PERFUSION-CULTIVATION OF ANIMAL CELLS AND EQUIPMENT THEREFOR

Abstract: Method and movable culture unit for perfusion-cultivation of animal or human cells for determination of effects of a test substance or other environmental parameter on the culture, whereby the culture is exposed to a continuous, preferably laminar flow of substrate. The culture unit comprises at least one celldish (6, 7) forming a closed culture chamber with turned cone (2, 3) of a transparent body (1) and channels (9-12) arranged for supply and discharge of substrate.

Description: Perfusion-cultivation of animal cells and equipment therefor The invention relates to a method of producing stable perfusion-culti- vations of animal cells in a specially designed system, thereby deter¬ mining toxicity, and apparatus for performing the method. Requirements for improved working environment, combined with the rapidly increasing number of chemical substances in working life and in society in general has increased the need for toxicological examination. Such exami¬ nation is normally performed by means of animal experiments which, how¬ ever, are expensive, time-con3uming and require considerable resources. The determination of acute toxicity (LD values) on all the substances in 50 question requires such vast resources that it is in practice difficult to achieve. The objection can also be raised that these experiments con¬ stitute cruelty to animals. Attempts have been made to achieve mathematical models and store infor¬ mation in computers as to how an organ or a system of organs reacts in various situations, the computer subsequently simulating the toxic action of the substances tested, on the basis of this information. In biochemical models, the action of the chemical substance is tested on isolated bio¬ logically active substances, i.e. substances such as enzymes which control the chemical process in plants and animals which is the basis of all life. However, these mathematical and chemical models are uncertain and it is difficult to obtain reliable values of acute toxicity with them. The microbiological methods (Ames tests) are mostly used to deterc-iπe the muta- genity of the substances, and thus their ability to cause cancer. A simpler and less expensive method is thus required for determining the toxicity, and such a method has been effected according to the invention by means of stable perfusion-cultivations of animal cells in a specially designed apparatus giving laminar flow of the substrate. Cultivation ' -of cell cultures is performed by keeping isolated cells from animals and humans alive and cultivating them outside the body. The most usual type of cultivation is when the cells are placed in a dish or glass or plastic and surrounded by a substrate similar in composition to the body liquids. A test substance can .be added to the cultivation substrate and changes in the cells be measured. The concentration of the substance which gives a certain defined alteration (in appearance, growth rate, size, 0 absorption, enzyme activity, or ratio between living and dead cells, for instance) constitutes a gauge of the toxic activity in the cell culture. A great advantage with cell cultures is that they enable tests on human material since cultures can be started from cells in blood samples, a - niotic fluid or surgically removed tissues, for instance. When a cell culture is used for studying biochemical processes, it is important that cultivation occurs in a well defined environment. Perfusion cultivation enables this, providing the cultivation equipment used .allows a uniform supply of nutrient. The flow properties of the cultivation chamber are important. If it were possible to keep the perfusion cultivations stable, they could be used for fundamental biochemical studies of various animal cells, in medical research and for determining acute toxicity, for instance. To enable cell cultures to be used for following up biochemical processes, the cultivation must take place under extremely stable conditions and so far it has not been possible to achieve this in a satisfactory manner. According to the invention, stable perfusion cultures are achieved by means of a specially designed apparatus which gives an extremely uni¬ formly, substantially laminar flow. In the following the method and ap¬ paratus will be described with reference to seven drawings in which Figure 1 shows a perfusion chamber, Figure 2 shows how the apparatus is connected, Figure 3 shows a schematic view of oxygen-measuring equipment, Figure 4 shows examples of physical measurement parameters, Figure 5 shows the effect of adding hexachlorophene, Figure 6 shows the effect of adding CN and Figure 7 shows the effect of Triton X-100 