and Maintenance of Cell Lines
work with the cell lines ensured that they are stable in
continuous culture, can be cultured to high cell concentrations on a
routine basis and produce consistent amounts of active specific
cell lines are tested for mycoplasma infection. Mycoplasma is a
common contaminant of cultured cell lines, and, if present, can adversely
affect the growth rate and antibody secretion rate of the cells, making it difficult to expand cell cultures into large growth vessels.
If a mycoplasma infected cell line is grown in a laboratory, it is very
difficult to avoid cross contamination into previously uninfected
cultures. All new cell lines are kept in "quarantine" (in a
separate incubator, and not handled at the same time as other lines) until
they have been tested and shown to be free of mycoplasma. Several methods
have been used for mycoplasma detection including a culture
method, a DNA hybridisation kit, PCR based method and most recently a
biochemical test which detects the activity of mycoplasmal enzymes. If a
cell line is mycoplasma positive, it undergoes a regime of treatment,
developed in the laboratory, with the antibiotic ciprofloxacin. This is effective in eliminating mycoplasma from cell cultures. Treatment
continues until the culture gives a negative result with the mycoplasma
test. Once a treated line is mycoplasma test negative, normal work
continues on the line, but, it is retested at later stages of growth.
of cell lines is monitored by limiting dilution cloning analysis1.
All lines are cloned at least three times after primary fusion, and
cloning continues until all clones isolated are secreting specific
antibody. At the final cloning stage, several clones are compared for
1. Growth rate
2. Growth capability of cells in larger culture vessels
3. Secreted antibody potency (measured by serological titration, flow
cytometry or intensity of staining immunoblots)
concentration measured by double antibody sandwich class specific ELISA2.
The best clones are selected (optimal growth/ high level of secretion of specific
antibody/highest potency for the lowest antibody concentration). Comparison of antibody concentration and potency is
important if the avidity of the secreted antibody is to be maintained
and/or improved. We have observed significant variation in antibody
avidity between different sister clones3.
specificity and immunoglobulin class and subclass of the antibody secreted
by the best clone are carefully confirmed. The clone is then expanded in
culture to provide sufficient cells to freeze a master cell bank and a
working cell bank which are stored indefinitely in liquid nitrogen. One
vial from each freezing is thawed to check the viability of the frozen
Harvesting of Cell Lines
antibodies are grown in in vitro cell culture conditions. Because we
have optimised conditions for producing antibodies at high levels there is
no need for in vivo ascitic fluids to be produced. When antibody is
required, one vial of frozen cells is taken from the working cell bank and
rapidly expanded in culture to the required final volume. If only small
volumes of supernatant are required, the cells are grown in static flasks.
If greater than 500 ml is required, the cells are grown in suspension
spinner vessels or other systems. Higher concentrations of antibody can be
achieved using hollow fibre technology . The concentration obtained varies
from cell line to cell line but concentrations 10 times those obtained by
static culture is normally achieved with hollow fibre.
The growth and secretion rate
varies for each cell line and the conditions for growth must be optimised
for each cell line. Growth and production of antibody is monitored daily by
cell counts and quantitation ELISA. Following the peak of antibody
production, cultures are harvested by centrifugation and/or ultrafiltration
followed by sterile filtration of supernatant. Each batch of supernatant is
assessed for specificity, potency and antibody concentration by various QC
tests ranging from ELISA, serology, flow cytometry, SDS PAGE and
immunoblotting. Supernatant is then aseptically distributed into aliquots at
a range of different volumes, and stored at -30°C until required. The
activity of the frozen material is monitored.