A Royal Victoria Hospital & McGill University Research Laboratory

dedicated to

Computer-based,

Real-Time monitoring of

In Vitro Human Cell Culture systems

InVitroPlus Laboratory, MicroSurgical Research Laboratories

L4.63, 687 Pine Avenue W., Montreal, Quebec, Canada, H3A 1A1.

Ph (514) 398-6988, Fx (514) 398-7435, http://www.invitroplus.mcgill.ca

 Paul Héroux PhD, Associate Professor, Faculty of Medicine,

McGill University and

Medical Scientist, Plastic Surgery, Royal Victoria Hospital

paul.heroux@mcgill.ca.

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, McGill University.

This text can be freely copied, distributed, printed and used

for formal and informal educational purposes.

  1. Tune your browser to the FTP site: http://www.invitroplus.mcgill.ca/Ftp/
  2. and click on the file "Toxicology Course Notes 2006w.pdf"

IN-VITRO PLUS SYSTEM

System Description | System Applications | Data Storage

Data Processing | Image Analysis | Biological Targets | Sample Results

Subjects proposed to PhD Students | Real-time Laboratory Results | References

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).

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 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.

DATA STORAGE

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.

DATA PROCESSING

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.

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

 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.

REAL-TIME

UpdateFE.txt

UpdateIM.txt

UpdateAN.txt

UpdateIN.txt

REFERENCES

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",

Paul Héroux,

in Sourcebook on Asbestos Diseases, Volume 15, Peters and Peters Eds.,

Lexis, 131-150, 1997.

RELATED WEB PAGES 

http://celox.bioreagent.com | http://www.toxicology.org | http://www.polyfiltronics.com