on the outflow of %-2- deoxy-D-glύcose. Description of test system The test system in question is based on cultivation of neuroblastoma cells C ' 1300, 41A, -and the effect on these cells measured as general biological cell changes or biochemical changes, as a function of the content of toxic substances in the substrate. The cell line used is suitable for such measurements since its biology and biochemistry is well documented. The parent culture is cultivated in what are known as Costar plastic bottles in a commercial substrate Ham's F10 which includes 9$ serum from new-born calf and 4? foetal calf serum. Description of the test apparatus The test apparatus according to Fig.lA comprises a body 1 made of poly¬ carbonate. The bod7 has a turned cone 2,3 at each end. At the top of each cone is a turned recess 4,5, the depth of this recess deter ing the culti¬ vation volume of the perfusion chamber. A cell dish 6,7 with the lid removed is pushed over the turned cone 2,3. The cone 2,3 has the same outer diameter as the inside of the bottom of the dish. An 0-ring 8, suitably of fluorine or silicon rubber, is provided to seal between the body 1 and the cultivation dishes 6,7. Medium is supplied to or removed from the chamber through holes 9,10,11,12 drilled in the periphery of the recess 4,5. Experiments with coloured solutions show that the flow is substantially laminar. Since the chamber is made of transparent material, it can be used in assembled state for light-microscopic studies. Connections 13, 1 of uer type are arranged to the chamber, and to these are connected hoses 15, 1δ of silicon rubber, to enable satisfactory acidi¬ fication of the substrate by means of diffusion. The following criteria are valid for the perfusion chamber: (1) The cells are sown and cultivated in their normal environmentj in this case in Falcon culture dishes in a culture incubator of conventional type. (2) The chamber is simple, practical and robust to handle. Loose washers, screws, gla≤s, etc, is eliminated. (3) It can be autoclaved. (4) It is inexpensive and simple to manufacture. (5) It is suitable for light-microscopic studies (6) It can be used in most types of perfusion applications. As mentioned previously, it is important that the flow in the perfusion cell is substantially laminar so that the cell's biochemical activity is not disturbed by turbulent flow. The flow in the cell is determined by the channels in the cell and their diameter and extension in the perfusion cell. The following diameters, for instance, have been found suitable. See Fig.1. 9 2.0 10 2.0 Tftfll 11 2.0 mπi 12 2.0 l^fl 13 4.0 ffltf^ 14 4.0 rπyη For good flow geometry, it is "also important that the channel 10-11 has an angle of about 50 - 55 to the horizontal plane. These measurements .are applicable for an apparatus with double chamber and a cell diameter of 46 mm as in Fig.1, and for the normal flow rate of about 1 ml/ in. A lamina flow can be ensured by regulating the flow rate. The body and the chamber may have a different appearance, deviating from Fig.1, but the apparatus shall include a closed space for the perfusion chamber. With the apparatus described, perfusion technique can be utilized in an extremely simple manner for biological cell studies and this is to a great extent because the dish forms a part of the assembled chamber. The cells need * not be removed and transferred to a different surface, but can grow on their normal surface, in an environment which can be kept constant thanks to the perfusion technique. Due to its simple design, it should be pos¬ sible to manufacture the apparatus relatively cheaply, thus enabling increased use of perfusion technique for biological cell studies. The apparatus is connected in a simple manner by means of hoses and can easily be moved to different measuring points. To summarize, it can be established that the apparatus described is simple in design, ordinary cell cultivating dishes are used, it should be possible to produce it relatively cheaply, it has been found to have good through- flow properties, it is suitable for microscopic studies and it has been found useful in practice in a number of toxicological and general bio¬ logical cell studies. Fig.133 shows a body 1 for perfusion of only one dish 2. Figure 2 shows how the apparatus is connected. The medium is pumped by a pump 18 from a store 17 through hoses 19 to the perfusion chamber 20 via connections 21 ,22. Figure 3 shows schematically the layout of equipment for measuring oxygen container, 24 a pump, 25 a means for o-rygena ion 26 an air-lock, 27,28 oxygen gas electrodes of Clark type, 29 a calibration loop, 30 an cultivation chamber (double), 31 a collecting vessel for used medium and 