Real-Time monitoring of
In Vitro Human Cell Culture systems
InVitroPlus Laboratory, MicroSurgical Research Laboratories
Ph (514) 398-6988, Fx (514) 398-7435, http://www.invitroplus.mcgill.ca
Paul Héroux PhD, Associate Professor, Faculty of Medicine,
Medical Scientist, Plastic Surgery,
Here is a free offer: you may download this Textbook on Toxicology
(322 pages, 20 Mb) used in the Occupational Health curriculum
at the Faculty of
This text can be freely copied, distributed, printed and used
for formal and informal educational purposes.
Tune your browser to the FTP site: http://www.invitroplus.mcgill.ca/Ftp/
and click on the file "Toxicology Course Notes 2009.pdf"
Cell division and survival (apoptosis-necrosis) are pivotal to many aspects of toxicology,
cancer biology and embryogenesis.
There is a need for a powerful and sensitive laboratory instrument capable of long-term
and simultaneous assessment of mitosis, cell stress and cell death.
Enhancement of mitosis is of interest in cancer progression, and cell death
is of interest in chemotherapy.
For example, scientists have known for years that many carcinogens are not mutagens,
behaving rather as promoters of mitogenesis.
In the same way, chemotherapy needs detailed information about the cell killing performance
of various drugs individually, and of drug combinations.
Our instrumental design allows rapid testing not only of single agents, but also
of combinations of 2 to 5 agents in various concentrations. We have assembled
a computer-controlled system which uses computer-vision and inverted
microscope inside an incubator to track continuously the proliferation of a large
number of cells in culture over days to weeks. Multiple separate tests can be followed
simultaneously in a standard 96-well dish format (below).
Environmental or pharmaceutical agents can be injected into the cultures
in scales of concentration and assayed by computer for positive or negative
reactions of the cultured cells.
For example, if two agents are studied simultaneously, a matrix of 12 x 8 concentrations
can be used in the 96-well dish format.
There are applications for this system in:
the investigation of environmental toxicants, singly or in combinations,
the assessment of basic toxicity of chemical exposures,
the investigation of tumor initiation, promotion and progression, and
the optimization of therapeutics-pharmaceutics, particularly in oncology.
The system is thus capable of tackling a wide range of problems from
environmental science through basic and applied cancer research, to high
throughput screening for drug discovery.
The data generated by the InVitroPlus system is heavily image-based.
A single test in the InVitroPlus laboratory typically produces thousands of images,
corresponding to the individual wells of the 96-well test plate.
Database files for convenient access to image analysis results are generated automatically.
Other data consists of files describing the multi-well-dish pattern used in a given test
and of ASCII files describing cell events that are automatically logged by computer analysis
during the test.
The data set constitutes a permanent, detailed record of cellular reactions
over time, which can be interpreted immediately by a human observer
(using specialized viewers) and by specialized software.
The same image record can later be re-interpreted according to new needs.
The general pattern of automated data treatment is shown below.
Computer-based data interpretation is heavily dependent of the techniques of
object contouring and recognition, as exemplified in the following micrograph.
A micrograph of HL60 cells near confluence. The computer circled living
cells in red, debris and dead cells in blue. Accuracy of cell recognition
is better than 95 %.
The InVitroPlus laboratory uses Human Erythroleukemia Cells (K562) grown in serum-free medium,
affording exceptional reproducibility.
The following cell lines have also been used in our laboratory in the past:
PC12, transformed mouse macrophages, fibroblasts, HL-60.
When the cell-killing action of chemotherapeutic agents are documented,
we use the variable Log(1/N) as a vertical axis. This display is specially
sensitive to the death of the last few survivors in a cell population, a concern
for the elimination of both reduced tumors and metastases. Thus, a shape
climbing to the maximum height (blue color) signals treatment success, while
depressions (red color) signal cell proliferation. These results were derived using
the erythro-leukemia K562 cell line and display the destiny of 50 cells which
were followed over more than 6 days.
In the first case below, comparable weights of doxorubicin and methotrexate
were most effective against K562 cells, while methotrexate alone stimulated
proliferation for 6 days.
Doxorubicin and cyclophosphamide (below), two of the drugs in the CAF
breast cancer protocol, display treatment success as well as unimpaired
proliferation when concentrations are insufficient (red, to the right of the graph).
Note that in this graph, concentrations of the two drugs increase in the same
direction, while they are opposite in the test above.
Optimal concentration of drugs for the control of cell proliferation can be investigated,
as shown below for doxorubicin and cyclophosphamide.
Influence of apobodies on the apoptotic and mitotic rates of K-562 cells.
Dynamics of apobodies.
Software development: cell and object recognition for living human cells.
Software development: time-series interpretation of imaging results.
More information on the techniques used in the InVitroPlus laboratory can be
found by consulting the following publications:
"Proliferation and Apoptosis Rates of Living Human Erythroleukemia Cells"
Paul Héroux, Igor Kyrychenko and Michel Bourdages
Microscopy and Analysis • May 2004, pp. 13-15.
"Customizing Cancer Treatment Options",
in Sourcebook on Asbestos Diseases, Volume 15, Peters and Peters Eds.,
Lexis, 131-150, 1997.
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