SFM for CHO Cells in Suspensions

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Serum-Free Medium for CHO Cells in Suspensions

Cat. #	Product Name	Size	Storage temp.
05-062-1	BIOCHO-2 SFM Base w/o L-Glutamine	B 100 ml A 500 ml	2-8ºC
05-062-5	BIOCHO-2 SFM Base Ten-Fold Conc. w/o L-Glutamine w/o Sodium Bicarbonate	B 100 ml A 500 ml	2-8ºC
05-620-1	BIOGRO-CHO Serum-Free Media Supplement 100-Fold Concentrate	F 1 ml H 5 ml E 50 ml	-20ºC

Instructions For Use

Introduction

The transfer of CHO cells growing in serum-supplemented culture to serum-free suspension culture can be carried out in two ways:

1. Single Stage

Direct transfer of the cells from serum-supplemented monolayer culture to serum-free suspension culture using BIOCHO-2 SFM Base and BIOGRO-CHO.

2. Two Stage

Transfer of the cells from serum-supplemented monolayer culture to suspension culture using BIOCHO-2 SFM Base and 5% serum, followed in the second stage by replacement of the serum-containing media with BIOCHO-2 SFM Base and BIOGRO-CHO.

In either case, it is important to use materials and techniques that were specifically developed for serum-free culture. Following the detailed procedures given here will ensure success in the transfer to serum-free suspension culture.

Growth Medium

BIOCHO-2 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-2 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.

This medium is intended for the growth of CHO cells of various kinds: CHO-KI, and transfected cells containing recombinant DNA related to the DHFR gene.

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

Trypsinization With Crystalline Trypsin

1. 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.

2. 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.

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.

Adaptation of the CHO Cells to Serum-Free Medium

As explained above, it is possible to try a single-stage approach in the transfer to serum-free suspension culture. However, sensitive recombinant cells may not adapt successfully using this approach, and at least 7-10 days will be required in order to reach reasonably high cell density with non-sensitive cells.

In the two-stage approach, the cells are first transferred to serum-supplemented BIOCHO-2 for a period of 7 days, and the subsequent transfer to serum-free culture will require from 3-4 days, or up to 10-15 days in the case of sensitive cells.

When transferring the cells to suspension culture, it is important to guarantee all of the following conditions:

Cell Concentration for seeding: At least 500,000 per ml

Volume of the Culture: No more than 40-50% of the volume of the spinner

Speed: 60-80 revolutions per minute

Viability of the Cells: at least 90%

After 3-4 days the cells will begin to form aggregates of 3-4 cells, and these aggregates will then grow to contain approximately 50 cells. After a cell density of 2x10⁶ per ml is reached, half of the culture medium should be replaced every 2 days. When the viability of the cells goes below 85%, or after several weeks, it is advisable to centrifuge the cells at 5-10ºC (500g).

Since the cells grow as aggregates, in order to follow their growth by counting, a sample must be taken while stirring is in progress, 1:1 dilution with crystalline trypsin is carried out, and the cells are left for 15 minutes at ambient temperature. After non-aggregated cells are obtained, they can be stained with Trypan Blue and counted.

September 2001