WO1993024607A1

WO1993024607A1 - Method for performing cytotoxicity tests and a system therefor, and a kit included in said system

Info

Publication number: WO1993024607A1
Application number: PCT/SE1993/000183
Authority: WO
Inventors: Anders Andrén; Anders Wieslander
Original assignee: Gambro AB
Current assignee: Gambro AB
Priority date: 1992-05-25
Filing date: 1993-03-03
Publication date: 1993-12-09
Anticipated expiration: 1994-11-25
Also published as: SE9201636L; EP0672109A1; SE9201636D0; JPH08502401A

Abstract

A system and kit for cytotoxicity tests comprising those substances (1-7) necessary for the test, such as necessary chemical compounds and test cells, and preferably also a test plate and other instruments necessary for the test, such as pipettes (9) or similar, characterized by means (10-12) for storing a mixture (2) of test cells and a cell-culture medium at a lowered temperature, preferably corresponding to normal refrigerator temperature. The invention further includes a method for storing cells for cytotoxicity tests during transport, which cells are intended for the system and kit with substantially maintained biological properties, characterized in that they are transported at a lowered temperature, preferably at substantially normal refrigerator temperature.

Description

TITLE

METHOD FOR PERFORMING CYTOTOXICITY TESTS AND A SYSTEM THEREFORE, AND A KIT INCLUDED IN SAID SYSTEM

TECHNICAL FIELD

The present invention relates to a method and a system for cytotoxicity tests comprising those substances necessary for the test, sv as necessary chemical compounds and test cells, and prt .--rably also a test plate and other instruments necessary for the test, such as pipettes or similar.

The invention also comprises a kit intended to be included in the above defined system.

BACKGROUND OF THE INVENTION

The applicant presently performs so called cytotoxicity tests (cell tests) in its laboratory. This implies that the toxicity of solid materials is tested on established cell cultures, preferably on the established mouse fibroblast cell line L-929, which is primarily a stage in the registration of new materials. These solid materials can be various types of plastics which are to be used in medical products, for example plastics included in dialyzers or similar. Tests are also carried out for overseas subsidiary companies which can lead to certain undesirable time delays because of long transportation and time-consuming reporting. It would therefore be desirable if these tests could instead be carried out on site. The idea of a so called kit is rather new. On the market there are a number of different test kits with which for example enzyme activity, presence of various chemicals, release of certain substances and so on can be measured and determined. These kits includes the equipment which is necessary to perform a small scale analysis. In other words no large laboratory apparatus is required.

THE IDEA BEHIND THE INVENTION

The idea behind the present invention is to provide a system and a kit respectively with which the same test which the applicant presently performs centrally can be carried out. The problem is however how the cells can be sent so that they survive transportation. The basic notion of cell storage is that parameters such as temperature, pH, osmolarity, humidity, etc. must be constant, i.e. they should mimic the environment from which the cells come. It is however generally known that cells can be rapidly frozen and stored in liquid nitrogen so that they may be defrosted at a later date for use. This method is however burdened with the disadvantage that the deep frozen cells take a long time to regain their normal metabolism.

According to the invention the temperature is instead reduced to "refrigerator-temperature". Preferably a so called single cell suspension with a certain cell density is hereby used. This suspension can then later be transferred to ampoules with a volume of say 500 ml. These ampoules should then be placed in an incubator for adjustment of pH to around 7,0 - 7,2 in the storage liquid. Thereafter they are suitably provided with sealing covers and can then be stored at "refrigerator-temperature". Preliminary studies have shown that cells could be stored in the refrigerator for up to four weeks. Within two weeks 80% - 90% of cells capable of life remained and within four weeks approximately 50%. Trials have also been performed for testing which cell density in the ampoule is best, which serum concentration is suitable, the sensitivity compared with normally stored cells and the viability and growth capability during four weeks.

DESCRIPTION OF THE INVENTION

The present invention thus relates to a method for performing cytotoxicity tests, a system and a kit therefor, comprising those substances necessary for the test, such as necessary chemical compounds and test cells, and preferably also a test plate and other instruments necessary for the test, such as pipettes or similar.

The system according to the invention is characterized by means for storing a mixture of test cells and a cell- culture medium at a lowered temperature, preferably corresponding to normal refrigerator temperature.

In order to facilitate transport, said means can include a heat insulating transport container which preferably also contains a cooling medium, for example a freezable cool block.