32 a printer with amplifier unit. Figure 4 shows examples of physical parameters which can be introduced in a multi-parametric system. The figure shows changes measured once complete cell death has occurred. An arrow indicates the addition of toxic sub¬ stance. The curves denote: 1 pH 2 D0 2 3 ORP 4 Trypan » The effect of the addition of hexchlorophene on the oxygen gas consumption can be seen in Fig.5, and Fig.6 shows the effect of the addition of KCN on the oxygen gas consumption. Fig.7 shows the effect of Triton X-100 on the outflow of 3 H-2-deoxy-D- glucose from cultivated neutroblastoma cells in the perfusion system. The curves specify control (1), 0.015? (2), 0.022? (3) and 0.035 Triton (4). The y-axis log in the diagram indicates remaining activity -in. $ of the total. The parameters which can be used to study the state of the cells during perfusion are primarily: DO see Fig.4 and 5 pH see Fig.4 Redox potential see Fig.4. Examples of other parameters which can be used are ATP (adenosine tri- phosphate) content and LDH (lactate dehydrogenase) activity in the per¬ fusion liquid-. These function as indicators of membrane leakage and their concentration can be expected to increase after exposure. Another way of measuring membrane leakage Is to study the outflow of an isotope-marked marker (see Example 5). It is also possible to measure the absorption of trypan blue in dead cells with the help of light absorption (see Example 4 and Fig.4). The invention will now be described in more detail with the help of the following examples. Example 1 Comparison between perfusion culture in the cell described above and a unperfu3ed culture The cell line used is Neuroblastoma C1300, substrate F10 according to Ham, R.G. Exp Cell Res, 29 (1963) P 515 87? Calf serum 9? Foetal calf serum 4? The perfusion was carried out in a recirculated system with a perfusion volume of 100 ml. The perfusion rate was 1 ml/min. The cultures were checked in a microscope during the perfusion period. After 7 days the concentration of cells in the perfused dishes was 14 times the original concentration. The equivalent figure for the control cultures, i.e. un- perfu≤ed cultures was 6 times. No signs of the growth curve starting to flatten out could be detected in the perfusion cultures. Growth had ceased in the control cultures, how- 2 ever. When the experiment was discontinued, the number of cells per cm was 350,000 in the perfusion culture. Example 2 The effect of hexachlorophene on the oxygen gas consumption in neuro¬ blastoma cells Cell line and substrate as in Example 1. Hexachlorophene was added on two occasions, marked in Fig.5. Six minutes after measurement 2.5,ug/ml (1) was added and after another 30 minutes The experiment layout is shown in Fig.3 and the pump rate of 1 ml/min. The eexxppeerriimmeenntt iinnddiiccaatteess tthhaatt tthhee ssyysstteemm πregulates the 0-, absorption in the cells in an extremely sensitive manner. ' OMFI Example 3 The effect of adding potassium cyanide on the oxygen gas consumption in neuroblastoma cells Cell line and substrate ' as in Example 2. The culture volume and. ump rate as in Example 1. 1 mM KCN was added 5 minutes after measuring was started. The result can be seen in Fig.6. Example 4 Determining LD values by means of cell death, measured as absorption of trypan blue The accumulation of trypan blue (a stain which is taken up in dead cells) can be calculated with the help of light-absorption measurements. It has been found that cell death in neuroblastoma cells correlates well with the j . n values obtained from animal experiments for the following tested substances: acetone, benzene, n-butanol, t-butanol, dioxane, n-propanol, HgCl 2 , CH HgCl, CdCl 2 , ^C^O and pp-DDT. A correlation coefficient of O.98 was obtained upon comparison. Example 5 Leakage of ^3-2-deoxy-D-glucose from cultivated cells as a measurement of membrane damage The substance 2-deoxy-D-glucose (2DG) is taken up by the cell over the membrane via a specific transport system. Inside the cell, 2DG is con¬ verted in such a way that its ability to pass through the cell membrane is drastically reduced. This means that if cultivated cells as placed in a solution containing radioactively marked 2DG, the radioactivity will be enriched in the cells, whereas cells in which the membrane has been damaged due to the presence of (milieu-)toxic substances, will release 2DG into the solution again. The radioactivity collected in the cell will then decrease. The degree of reduction can be taken as a measurement of the extent of cell membrane damage. This fact has been utilized in the present method. After being allowed to absorb radioactive 2DG for two hours, the radioactivity not absorbed was washed off, and the cells placed in the perfusion system and perfused at a rate of 1 ml/min. The perfusate was collected with the help of. a fraction collector which changes collecting vessel every fifth minute. When the radioactivity was measured in the perfusion liquid it was found that the activity leaks slowly out of the cells. The detergent Triton X-100 was used as model substance for membrane-damaging substances and it was found that the outflow of radioactivity from cultivated neuro- blastoma cells increases in the presence of Triton. The extent of the increase is correlated to the Triton concentration (see Fig.7). TZ A

