CA2139782A1 - Process for co-cultivating a type of cells with liver cells - Google Patents
Process for co-cultivating a type of cells with liver cellsInfo
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- CA2139782A1 CA2139782A1 CA002139782A CA2139782A CA2139782A1 CA 2139782 A1 CA2139782 A1 CA 2139782A1 CA 002139782 A CA002139782 A CA 002139782A CA 2139782 A CA2139782 A CA 2139782A CA 2139782 A1 CA2139782 A1 CA 2139782A1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
- C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
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- 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
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2503/00—Use of cells in diagnostics
- C12N2503/04—Screening or testing on artificial tissues
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A process is disclosed for co-cultivating a type of cells. Liver cells (3) are cultivated on a support (1) according to a sandwich process, and the second type of cells to be analysed (5) is applied thereupon or therebetween.
Description
TransIation of the amended sheets of the international preliminary examination report PROCESS FOR CO-CULTIVATING A TYPE OF CELLS WITH LIV:ER CELLS
==_=_====_====.======_=======
This invention concerns a process for the in-vitro production of hepatocellular products and the provision of the means for these products to act on a second type of cells in a stable cellular test system.
Up to now, there has been a lack of a functional test system for eucaryotic cells which was stable in vitro and in which the influence of drugs or chemical substances and, at the same time, their metabolites on any particular cell type could be i`nvestigated with regard to their action as a whole.
However, a test system that could be used under in-vivo conditions would, for example, be of particular importance for:
1. The development of precursor drugs. One example to be mentioned here are the procytostatics which are converted by metabolically, i.e. enzymatic transformation in the - li~er, into more easily secretable substances, into a form that is toxic to tumours. This type of prècursor has few forganic side effects when administered to patients. In other words, such substances are the ideal kind of anticancer drug.
==_=_====_====.======_=======
This invention concerns a process for the in-vitro production of hepatocellular products and the provision of the means for these products to act on a second type of cells in a stable cellular test system.
Up to now, there has been a lack of a functional test system for eucaryotic cells which was stable in vitro and in which the influence of drugs or chemical substances and, at the same time, their metabolites on any particular cell type could be i`nvestigated with regard to their action as a whole.
However, a test system that could be used under in-vivo conditions would, for example, be of particular importance for:
1. The development of precursor drugs. One example to be mentioned here are the procytostatics which are converted by metabolically, i.e. enzymatic transformation in the - li~er, into more easily secretable substances, into a form that is toxic to tumours. This type of prècursor has few forganic side effects when administered to patients. In other words, such substances are the ideal kind of anticancer drug.
2. Identification of substances that cause mutations, or carcinogens. As we know, certain substances that occur in , .:
amended sheet 213~78~
our environment are not primarily carcinogenic, but are activated when they are taken up by the human organism and metabolised, and thus become carcinogenic.
amended sheet 213~78~
our environment are not primarily carcinogenic, but are activated when they are taken up by the human organism and metabolised, and thus become carcinogenic.
3. Toxicity studies. As we know, newly developed drugs or chemical substances have to be tested for any potential toxic effect. However, a large ~umber of drugs have toxic effects, mainly because of their metabolism and their varying species-dependent detoxification capacity.
4. The study of interactions. This is understood to mean the interactions occurring between different types of cells.
The cases mentioned under points 1 to 3 particularly involve substances that are not converted into their "active form"
until after metabol~sm. What is known about testing these substances is that they can either be administered to animals, or applied to particular cell types in an enzyme mixture after metabolism, or examined in a conventional monoIayer co-culti-vation system between liver cells and another cell type.
However, the disadvantage of these known methods is, among other things, that there are extreme differences in the pattern of metabolite production, not only between different animal species, but especially between animals and humans as well. This means that all substances, the active or toxic form of which can only occur after being metabolised by a human , ~ . . .
metabolism, cannot be usefully determined in animal experi-ments.
,, . .
~- ~ For this reason, attempts have already been made to study -- various cell types using co-cultivation methods with liver - cells (see e.g. WO 84/04325 and "Annals of the New York Academy of Sciences" 349, 1980, pages 264-272). However, all , ; ` .
amended sheet , of these known co-cultivation methods have the inherent proh lem that the function of the hepatocytes, i.e. the liver cells, starts to deteriorate immediately after preparation of the culture, since basic hepatocyte-specific and cytobiologi-cal requirements were not and could not be taken into account in the method applied. Longer-term studies which use such systems do not therefore make any sense. However, such studies would be very important, since it is a well known fact that the properties of carcinogenic substances only manifest them-selves after a longer period.
In the known co-cultivation methods, the cells were mixed in a two-dimensional layer, which meant that there was no longer an easy way to separate them selectively. In other words, the test system is not reusable.
Particularly due to the unstable function experienced with the known test systems and the fact that their reactions do not reflect in-vivo conditions, the application of substances to the respective cell types in an enzyme mixture following meta-bolism has already been proposed for studying such effects.
The disadvantage of such a test system, however, is that again onl~ a certain percentage of the metabolites that occur in vivo can be detected, since complete metabolism is bound to an intact celLular system. This means that substances that may ~e .
therapeutically effective or toxic remain undetected, since there is no way that they can even form. However, the main disadvantage is that potential carcinogens or cytostatics can-not be detected in this way.
An_investigation of interactions between different cell types, in-pa-rticular between hepatocytes and a further cell type in co-culture, e~uivalent to case 4, would only make sense if the amended sheet hepatocytes were able to maintain stable function in the culture. However, this is not the case in the known methods.