Said cell-culture medium is preferably a typical such medium, for example Minimum Essential Medium (MEM) with Earles salts (Gibco BRL England) , together with a serum, preferably fetal calf serum. In order to ensure the cell survival, it has been shown that said cell-culture medium should be supplemented with at least 10%, preferably approximately 30%, serum.

So that the cells will not be damaged by the cooling medium, the transport container should include a preferably weak-insulating partition wall which separates said cooling medium from the remaining contents of the container.

The partition wall is preferably made in the form of a holder, made from foam rubber or similar, for retaining and protecting the remainder of the components in the container, such as test tubes, bottles, pipettes etc.

In practice it has been shown to be suitable that said test cells consist of an established cell-culture, preferably the established mouse-fibroblast cell line L-929. This cell-culture can preferably contain more than 200 000 cells/ml, preferably in the order of 1 000 000 cells/ml.

The invention also relates to a kit for cytotoxicity tests included in a system of the above defined type. This kit is characterized in that it includes a transport container of heat-insulating material which contains essentially all the substances necessary for the test, such as necessary chemical compounds and test cells, and preferably also a sampling plate and any other instruments necessary for the test, such as pipettes of similar. The kit suitably also includes a cooling medium, for example a freezable cool block, which is preferably separated from the remaining components in the container by means of a suitably weak- insulating partition wall. The invention also relates to a method for storing cells for citotoxicity tests during transport, which cells are intended for the system as defined above and/or the above defined kit, with substantially maintained biological properties. The method is characterized in that the cells are transported at a lowered temperature, preferably at substantially normal refrigerator temperature.

The cells' survival capability is increased for example if they are transported in a heat-insulating container, for example of frigolite or similar, together with a cooling medium, for example a frozen cool block. After transportation the cells are suitably stored in a refrigerator until they are to be used.

DESCRIPTION OF THE DRAWING

The attached drawing shows a kiτ_ according to the invention. By way of example, the kit can contain the following:

1. 20 ml Minimum Essential Medium (MEM) with Earle's salts (Gibco BRL England) . Supplemented with 10% fetal calf serum, L-Glutamine, Gentamycin and NEAA (Non essential A inoacids) .

2. Cell suspension consisting of 0,5 ml Minimum Essential Medium (MEM) with Earle's salts (Gibco BRL England) . Supplemented with 50% fetal calf serum, L- Glutamine, Gentamycin and NEAA (Non essential Aminoacids) + cells 900 000 cells/ml.

3. 50 ml. Otherwise identical to No. 1.

4. Identical to No. 1. 5. 0,5 ml concentrated neutral red solution (4 g/100 ml H₂0. E. Merch Germany) .

6. 50 ml PBS solution. Phosphate Buffered £3alin. (Physiological buffer) .

7. 50 ml EtOH-Hac. (50% ethanol in water acidified with 1% concentrated acetic acid) .

8. 96-hole cell-culture plate.

9. Sterile pipette tips.

10. Cool block.

11. Test tube holder of foam rubber.

In practice is has been shown to be suitable to select an outer container with dimensions 30 x 30 x 30 cm with the weight thereby being approximately 1 kg.

SUGGESTION FOR INSTRUCTIONS FOR USE OF A CITOTOXICITY TEST ACCORDING TO THE INVENTION.

Day 1 A: It is best to start the experiment on a

Monday of Thursday.

NB: Use protective gloves and apply disinfectant (Gevisol) to all used material. The plate should contain two columns with empty wells (column 1 and 12) without cells. All remaining wells should contain cells. Two columns (column 3 and 11) are recommended as control columns. Column 2 can be used for positive control. The remaining seven columns with eight wells respectively are intended for the unknown

10 samples.

B: Addition of the L-929 cells to the 96-hole plate.

15 Heat tube No. 1 containing cell-culture medium for ten minutes in a water bath at 37°C.

Suspend the L-929 cells in tube No. 2 to a

20 one cell suspension by first pipetting it up and down 10 - 15 times and thereafter 10-15 times with the pipette tip on the base of the ampoule (use a 300 μl pipette, avoid air bubbles) .

25

Pipette 200 μl of the single cell suspension in tube 2 to the pre-heated culture medium in tube 1. Mix carefully, avoid air bubbles.

30

Distribute 200 μl of the diluted cell suspension to each well, with the exception of the blank-columns.

