NIIF PARA PYMES

Because of the large size of mammalian cells and the relatively low cell densities reached, it is possible to make a direct count of cell numbers using a haemocytometer, a suitable dye and a magnification of x200 on a microscope (Olympus BH- 2 ) . On some occasions, photographs were taken of the samples using an Olympus 0M-2fj camera attached to the microscope.

100 ^.l of a well-mixed cell culture sample was placed in a Treff tube and an equal volume of a 0.4% (w/v) solution of trypan blue (Sigma, T614 6) was added. A haemocytometer with Modified Fuchs-Rosenthal grid (Philip Harris Scientific, London) was prepared and cell/dye mixture introduced under the cover slip, allowing the chamber to fill by capillary action. A hand counter was used to record the number of cells in three sections of the haemocytometer grid, and a simple calculation was then used to find the number of cells per millilitre of broth.

It is also possible to convert between dry weight and cell count using the following equation

Absolute cell number x (4.9 x 10'^°) = dry weight in grams. (Pers. comm: S. Froud, Celltech Ltd., Slough, Berkshire.) 6.1.4 Cell viabilitv.

6.1.4.1 Dye exclusion.

This method was used as an extension of the measurement of total cell number. It is based on the principle that live cells, having an intact, functioning membrane, will exclude certain dyes, while dead cells will become stained as their membranes become permeable. Dyes commonly used include trypan blue, eosin yellow and erythrosin B. The latter is particularly useful for culture media containing serum as, unlike trypan blue, it does not stain serum proteins (Griffiths, 1985; Phillips, 1973). However, no particular problem was encountered with the use of trypan blue provided that the cells were counted within five minutes of mixing with the dye, as viable cells had a tendency to take up the dye if left for longer. Results were recorded as a percentage of the total cell count. The main drawback with the dye exclusion method is that it is a measure of membrane function, while the strict definition of a viable cell is one that will replicate in culture (Petersen et al, 1988). Furthermore, this method does not take into account cells which have lysed (Marc et al, 1990) . The permeability of a membrane can be affected by recent trypsinisation or by freezing and thawing, especially in the presence of dimethyl sulphoxide, so cells recently revived from frozen storage in a cell bank must be counted with care; many cells indicated as 'non-viable' by the dye exclusion method will recover and replicate normally. The pH and concentration of the surrounding media also affects the membrane characteristics (Griffiths, 1985). It is therefore usual to combine this method with another, and take account of the results of both tests.

6.1.4.2 Measurement of the extent of cell Ivsis.

This can be determined by monitoring the extent of release of the cytoplasmic enzyme lactate dehydrogenase into the surrounding medium. Problems with the assay method, and degradation of the enzyme standard meant that the method was not included in these studies. The method is described in the

section concerning future work (Section 8.1.3). 6.2 Tests for contaminating microorganisms.

The following tests are routinely used to check the sterility of mammalian growth media and can also be used to test for the presence of contaminants during a fermentation.

The earliest sign of contamination with aerobic bacteria is an increase in the oxygen demand of the cell culture. This is followed by a colour change of the phenol red indicator in the growth media from red to yellow as a result of the acidic conditions generated by the bacteria, usually B a c i l l u s .

A further test is the inoculation and incubation at 20-25°C of tryptone soya broth (Oxoid CM129). The culture should be examined after 5-7 days, and again after 14 days. Cloudiness indicates the presence of aerobic bacteria.

To detect the presence of moulds and fungi, a sample of culture media should be used to inoculate a vial of sabouraud liquid media (Oxoid CM147), which is then incubated at 20-25°C and examined after 5-7 days, and after 14 days. Again, cloudiness indicates the presence of contaminants.

Fluid thioglycollate medium (Difco 0256-17) is used to test for anaerobic bacteria. The inoculated vial is incubated at 30-35°C and examined for cloudiness as previously described. During the course of this study, such tests were used only to confirm the sterility of media, and when specific contamination problems necessitated the identification of the contaminating microorganism in order to trace its source. 6.3 Tests for viral contamination.

