Serum-Free Medium for Adherent Cells

BIO-MPM-1 Cat. no.: 05-060-1

Introduction

A successful transition from cell culture work utilizing serum-containing media to serum-free cell culture often requires the use of techniques which were specifically developed for this purpose. For example, 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 BIO-MPM

Growth Medium

BIO-MPM-1 is a ready-to-use serum-free medium for adherent cells, after the addition of 2 millimolar glutamine. The formulations contains no albumin which has been found to be non-essential for cell growth, and even prevents efficient adhesion in some cases. The protein content of BIO-MPM-1 is therefore less than 30mg per liter, and the medium contains no growth factors or hormones other than insulin. The formulation also contains no attachment factor, which in many (but not all) cases must be added for successful use.

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.

Testing for the Necessity to Use Fibronectin or Other Additives

Since BIO-MPM-1 is intended for use with adherent cells, in many cases it will be necessary to use fibronectin for the initial adhesion of the cells to the growth surface. This must be checked individually for each specific type of cell. Some cells will not grow at all without fibronectin; others require no fibronectin addition; while others grow without fibronectin, but performance will be improved is fibronectin is added.

In the cases of a few unusual types of cells, it may also be necessary to add the growth medium BIO-MPM-1 a specific growth factor or hormone, for which the cells have a specific requirement.

Adaptation of the Cells to BIO-MPM-1

In most cases it is possible to seed the cells that have been removed from freezing medium directly in BIO-MPM-1, when the cell concentration is at least 5x105 cells per 25cm2. The cells will begin to grow in BIO-MPM-1, 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 BIO-MPM-1 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 be obvious whether direct adaptation has been successful, and if not, only the weaning experiments are continued. As part of these experiments it is also necessary to test for the possible requirement for fibronectin.

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 BIO-MPM-1. When the medium is replaced in the days following initial seeding, no further fibronectin is required. Instead of fibronectin, it is, of course, possible to use other adhesion factors such as poly-lysine, collagen, etc.

Summary

In order to succeed in the serum-free growth of adherent cells, 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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