35 Incubate the plate at 37°C with 5% C0₂ for 24 hours. Day 2 C: Addition of the test substances to the 96- hole plate.

NB: Prepare the test substances before the start (use the medium in tube No. 3) .

1. Remove the culture medium from the plate by turning the place to and fro on an absorbing paper. Immediately place the paper with the culture medium in a plastic bag which will later be discarded. The cells hereby remain attached to the walls of the plate.

2. Add 200 μl/well of controller, test substances and positive control to eight parallel wells. Add only culture medium to column 1 and 12.

3. Incubate the plate at 37°C with 5% C0₂ for

72 hours.

Day 5 D: Determination of growth inhibition

1. Temper tube No. 4 with cell-culture medium in a waterbath at 37°C for ten minutes.

2. Add 250 μl concentrated neutral red solution from tube 5 to the pre-heated medium i tube 4.

3. Remove the medium from the plate according to C:l.

4. Add 200 μl of the prepared neutral red solution to all wells on the plate. 5. Incubate the plate at 37°C with 5% C0₂ for 3 hours.

E: Determination of neutral red adoption. 5

1. Remove the solution with neutral red according to C:l.

2. Rinse all wells on the plate once with the 10 PBS-solution from tube No. 6 (200 μl/well) .

3. Add 200 μl EtOH-HaC from tube No. 7 to each well on the plate.

15

4. Incubate at room temperature for approximately 20 minutes so that the colour will be extracted from the cells.

20 5. Shake the plate for approximately 60 seconds and measure the absorbance at 540 nm.

Calculations,

25

1. Calculate the mean absorbance from blanks, controls, posif ve control and unknown samples. Subtract the mean value of the blank from all other values. Growth

30 inhibition is calculated according to the following: 100-(Y/X x 100)

Y — mean value of respective unknown samples.

35 X = mean value of controllers. PRELIMINARY PERFORMED TESTS

Storage fluid

Object:

Preliminary tests have been performed to establish which type of storage solution should be used.

Two ampoules, one with complete cell-culture medium and one with fetal calf serum (FCS) were prepared. A certain quantity of cells (180 000 cells) were divided into the two ampoules. The cells were stored for seven days in a refrigerator. Thereafter the growth was analyzed after three days compared with normal cells after the same time. (Neutral red analysis (SF/GTI 014) .

Results: Absorbance %growth compared with control

Control (normal cells) 1,46 100 Cold cells in medium

(10% FKS) 1,05 72

Cold cells in serum

(100% FCS) 1,04 71

I.e. no difference between the various storage solutions. However difference between normal cells and cold cells.

We thus tried to modify the storage solution. Normally the cells grow in a culture medium which is supplemented with 10% fetal calf serum. We tested two new serum concentrations of 30% and 50%.

1. Culture medium supplemented with 10% serum + cells.

2. Culture medium supplemented with 30% serum + cells. 3. Culture medium supplemented with 50' serum + cells.

Absorbance

Serum cone. 10 days 17 days 28 days

10% 1,184 0,858 0,242

30% 1,515 1,174 0,416

50% 1,467 1,187 0,634

Serum concentration 10% implies 10% FCS and 90% MEM. The table shows the absolute absorbance. Survival with 10% serum concentration is acceptable though it can be seen that an increased serum concentration in the storage liquid improves the survival of the cells when they are stored in a refrigerator. The table does however show that in principle it is sufficient to increase the concentrarion up to 30% serum concentration. The cells do not need normally to be stored for longer than one week in a refrigerator.

Viability

Serum cone.

10%

30%

50%

The table shows the viability (life capability) of the cells which were stored in the refrigerator. The results show that an increased serum content in the storage liquid retains the viability in the long term (cf. 10%/17 days with 30% and 50%/17 days respectively) . The measurements are based on Tryphan blue colouring (colouring dead cells blue, living cells are not coloured) , after which the cells were counted i Bύrker chambers under a microscope. Cell concentration Ob ect:

The object of this test was to see if there were any differences between a low and a high concentration, and which cell concentration one should have in the storage solution.

The following test was performed. Two cell suspensions with various cell concentrations were prepared.

A: 234 000 cells/ml

B: 1 600 000 cells/ml

The ampoules were filled with respective cell suspensions and placed in a refrigerator. The viability was determined after 4, 7, 11, 14, 18 and 21 days.