The cell lines used in the course of this study were all in commercial use, and had undergone the rigorous tests described in Section 2.3.6. For this reason no further tests for viral contamination were carried out.

6.4 IgM assay.

To decide upon a suitable harvesting point in the culture cycle, the level of the required product should be monitored. This can present problems if the product has not been analysed with regard to chemical composition (eg. antibodies or v i r u s e s ) . Indeed, some products can be evaluated only by applying them to animals or humans and measuring their effects as "a definable non-specific appearance or increase of a biological property (production of antibodies, resistance) specifically characteristic of the biological” (Fontaine et

al, 1985). More usually, some specific characteristic of the product can be assayed and quantified. The cell line which was predominantly used in this project is a mouse hybridoma which produces immunoglobulin M (IgM). It can be assayed using an enzyme-linked immunosorbent assay (ELISA). In this case, a double antibody sandwich ELISA was used, in which the antibodies in the culture media are exposed to specific antigens which have been adsorbed onto the plastic ELISA plate. To detect the antibodies which have been bound to the antigens, a conjugate is added. This is another antigen, to which a particular enzyme has been linked. The test system is then washed clean of any uncombined conjugate and incubated with an appropriate substrate for the enzyme, and the product of the enzymic cleavage is assayed, for example by spectrophotometry.

Each sample was prepared to give a total of seven dilutions ranging from 1/10 to 1/1000, as was the IgM standard (Purified mouse IgM k, TEPC 183, obtained from Organon Teknika

(Cat.#50336) or Sigma (M2770)). The standard concentrations ranged from 10 to 1000 ng ml'^. 100 /Ltl volumes were added in duplicate to 96-well, flat-bottomed microtiter plates (EIA II microplates, ION Flow, High Wycombe, Bucks.) that had been coated overnight in a buffer containing coating antisera (Goat antimouse IgM, Tago Immunologicals Inc. (Cat.#4142)) . After a period of time the plate was washed to clear it of any unbound antibody, using a Titertek Handiwash 110 plate washer (ICN Flow, High Wycombe, Bucks.), then a conjugate was added.

This consisted of a further antibody conjugated to the enzyme horseradish peroxidase, type VI (Goat anti-mouse IgM HRPO, Tago Immunologicals Inc. (Cat.#6442) or Anti-mouse IgM (ju- chain specific) peroxidase conjugate, Sigma (A8786)). Again after a period of time, the plate was washed clean of any excess conjugate and a substrate was added. Reaction with the horseradish peroxidase caused the development of a blue colour, the absorbance of which was read at 570 nm using a Titertek Multiskan Plus Mk. II microplate reader (ICN Flow, High Wycombe, Bucks.). A standard curve was constructed for each plate and the concentration of IgM in each sample was calculated from this. Studies of cell lines producing IgG in the course of this thesis were assayed using protocols and reagents supplied by Celltech Ltd. (Slough, Berkshire). Ideally, another method would have been included to verify the results as the variance of the ELISA is ±25%. However, the options were limited by the small, dilute samples available. The use of a Protein A column to bind and release IgG is common, but Protein A does not bind IgM. The possibility of using thin layer chromatography was also explored, but the antibody is denatured by the sample preparation procedure, and appears as two faint bands — light and heavy chains — which are difficult to see even with silver staining. Quantification of such bands using densitometry was considered too inaccurate to be used. As a result the findings of the ELISA were not verifiable except by duplication, which was used both per plate and between plates to reduce error as much as possible. In a limited number of cases it was possible to verify the ability of the IgM to agglutinate blood cells by sending the samples to the Protein Fractionation Centre at the Scottish National Blood Transfusion Centre in Edinburgh. The samples were assayed using a method known as 'titre/immediate spin' which assesses the activity of the sample over a series of doubling dilutions from neat to 1/512. Such samples were of necessity restricted in number, and could by no means be used as a routine method of verifying ELISA results.