2.11.7 Assay o f Antibody Production in transfected CHO dhff cells.
Following the growth of selected CROdhfr' cells to just below confluent level in CROdhfr' (selective) growth medium B, the cells were trypsinised and spun down in a bench top centrifuge (250g for 5 minutes at room temperature). The cells were then resuspended in 20ml of pre-warmed CROdhfr' (selective) growth medium B and a viable cell count was carried out (see section 2.9.5). The cells were pelleted again as before and resuspended in sufficient pre-warmed CROdhfr' (selective) growth medium B to produce a viable cell count of 1 x 10^ cells/ml. 1ml (i.e. 1 x 10^ cells) was then added to a 100mm diameter tissue culture dish containing a further 9ml o f pre-warmed CROdhfr' (selective) growth medium B. The cells were grown to near confluency by incubating the cells for 3 days in 5% CO2 at 37°C. After three days, the supernatant of
using an ELISA, as in section 2.10.1. The cells from each 100mm diameter tissue culture dish were trypsinised and counted using a Trypan blue (see section 2.9.5).
From the new viable cell count and the antibody concentration in the decanted supernatants, it was then possible to calculate the level of antibody production in ng/10^ cells/day. A cell bank was created of those cell lines producing the highest amounts of whole IgG and further selection was carried out using methotrexate amplification.
2,11,8 Amplification o f transfected CHO dhff cells following electroporation and selection.
Following the growth of the selected CROdhfr cells in CROdhfr' (selective) growth medium B in a 175cm^ flask, the cell lines producing the highest levels o f IgG were trypsinised and spun down at 250g for two minutes at room temperature in a RTH-750 Sorvall rotor. The cells were resuspended in 20ml o f CROdhfr (selective) growth medium B to produce a final viable cell count of 1 x 10^ cells/ml. 0.5ml (i.e. 5 x 10^ cells) o f this solution was diluted in a further 49.5ml of pre-warmed 1 x lO'^M amplification medium (i.e. CROdhfr' (selective) growth medium B supplemented with 1
X lO'^M methotrexate [Amethopterin A6770, Sigma, Poole, UK]). The resulting culture
was then divided equally between five 1 OOmm-diameter tissue culture dishes, (i.e. 1 x 10^ cells/culture dish). This dilution was required in order to produce pure foci that could be easily identified from one another on the plate.
In addition, 0.5ml aliquots of the above cells were also plated out in amplification medium supplemented with 1 x lO'^M methotrexate. This second higher concentration of methotrexate was used in case the initial 1 x lO'^M concentration was too low for the cell line to produce single discrete foci.
The cells were incubated at 37°C in 5% CO2 for 10-14 days. The amplification medium
(supplemented with the appropriate concentration of methotrexate) was changed every 3-4 days. After 10-14 days, foci of transfected cells were easily visible in most, but not all of the culture dishes. If all the dishes, at a particular methotrexate concentration failed to produce any discrete visible foci, the dishes were incubated for a further 7 days to allow any slow growing foci to appear and were then discarded.
Materials and Methods
Approximately 48 foci were picked for each transfection using the same method as before (see section 2.11.5) and were then transferred into individual wells o f a 24-well tissue culture plate containing 1ml of pre-warmed CWOdhfr' amplification medium. The “picked” cells were allowed to grow in the amplification medium until almost confluent (usually after 7-14 days), whilst the medium was changed every 7 days or as required. Once almost confluent, the medium from the individual wells was tested for antibody production as before, using the Whole IgG ELISA (see section 2.10.1).
Those clones producing the highest levels of antibody were selected for expansion in CYiOdhfr (selective) growth medium B until the cells were growing in 175cm^ tissue culture flasks. The antibody production rates of individual clones (see section 2.11.6) were then determined in order to identify those clones worthy of further analysis and development.
A cell bank was created o f the selected amplified cell lines and a second round of methotrexate amplification using a higher concentration of methotrexate was considered. Normally the concentration of methotrexate used in a second round of amplification should be no greater than 10-fold the concentration o f methotrexate that was used to derive the amplified cells in the first round. When creating a cell bank, selective pressure was maintained when fi-eezing cells i.e. concentration of methotrexate was kept the same on freezing as that used in culture.
2,11.9 Production o f CHOdhff control line-containing pG lD 210 only (no Vx region sequence).
The ultimate aim of producing cell lines that would stably produce whole IgG was to use them to produce IgG not only for functional tests in vitro but also in tests to determine the pathogenicity of this IgG in vivo. If the pathogenic effects o f these cell lines were to be investigated in vivo, a negative control cell line that had undergone the same procedures and stresses as the IgG-producing cell lines but that would not produce IgG would be required. This was achieved by transfecting the CROdhfr' cells with empty (i.e. no Vh or encoding regions) expression vector pGlD210. Since neither
control cell line was treated exactly as the IgG producing cell lines. The clones were treated (i.e. selected and amplified with methotrexate) exactly the same as the test IgG- producing cell lines.
Non-transfected CYiOdhfr cells were potentially insufficient as a control for these in vivo experiments for two reasons. Firstly it could be argued that the presence o f the expression plasmid DNA inside the cells, regardless of the IgG produced, could possibly be toxic and cause pathogenic effects to the mice. Secondly, the non- transfected cells CHOdhfr' do not contain a functional DHFR gene as they do not contain the expression vector containing the dhfr gene as do the test cell lines. Therefore this cell line may not grow as well in vivo due to the fact that the concentrations and availability of the deoxyribonucleosides and ribonucleosides required for growth might not be sufficient and these CYiOdhfr cells would not be able to assimilate these essential nutrients.