GCSS - General Cell Screening System

A new system for the determination of cell numbers, cell growth rates and cell growth kinetics in 96 well plates

red line Franz Steindl*, Josef Atzler**, Alex Livingston**, Karl Puchegger**, Christine Schmatz*Willibald Steinfellner*, Karola Vorauer-Uhl*, Peter Rubenzer**, Hermann W. D.Katinger*

* Institut für Angewandte Mikrobiologie, (IAM) Universität für Bodenkultur, (BOKU) Nußdorfer Lände 11 A-1190 Wien
** SLT Labinstruments GmbH Untersbergstr. 1 A-5082 Grödig

Principle-Materials-Test protocol-Advantages-Discussion-Literature

Introduction

Modern and effective biological research needs new and effective methods, that are :
1) ecologically compatible,
2) drastically minimize animal trials and
3) are economical.
To meet all these requirements and some additional facts there is a common trend toward reproducible "in vitro " assays and production systems. Although the use of 96 well-microtiter plates allows the performance of many experiments and samples under the same conditions (time, temperature, etc.) the task to statistically relevant data in the field of cell culture (e.g. biology, biotechnology, oncology, pharmacology, toxicology, virology, etc.) is still difficult and time consuming. Today, standard methods to determine cell numbers in 96 well- microtiter plates are all endpoint methods or prerequisite sampling. Methods for determination of cell numbers such as measuring the incorporation of radioactive nucleotides like thymidine (1), or measuring the activity of "marker-enzymes", like mitochondrial dehydrogenases using MTT (2), XTT (3), or MTS (4) as chromogenic substrates, or lactate dehydrogenase (5), or acid phosphatase (6), etc. allow only one determination per plate and the cells are lost. Additionally, the results are not dependent on the actual cell number only, but also on the grade of cell activity and the conditions of preculturing the cells, which can also influence these results. All kinetic parameters and the whole development within an experiment until the endpoint is measured, remain as a "black box" by using these methods. In contrast, the procedure of the General Cell Screening System (GCSS) (7) enables for the first time to quantify cell numbers in 96-well cell culture plates without any influence on the cells. As the cell numbers can be repeatedly determined within one 96 well plate, also all kinetic parameters can be performed and quantified. The method is based on a high resolution turbidy measurement (700 nm) performed directly in the 96 well cell culture plate. The system consists of the GCSS-reader (an 8 channel photometer, the beam has the form of a narrow rectangle Fig.1 and 2 ), the GCSS- plate (Fig.4 and 5.) with a new form of the wells (Fig.3 ), an Apple Macintosh computer and the GCSS- Software. The wells of the GCSS-plate have a rectangular bottom (6x2mm) and they are arranged in the same way as in common 96 well microtiter plates. The content of the wells (in the dimension of the bottom) is measured quantitatively by additive measurements with the rectangular beam of the GCSS-reader. Calculation of cell numbers per well is done with an established and stored calibration curve for the used cell line or cell type. With the GCSS-Software calculation (including plotting on the screen) of cell numbers, increase of cell numbers, cell growth rates and cell growth kinetic curves can be done within a few seconds for each measurement or each freely choosen interval of measurements, respectively.
The quickness of this method ( time for measuring = 8 sec./plate; including data transfer, saving and showing the results on the screen = 40 sec/plate) and the exact protocol of the data are further advantages compared to common methods. An integrated powerful database assures the management of enormous data volumes. Up to thousands of plates from different experiments can be handled at the same time with GCSS.

Principle of the GCSS-method for determination of cell numbers

The GCSS-method is based on the measurement of transmittance at 700nm. At 700 nm transmittance is either influenced by the colour of the pH- indicator (Phenolred), nor by the protein content nor by the composition of common cellculture media. The decrease of transmittance due to light scattering is caused by particles is the basic parameter. How much light is scattered by particles like cells depends on the number, form and size. So, for all cell lines or at least types of cells used an own calibration curve should be established. Transmittance is a time independent parameter and therefore the calibration curves can be used over time. To quantify inhomogenious distributed particles the whole relevant volumne has to be measured, the rectangular bottom of the wells from the GCSS plate enables to do this. The GCSS-reader measures the whole area of the well bottom in additive measurements with a rectangular beam (Fig.3).
To get more easily to read reports the measured transmission values are shown as "opacities = 1-transmission" on the screen, so higher values mean a higher cell number in this reports.

Materials and methods

Cell culture
RPMI 1640 Fa. Biochrom
DMEM/HAM`s F12 1:1 (Fa. Biochrom)
FCS (Fetal Calf Serum) (Fa. Gibco)
Ricin RCA60 Fa.Sigma Catalog Nr. L 8508
Trypsin Fa. Gibco
EDTA Fa. Biochrom
Cell lines
Adherent cell lines: CHO-K1 (ATCC, Nr. CCL 61),HeLa (ATCC, Nr. CCL 2)

Suspension cell lines: CEM-T4 (ATCC, Nr. CCL 119), IAM- 4F12: a human B-Cell hybridoma linie, IAM- 2G12/2H5: a human B-Cell hybridoma linie

