| Recommended seeding

(total cells)

---|---|---|---
24-well| 1 .9| 0 .5| 1 .52 × 104
12-well| 3 .9| 1| 3 .12 × 104
6-well| 9 .6| 2| 7 .68 × 104
T25| 25| 5| 20 × 104
T75| 75| 10| 60 × 104

Subculturing Vero cells 4Cell®
NutriVero™ Flex 10 Medium was developed for the optimal proliferation of Vero cells . We recommend seeding the cells at a concentration of 8000 cells/cm2 (Table 1), re-feeding the cells with fresh warmed to room temperature 4Cell® NutriVero™ Flex 10 Medium every 2 – 3 days, and subculturing when the cells reach approximately 80 – 100% confluence.

Procedure

• Remove culture medium and gently wash once with DPBS w/o Ca, Mg (Cat. No. 02-023-1).
• For a T25 culture flask, add 1 mL of Recombinant Trypsin Solution with EDTA (Cat. No. 03-079-1) at room temperature (for any other cultureware, the appropriate volume should be adjusted).
• Incubate for 2-4 minutes at 37°C and verify cell detachment using an inverted microscope.
• Note: The more the culture is confluent, the slower the detachment will be, and the higher the volume of trypsin recommended.
• Following detachment, add 5 mL of pre-warmed Soybean Trypsin Inhibitor (SBTI, Cat. No. 03-048-1, diluted 1:50 in DPBS according to instructions)
• Collect cell suspension into a sterile tube and re-wash the cultureware as necessary to collect the entire cells.
• Centrifuge the cells for 4.5 minutes at 400 gat room temperature. Carefully discard the supernatant.
• Resuspend the cell pellet in a minimal volume of pre-warmed 4Cell° Nutri Vero” Flex 10 Medium. Take a sample to perform a viable cell count. For cryopreservation continue to Section 3.
• Seed cells into the desired culture are. Seeding densities and the required volume of 4Cell NutriVero” Flex 10 Medium to be added should be calculated (see Table 1).
• Incubate in a humidified CO, incubator (37°C).
• Feed cells with fresh warmed 4Cell° NutriVero” Flex 10 Medium every 2- 3 days.

Cryopreservation of serum-free cultures

• Grow desired quantity of cells in T-flasks, and harvest when flasks are 80% confluent.
• Following centrifugation, re-suspend the cell pellet in cold NutriFreez™ D10 Cryopreservation Medium at a concentration of 0 .5 – 5 × 106 cells/vial, 1 mL/vial .
• Freeze the cells gradually (1 – 2°C/minute using a freezing container [Mr . Frosty™] or controlled cryopreservation system), and store them in liquid nitrogen storage tank (vapor phase).
• Viability and recovery of cryopreserved cells should be checked 24 hours after storage of vials in liquid nitrogen by following the thawing procedure outlined above

Spinner Flasks or bioreactors: Microcarrier cultures
During cell culture, the rotational speed of the spinner flask should be 30 – 60 rpm . Seeding densities and attachment conditions for microcarrier cultures should be optimized to provide sufficient time for cell attachment.
Sample procedure
Vero cells can be seeded on Cytodex 1 microcarriers in spinner flasks or bioreactors . For a 250 mL spinner flask with a working volume of 100 mL, add 3 gr/L cytodex 1 and 1 × 105 Vero cells/mL and stir at 30 rpm . For 2L bioreactors with a working volume of 2L, add 3 gr/L codex 1 and 1 .3 × 105 Vero cells/mL and stir at 100 rpm.

Quality Control

The performance of 4Cell® NutriVero™ Flex 10 is tested using Vero cells pre- adapted to serum-free culture in 4Cell® NutriVero™ Flex 10 in Vero culture systems . Additional standard tests are pH, osmolality, sterility and endotoxins. For full specifications please check the lot-specific Certificate of Analysis (CoA).

Quality Assurance

• For research use & further manufacturing
• Manufactured under ISO 13485 QMS and in compliance with applicable cGMP guidelines.
• Manufactured under controlled environments and processes in accordance with:
  • ISO 13408 – Aseptic Processing of Health Care Products
  • ISO 14644 – Cleanrooms and associated controlled environments

Product Label Symbols

• Indicates the manufacturer’s catalog number so that the product can be identified.
• Indicates the manufacturer’s batch code so that the batch or lot can be identified. Note: Synonyms for batch code are lot number and batch number.
• Indicates the date after which the product is not to be used.
• Indicates the temperature limits to which the product can be safely exposed.
• Indicates a product that has been manufactured using accepted aseptic techniques

Auxiliary products

• Product Cat. No.
• Recombinant Trypsin EDTA Solution 03-079-1
• Soybean Trypsin Inhibitor (SBTI) 03-048-1
• NutriFreez™ D10 Cryopreservation Medium 05-713-1

Ordering Information

*Other sizes are available on request

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The information and figures contained in these instructions correspond to the version date specified below . Sartorius reserves the right to make changes to the technology, features, specifications and design of the equipment without notice . Masculine or feminine forms are used to facilitate legibility in these instructions and always simultaneously denote the other gender as well.
Copyright notice:
This instruction manual, including all of its components, is protected by copyright . Any use beyond the limits of the copyright law is not permitted without our approval . This applies in particular to reprinting, translation, and editing irrespective of the type of media used.

Germany
Sartorius Stedim Biotech GmbH
August-Spindler-Strasse 20
37079 Goettingen
Phone +49 551 308 0
www.sartorius.com
USA
Sartorius Stedim North America Inc .
565 Johnson Avenue
Bohemia, NY 11716
Toll-Free +1 800 368 7178

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