Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L7/00—Heating or cooling apparatus; Heat insulating devices
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L9/00—Supporting devices; Holding devices
  • B01L9/06—Test-tube stands; Test-tube holders
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F25—REFRIGERATION OR COOLING; COMBINED HEATING AND REFRIGERATION SYSTEMS; HEAT PUMP SYSTEMS; MANUFACTURE OR STORAGE OF ICE; LIQUEFACTION SOLIDIFICATION OF GASES
  • F25D—REFRIGERATORS; COLD ROOMS; ICE-BOXES; COOLING OR FREEZING APPARATUS NOT OTHERWISE PROVIDED FOR
  • F25D3/00—Devices using other cold materials; Devices using cold-storage bodies
  • F25D3/02—Devices using other cold materials; Devices using cold-storage bodies using ice, e.g. ice-boxes
  • F25D3/06—Movable containers
  • F25D3/08—Movable containers portable, i.e. adapted to be carried personally
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F25—REFRIGERATION OR COOLING; COMBINED HEATING AND REFRIGERATION SYSTEMS; HEAT PUMP SYSTEMS; MANUFACTURE OR STORAGE OF ICE; LIQUEFACTION SOLIDIFICATION OF GASES
  • F25D—REFRIGERATORS; COLD ROOMS; ICE-BOXES; COOLING OR FREEZING APPARATUS NOT OTHERWISE PROVIDED FOR
  • F25D2303/00—Details of devices using other cold materials; Details of devices using cold-storage bodies
  • F25D2303/08—Devices using cold storage material, i.e. ice or other freezable liquid
  • F25D2303/082—Devices using cold storage material, i.e. ice or other freezable liquid disposed in a cold storage element not forming part of a container for products to be cooled, e.g. ice pack or gel accumulator
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F25—REFRIGERATION OR COOLING; COMBINED HEATING AND REFRIGERATION SYSTEMS; HEAT PUMP SYSTEMS; MANUFACTURE OR STORAGE OF ICE; LIQUEFACTION SOLIDIFICATION OF GASES
  • F25D—REFRIGERATORS; COLD ROOMS; ICE-BOXES; COOLING OR FREEZING APPARATUS NOT OTHERWISE PROVIDED FOR
  • F25D2331/00—Details or arrangements of other cooling or freezing apparatus not provided for in other groups of this subclass
  • F25D2331/80—Type of cooled receptacles
  • F25D2331/804—Boxes

Definitions

• 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.
• cytotoxicity tests cell tests
• cell tests cell tests
• 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.
• 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 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.
• the temperature is instead reduced to "refrigerator-temperature".
• 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.
• 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.
• 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.
• MEM Minimum Essential Medium
• Gibco BRL England Eagle BRL England
• serum preferably fetal calf serum.
• said cell-culture medium should be supplemented with at least 10%, preferably approximately 30%, serum.
• 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.
• 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.
• a heat-insulating container for example of frigolite or similar
• a cooling medium for example a frozen cool block.
• the attached drawing shows a ki ⁇ _ according to the invention.
• the kit can contain the following:
• 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.
• MEM Minimum Essential Medium
• NEAA Non essential Aminoacids
• Test tube holder of foam rubber 11.
• 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
• NB Prepare the test substances before the start (use the medium in tube No. 3) .
• 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) .
• Serum cone 10 days 17 days 28 days
• 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.
• 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 B ⁇ rker 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 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.
• 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 ⁇ l ampoules were filled and placed in a refrigerator. Days in refrigerator %growth after 3 days Viability compared with control
• ICG Inhibition of Cell Growth Results: The refrigerated cells appear to possess substantially the same sensitivity for acrylamide as do normally stored cells.
• the cell growth determines the function of the cells, i.e. in what condition they are in after being stored under refrigeration.
• 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.
• 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.
• 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.
• MEM minimum essential medium
• 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 B ⁇ rker 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.
• MEM Minim Essential Medium
• Table 3 Shows the ED value (Effective Dose) for the chemical Acrylamide which creates 20, and 80% respectively cell growth inhibition, expressed in ⁇ g/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.
• MEM Minimum Essential Medium

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• 1992
  • 1992-05-27 SE SE9201636A patent/SE9201636L/sv not_active Application Discontinuation
• 1993
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  • 1993-03-03 WO PCT/SE1993/000183 patent/WO1993024607A1/en not_active Application Discontinuation
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