CHO-Cells were cultivated in DMEM/HAM`s F12 1:1 +5% FCS, HeLa-Cells and all suspension cell lines were cultivated in RPMI 1640 + 5% FCS

Test protocol

Examination of the toxic effect of RCA60 on different cell lines: All cells were precultivated in Roux flasks. For these experiments cells (CHO- und HeLa-cells after treatment with trypsin) were sedimented in a centrifuge at 180g for 5 minutes, and the pellet was resuspended in fresh cell culture medium. The cell density was adjusted to 1,5.105cells/ml. For each cell line one GCSS plate was seeded using a multi channel pipette; 100µl/well of these cell suspensions were transferred to each well of a plate. Than the plates were incubated over night at 37°C in a 7% CO2-atmosphere. The toxic substance, Ricin Communis Agglutinin (RCA60), was diluted in cell culture medium 1:3,16 10 times (concentration range 2 ng - 63,2 fg/ml). Aliquots of these dilutions were transferred (50µl/well) to the cell culture plates, rows 3 - 12 starting with the highest RCA60 concentration in row 3 (100 pg/well) decreasing to a concentration of 3,16 fg/well in row 12). Each concentration was transferred to all eight wells in a row from A-H. To the 16 control wells (in row 1 and 2) 50 µl cell culture medium were added and were defined as reference values. Immediately afterwards the GCSS-plates were measured with the GCSS-reader. Then the plates were incubated at 37°C and measured after 2, 4, 8, 28, 53 und 78 h . After the last measurement a MTT-assay (2) was performed in the same plates. ( Comparison of results obtained with GCSS and the MTT-assay shown in Fig.20a ) Subcloning of Hybridoma cells: Cells from the B-cell hybridoma line IAM-2G12/2H5 were precultivated and harvested as described above. The resuspended cell suspension was adjusted to a cell density of 13 cells/ml (= 2 cells/150µl /well) and plated on 5 GCSS-plates. For seeding the cells the Pro/Pette (Fa. Cetus) with a 12-fold manifold was used. To get a homogeneous cell distribution in the plates the cell suspension in the flask was slightly stirred during the plating procedure. Then the plates were incubated at 37°C in a 7% CO2-atmosphere. Plates were measured after 2h, after 11, 15, 16, 20, 21, 27 and 28 days. On day 12, 50 µl fresh cell culture medium were added to each well. On day 15 and 20 samples of culture supernatant were taken from wells with a cell density higher than 4000 cells/well at this time and refilled with fresh medium. (Fig.18, 19, 21) The samples were tested for there antibody content.

Advantages of the GCSS Method

Discussion

The GCSS method enables for the first time to quantify cell numbers, without any influence on cells, within 96 well plates. As this method can be performed repeatably within one cell culture plate, parameters like cell number, cell increase or cell growth rates can be quantified always immediately using a once established calibration curve. Graph representations of cell growth kinetic curves in the 96 well format can be seen for the first time within a few seconds on the screen. This option enables an overview within a very short time about the kinetic development within the plates (fig. 6, 7, 9,10 and 18). For detailed analysis the repesentation of single wells (fig.21) or the mean of selected wells (fig. 15a and 15b) can easily be zoomed. This method always allows to watch and control results by microscope viewing in contrast to methods used to date, which give results long term after the cells are killed by the procedures. Exact data protocols and automatic data managing are additional advantages of the GCSS. As the GCSS method saves time, material and labour on one side and simultaneously results in more information on the other side, it may open an new dimension in cell culture and in vitro screening.

Literature:

1.Hewlett, G., Stünkel, K. G., Schlumberger, H. D.; (1989) A method for the quantitation of interleukin-2 activity. J. Immunol. Meth. 117, 243-246.

2.Mosmann, T.; (1983) Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays J.Immunol. Methodes 65, 55-63.

3.Paull, K. D. et.al.; (1988) The synthesis of XTT: a new tetrazolium reagent that is bioreduceable to a watersoluble formazan. J. Heterocyclic Chem. 25, 911-914.

4.Baltrop, J.A., Owen T. C., Cory, A. H., Cory, J. G.; (1991) 5-((3-Carboxyphenyl)-3-(4,5-dimethylthiazolyl)-3- (4-sulfophenyl) tetrazolium, inner salt (MTS) and related analogs of 2-(4,5-dimethylthiazolyl)-2,5-diphenylterazolium bromide (MTT) reducing to purple water soluble formazans as cell-viabilty indicators. Bioorg. and Med. Chem. Lett. 1, 611.

5.Racher, A.J., Looby, D., Griffiths, J.B.; (1990) Use of lactate dehydrogenase release to assess change in culture viabilty. Cytotechnology 3, 301-307.

6.Connolly, D. T., Knight, M. B., Harakas, N. K., Wittwer, A. J., Ferder, J. (1986) Determination of the Number of Endothelial Cells in Culture Using an Acid Phosphatase Assay. Anal. Biochem. 152, 136-140.

7.Steindl, F.; (Dez. 1990) General Cell Screening System - A New Dimension in Screening & Cell Culture. Int. Biotech. Laboratory News Edition

Acknowledgments: The GCSS developement was supported by the Austrian Forschungsförderungsfond für Gewerbliche Wirtschaft.
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