Result: No noticeable difference could be observed. Presently 900 000 cells/ml are used. Growth and viability with time Object:

To further determine the properties of the cells with time. The viability was measured to determine how many survived with time. The growth was determined to see if and how they grow. A cell suspension with 900 000 cells/ml and supplemented with 50% serum was prepared. 500 μl ampoules were filled and placed in a refrigerator. Days in refrigerator %growth after 3 days Viability compared with control

3 88% 97%

10 89%

14 76% 88%

17 61% 80%

21 45% 74%

24 40% 68%

28 22% 53%

Result: Both growth capability and viability is reduced with time.

Cell response/sensitivity Object:

To compare the sensitivity to acrylamide between refrigerated cells and normal cells.

Acrylamide has previously been thoroughly tested on L 929. The refrigerated cells were laid out on a 96-hole plate according to instructions. One day later the culture medium was replaced by a medium with acrylamide. After three further days the following results were obtained.

Refrigeratedcells 1 week old

9

11

14

40

80

97

100

100

ICG = Inhibition of Cell Growth Results: The refrigerated cells appear to possess substantially the same sensitivity for acrylamide as do normally stored cells.

SUPPLEMENTARY TESTS

We have chosen to perform three tests on our cells stored under refrigeration to determine the life capability of the cells.

l Growth: The cell growth determines the function of the cells, i.e. in what condition they are in after being stored under refrigeration.

2. Viability: The viability determines the survival of the cells after being stored under refrigeration, i.e. how long it is practicable to store them in the refrigerator. Results: The results can be seen from the annexed tables l and 2 respectively which show that there should be fetal calf serum in the storage solution. However, the choice of the serum concentration is of less importance. For practical and financial reasons we have chosen 30%. It is possible to store the cells for up to 18 days in the refrigerator with maintained life capability.

3. Dose-response: Dose-response tests are previously performed on normal cultured/stored L-929 cells. The chemical Acrylamide was prepared in seven different concentrations. These seven different concentrations were added to the cells on the 96-hole plate in seven various columns with eight wells in each column.- Cell growth inhibition, depending on the chemical concentration, was determined with neutral red colouring.

Dose-responsee curves were drawn from the values for these determinations in order to be able to calculate the ED value (Effective Dose, cf. LD, Leathal Dose), i.e. the dose/concentration of acrylamide which causes 20%, 50% and 80% cell growth inhibition respectively. This method was repeated on nine various occasions and the mean value calculated. The mean values are used as control in Table 3. A corresponding procedure was used for the L-929 cells which had been stored under refrigeration in order to determine their cell response/sensitivity.

Results: The results are presented in appended Table 3 which shows that no marked differences could be seen after storage under refrigeration. The invention is of course not restricted to the above described examples but can be varied within the scope of the appended claims. For example there is a large number of other cells and culture mediums which can be used instead of those mentioned above.

Table 1 Four days growth for L-929 cells which were stored under refrigeration for vario periods of time.

7 da s 14 days 18 days 31 days

15% 33% 43% 39%

not measured

Table l. Shows the relative growth for L-929 cells which were stored at a maximum 8°C in normal refrigerator. After storage in the r frigerator the cells were transferred to a 96-ho plate according to operating instructions. After 4 days in the incubator the growth w determined with Neutral red colouring. This process was repeated on cells which were stor for 7, 14, 18 and 31 days in the refrigerator. Two columns with normal cultured L-929 cel which were not stored in the refrigerator were used as cont-rol. The cells were stored in solution consisting of minimum essential medium (MEM , Gibco BRL>

with glutamine, Gentamycine, NEAA according to normal prescription. Finally the storage soluti was supplemented with 0%, 10%, 30%, 50% fetal calf serum and one sample containing only fet calf serum.

Table 2 Viability for L-929 cells which were stored under refrigeration for various perio of time

7 days 14 days 18 days 31 days

Serum con¬ centration 0% serum 94% 90% 86% 60% 10% serum 99% 97% 89% 67% 30% serum 99% 94% 97% 66% 50% serum 99% 92% 87% 60% 100% serum 99% 92% 85% not measured

Table 2. Shows the viability for L-929 which were stored at max 8"C in a normal refrigerato The cells' viability after 7, 14, 18 and 31 days storage was determined with Tryphan blue a cell-counting in a Bϋrker chamber. The cells were stored in a solution consisting of Minim Essential Medium (MEM, Gibco BRL) supplemented with L-glutamine, Gentamycine, NEAA accordi to normal prescription. Finally the storage solution was supplemented with 0%, 10%, 30%, 5 fetal calf serum and one sample containing only fetal calf serum.

