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A dedicated to
Computer-based, Real-Time monitoring of In Vitro Human Cell Culture systems InVitroPlus
Laboratory, MicroSurgical Research Laboratories L4.63, Ph (514) 398-6988, Fx (514) 398-7435, http://www.invitroplus.mcgill.ca
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
Medicine, 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"
IN-VITRO
System Description | System Applications | Data Storage Data Processing | Image Analysis | Biological Targets | Sample Results Subjects proposed to PhD Students | Real-time Laboratory Results | References |
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SYSTEM
DESCRIPTION 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).
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SYSTEM
APPLICATIONS 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 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.
IMAGE
ANALYSIS 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 %. BIOLOGICAL
TARGETS 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. SAMPLE
RESULTS 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.
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Optimal concentration of
drugs for the control of cell proliferation can be investigated, as shown below for
doxorubicin and cyclophosphamide.
SUBJECTS PROPOSED TO PHD
STUDENTS
REFERENCES More information on the
techniques used in the InVitroPlus laboratory can be found by consulting the
following publications:
Paul
Héroux, Igor Kyrychenko and Michel Bourdages Microscopy and Analysis •
May 2004, pp. 13-15.
Paul Héroux, in Sourcebook on Asbestos
Diseases, Volume 15, Peters and Peters Eds., Lexis, 131-150, 1997. OCCUPATIONAL
HEALTH PROGRAM AT Companies who contacted the
Department in recent years, looking for graduates. 3M Bombardier Concordia University Coca-Cola Canadian Fertilizers Limited Département de Santé Publique Health Merck Frosst Pratt & Whitney QIT – Fer et Titane Rio Tinto Alcan Toyota
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