Serum-Free Medium for CHO Cells

Instructions For Use

BIOCHO-1 SFM Base is the basic formulation for CHO cells. The solution contains amino acids, vitamins, salts, lipids and trace elements. This solution should be stored at 4ºC, and prolonged exposure to light should be avoided.

BIOGRO-CHO SFM Supplement contains proteins and other components that require storage at -20ºC.  This product is a 100-fold concentrate. Repeated thawing and re-freezing should be avoided by thawing, dividing into useful portions, and re-freezing.

Preparation of the complete medium is carried out by adding 1% BIOGRO-CHO SFM Supplement to BIOCHO-1 SFM Base. Two millimolar glutamine is then added.  The complete medium does not contain albumin, growth factors or hormones, other than insulin.  Total protein concentration is less than 30mg per liter.

After preparation, the complete medium can be stored for up to 30 days at 4ºC.  Prolonged exposure to light should be avoided.

Applications

These media are intended for the growth of CHO cells of various kinds: CHO-KI, and transfected cells containing recombinant DNA related to the DHFR gene.

Auxiliary Solutions

  • Crystalline Trypsin (Cat. No. 03-047-1)
    This is a ready-to-use solution with a concentration of 200mg per liter, and is shipped and stored at -20ºC.  Besides crystalline trypsin, it also contains additives which protect the cell wall.
  • Soybean Trypsin Inhibitor (SBTI) (Cat. No. 03-048-1)
    This solution is at a concentration of 5mg per ml (50-fold concentrate), and is also shipped at -20ºC.  Before use, the solution is diluted by a factor of 50 with sterile PBS solution, and can then be stored at 4ºC for up to 60 days.
  • Fibronectin (Cat. No. 03-090-1)
    This solution is at a concentration of 1mg per ml and is stored at 4ºC.
  • Serum-Free Cell Freezing Medium (Cat. No. 05-065-1)
    This solution is used for the cryopreservation of cells, which are to be grown in serum-free growth medium.  The use of this medium, rather than serum-containing freezing media, avoids the necessity of repeated passages in order to remove serum residues.

Adaptation of the CHO Cells to Serum-Free Medium

In most cases it is possible to seed CHO cells that have been removed from freezing medium directly in the serum-free medium, when the cell concentration is at least 5x105 cells per 25cm2.  The cells will begin to grow, and after a few passages the adaptation will be complete.

However, in those cases where the cells do not adapt successfully after direct transfer, it will be necessary to perform gradual adaptation (weaning). The cells should be seeded with serum-free medium 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 transfer, it will be obvious whether direct adaptation has been successful, and if not, only the weaning experiments are continued.

After successful adaptation, it is recommended to cryopreserve the cells in Serum-Free Freezing Medium, in order to avoid the necessity of any further adaptation in the future.

Trypsinization With Crystalline Trypsin

Compared to solutions of crude trypsin, crystalline trypsin solution does not damage the cells even after prolonged exposure. In addition, the excess trypsin can be neutralized with SBTI, thus avoiding the introduction of serum. The crystalline trypsin solution should be thawed, divided into smaller portions and then re-frozen, in order to avoid the necessity of repeated thawing-freezing cycles.

For trypsinization of the cells, use 1ml solution per 25cm2, after washing the cells with PBS solution (without calcium and magnesium). The culture should be left at room temperature, even if the trypsinization required as long as 30 minutes.  After the cells have been removed from the growth surface, suspend them in diluted SBTI solution (0.1mg per ml) at a ratio of 5ml SBTI solution for each ml of trypsin solution.  Then centrifuge, suspend the cells in serum-free medium and transfer them in the desired concentration.

The Use of Fibronectin

The fibronectin solution should be added to the growth medium in the growth vessel, which is then placed in an incubator 30-60 minutes before seeding.  The recommended concentration of the fibronectin is 5 micrograms per ml of serum-free medium.  When the medium is replaced in the days following initial seeding, no further fibronectin is required.

Summary

In order to succeed in the serum-free adherent cell culture, it is necessary to follow proper procedures and techniques carefully. Avoid the storage of the growth medium for long periods at room temperature, and also avoid extended exposure to light. We also recommend the use of minimal concentrations only of antibiotics. Remember that cells growing in serum-free culture are fragile, and are easily damaged by faulty techniques. However, careful attention to the recommended concentrations and procedures will lead to success in the great majority of cases.

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