Table 3 Effective dose for acylamide determined with L-929 cells which were stored und refrigeration for various periods of time.

Effective dose (ED) Control 4 days 7 days 11 days 14 days 18 days 21 days

ED value which creates

20% growth inhibition 40 43 33 26 44 28 25

ED value which creates

50% growth inhibition 57 66 51 44 69 58 45

ED value which creates

80% growth inhibition 81 99 73 74 105 119 78

Table 3. Shows the ED value (Effective Dose) for the chemical Acrylamide which creates 20, and 80% respectively cell growth inhibition, expressed in μg/ml. The ED values are determin for normally stored L-929 cells (control), and for L-929 cells which were stored at max. 8 in a normal refrigerator for various periods of time. The refrigerator cells were stored a solution consisting of Minimum Essential Medium (MEM, Gibco BRL) supplemented with glutamine, gentamycine, NEAA according to normal prescription and supplemented with 30% fet calf serum. After storage in the refrigerator the cells were transferred to a 96-hole pla according to operating instructions and thereafter placed in an incubator. After 24 hours the incubator seven different concentrations of the chemical were applied to the 96-ho plate. After a further 3 days in the incubator the cell growth was determined using Neutr red colouring.

Claims

1. System for cytotoxicity tests comprising those substances (1-7) necessary for the test, such as necessary chemical compounds and test cells, and preferably also a test plate (8) and other instruments necessary for the test, such as pipettes or similar (9) , characterized by means (e.g. 10-12) for storing a mixture (2) of test cells and a cell-culture medium at a lowered temperature, preferably corresponding to normal refrigerator temperature.

2. System according to claim 1, characterized in that said means includes a heat-insulating transport container (12) which preferably also contains a cooling medium, for example a freezable cool block (10) .

3. System according to claim 1 or 2, characterized in that said cell-culture medium is a typical such medium, for example Minimum Essential Medium (MEM) with Earle's salt (Gibco BRL England) , together with a serum, preferably fetal calf serum.

4. System according to claim 3, characterized in that said cell-culture medium is supplemented with at least 10%, preferably approximately 30%, serum.

5. System according to claim 2, characterized in that said transport container (12) comprises a preferably weak-insulating partition wall (11) which separates said cooling medium (10) from the remaining content (1-9) of the container.

6. System according to claim 5, characterized in that the partition wall (11) is in the form of a holder, made from foam rubber or similar, for the remainder of the components (1-9) in the container, such as test tubes, bottles, pipettes, etc.

7. System according to any one of the preceding claims, characterized in that said test cells consist of an established cell-culture, preferably established mouse- fibroblast cell line L-929.

8. System according to claim 7, characterized in that the cell-culture contains more than 200 000 cells/ml, preferably in the order of 1 000 000 cells/ml.

9. Kit for cytotoxicity tests included in a system according to any one of the previous claims, characterized in that it includes a transport container (12) of heat- insulating material which contains essentially all the substances (1-7) necessary for the test, such as necessary chemical compounds and test cells, and preferably also a sampling plate (8) and any other instruments necessary for the test, such as pipettes (9) or similar.

10. Kit according to claim 9, characterized in that it also contains a cooling medium, for example a freezable cool block (10) , which is preferably separated from the remaining components (1-9) in the container by means of a suitably weak-insulating partition wall (11) .

11. Method for storing cells for citotoxicity tests during transport, which cells are intended for the system according to claims 1-8 and/or the kit according to claims 9 and 10 with substantially maintained biological properties, characterized in that they are transported at a lowered temperature, preferably at substantially normal refrigerator temperature.

12. Method according to claim 11, characterized in that the cells are transported in a heat-insulating container (12) , for example of foam rubber or similar.

13. Method according to claims 11 or 12, characterized in that the cells are transported simultaneously with a cooling medium, for example a frozen cool block (10) .

14. Method according to any one of claims (11-13), characterized in that the cells are stored in a conventional cell-culture medium, for example Minimum Essential Medium (MEM) with Earle's salt (Gibco BRL England) together with a serum, preferably fetal calf serum.

15. Method according to claim 14, characterized in that said cell-culture medium is supplemented with at least 10%_A preferably approximately 30%, serum.