Claims: AMENDED CLAIMS (rece i ved by the International Bureau on 5 July 1982 (05.07.82)) Original claims 1. to 19. (cancelled) 20.Apparatus for performing perfusiσn-cultivation of one or more cell cultures of animal or human origin, characterised by a preferably cylindrical body (1), preferably of transparent ma terial, especially plastic, having a turned cone (2,3) at one or both ends, said cone having the same outer diamater as the inner diameter of the bottom of a standard circular cell dish (6,7) l-jith the lid removed, designed to be placed over the turned cone and form a closed chamber constituting the culti¬ vation space, said cone (2,3) having a turned recess (4,5) at the top, ωith drilled holes (9-12) in the periphery of the re¬ cess for the supply or removal of substrata through channels communicating ωith connections ( ' 15,15) to pipes such as hoses. 21. Apparatus according to claim 1, characterised in that the body (1) is in the form of a double cone, with a cone (2,3) at each end and a channel .(10—11) joining the tωα recesses. 22. Apparatus according to claim 2, characterised in that the chan nel (10-11) forms an angle of about 50-55 through the body (1 to " the horizontal plane. 23. Apparatus according to ' one or more of claims 1-3, characterise in that the body (1) is manufactured of polycarbonate plastic. 24. Apparatus according to one or more of claims 1—4, characterise in that the body (1) is connected to members (27,28) for measuring oxygen gas. 25. Apparatus according to one or more of claims 1-5, characterised in that the chamber is connected to a pump (18-24) providing a flou adjusted to give laminar floω. 26. Method of perfusion-cultivating one or more cell cultures of animal or human origin, characterised in that said cell culture( is (are) kept in a cultivation container in an apparatus in accordance ωith one or more of claims 1-4 and that said cell culture(s) is (are) " subjected to a preferably laminar floω of substrate so that cultivation is carried out under stable con¬ ditions. 27. Method according to claim 7, characterised in that a cell cul¬ ture containing neuroblastoma cells is used. 8 Method according to claims 7-8, characterised in that a test substance is added to tie substrate floω. 29. Method according to claim 9, characterised in that the change in oxygen absorption in the calls is determined. 30. Method according to claim 9, characterised in that the change in the number of living cells is determined. 31. Method according tα claim 11, characterised in that the number of dead cells is measured as a function of light absorption after the addition of a -suitable colouring agent, preferably trypan blue. 32. Method according to claim 10, characterised in that the change in pH value of the substrate is determined upon passage of the cells. 33. Method according to claim 10, characterised in that the change in redαx potential is determined. 34. Method according to claims 7-9, characterised in that culti¬ vated cells are placed in a solution containing a radioactively marked substance ωhich is absorbed by the cells and concentrated in them, but is released again into the solution from cells ωhose membrane has been damaged by toxic substances, after ωhich .radioactive substance ωhich has not been absorbed is removed, the cells are placed in the perfusion system and perfused, a membrane-damaging substance is added, the αutfloω of radioac¬ tivity is measured in the perfusion liquid and the increase in outfloω of radioactivity is used to determine the toxic action of the membrane-damaging substance added. 35. Method according to one or more of claims 7-15, characterised in that changes noted are used to determine the toxicity of the substances added to the substrate. 36. Method accqrding to claim 16, characterised in that changes noted are used to collect data concerning acute toxicity cor¬ responding to the LD go values. OMPI

Inventors: PETERSON ANDERS, WALUM ERIK
Applicants: PETERSON ANDERS, WALUM ERIK
Application year: 1982
Main classification: C12N5
IPC codes: C12M1, C12M3, C12N5, C12Q1, G01N33