The invention submitted is therefore based on the task of creating a method for co-cultivating a cell type in which at least an approximation to in-vivo conditions can be achieved over a longer period in order to investigate this cell type.
In the sense of this invention, this task is solved by culti-vating liver cells on a support above which a matrix layer is arranged and above which a second cell type to be examined is applied.
i In the sense of this invention, the problems outlined above are solved by a kind of "sandwich culture technique" for hepatocytes by cultivating the second cell type to be examined above this structure in a three-dimensional culture system.
One of the advantages of the type of co-cultivation method embodied in this invention is that a polarised anchorage of - the hepatocytes is thus maintained.
Since this kind of cell stacking or cell density wauld normal-ly very rapidly lead to an oxygen deficiency for the liver cells in the lower layer, a very advantageous and not obvious further development of the -invention is proposed in order to supply oxygen to the liver cells via the support in tha form of membrane oxygenation.
, In this way, the -support is appropriately constructed to achieve so-called membrane oxygenation, thus pro~iding the er cells with a~ sufficient oxygen supply.
In practice, three techniques are combined, making it possible amended sheet PCT/EP93/017~j6 to exploit the functional advantages of a sandwich hepatocyte culture technique for the production of hepatocellular pro-ducts and their immediate action on any chosen co-cultivated cell type above them, and vice versa.
The method used in the sense of this invention thus comprises three/ to some extent known, techniques, namely membrane oxy-genation via a support, a sandwich technique for cultivating the liver cells and a co-cultivation method which is new in this form.
This method eliminates oxygenation problems, provides a stable liver cell culture and can be used to simultaneously co-culti-vate a cell type with which one can investigate effects that, for example, are produced by the liver cells and their metabo-lic capacity.
In contrast to known test systems, especially of known co~cul-tivation methods, the invention presented provides a function-ally stable test system in which metabolites are produced over a period of several weeks and their effects studied. The same applies to cellular interactions. In this way, medium to long-term toxicity studies are-now possible for the first time. ~~ ~
Since tha arrangement of the liver cells and the second cell type to be investigated one above the~:other can lead to their separation, e.g. by a matrix layer, both cell types can be selectively separated from each other again as required. This can be done, for example, by adding -an enzyme to the culti-vated cell type located uppermost.- Trypsin has proved to be a suitable enzyme for separating the cells.
The longer-term functional stability of the liver cells and amended sheet -~1~978~
the capacity of a cell type to be separated while maintaining the liver cells immobilised in the sandwich system also makes the test system reusable.
For example, another cell type which is to be tested can be re-cultivated after the system has been washed. This is of particular importance because human cells, such as human liver cells, are not limitlessly obtainable.
Another very im~ortant advantage of the invented method is also the feasibility of using human hepatocytes which achieve a very high degree of relevance with regard to their results in humans, which is after all the object of all human pharma-ceutical and human toxicological studies.
There is also a higher level of relevance compared with methods that use enzyme mixtures, since the invented method provides an intact cellular system and thus all metabolites can be tested as a whole. Known co-cultivation methods only have very limited relevance for medium and longer-term studies in this case, since the liver cells in such known systems very rapidly lose metabolic enzymes.
In the following, one~example will be presented in more-detail to illustrate the principle of the invented method.
The figure shows a method for co-cultivating a celi type-with the cell types arranged one above the other.
I
!~ ' - For co-cultivation, an at least gas-permeable me~brane 1 is first coated with a matrix 2 for use as a support. As the gas-permeable membrane 1, the support used can ~e- made of a material with appropriately large pores or in which holes have been drilled and through which oxygen is passed or diffused.
amended sheet -213978~
l?CT/EP93/01756 For example, sintered metal discs are suited for this purpos~
into which oxygen deriving from an oxygen source is passed into channels or drilled holes in the interior of the dlsc, from where the oxygen is conveyed via appropriately drilled holes or pores to the surface of membrane 1. Porous plastics of cellulose are also suitable as membrane 1.
A layer containing protein, for example, such as collagen, can be used as matrix 2. If necessary, further components such as glycosaminoglycans or glycoproteins can be admixed as required. Such components exert inductive functions on hepa-tocytes. Hepatocytes, i.e. liver cells 3, are polarised and embedded in or above matrix 2.
V-79 cells of lymphocytes can be used as a celI type for co-cultivation with the hepatocytes. These cells are particularly advantageous for muta~enicity tests.
.
Matrix 2 also serves to improve the adhesion of the liver cells 3. If necessary, the liver cells 3 can also be applied directly to membrane 1 without the intermediately positioned matrix 2 if this membrane promotes the adhesion of the liver cells 3, as for example polyurethane foil, polypropylene or- -organic membranes, such as human or animal connective tissue. ~~
,:: , -The Iiver cells 3 can be isolated using the standard method of collagenase digestion of the connective tissue structure of a liver and then washed.
About 30 minutes to one hour after the liver ~ells 3 have been - -~ . .
applied to the first matrix layer 2, the application of a ``-second matrix layer 4 above the liver cells 3 can be started `~ using collagen as a basal adhesion structure. The second-~-~
matrix layer 4 serves to anchor the liver cells 3 along with ; ,,. ~, .
~: .
1~ amended sheet ,~
~, their upper cell membranes to this structure. As is the case for the first matrix layer 2, if necessary, further substances can be admixed to the second matrix layer 4.
After adhesion of the second, upper matrix layer 4, any second cell type or even several cell types 5 can be applied on top of this.
Finally, culture of nutritive medium 6 is applied in a layer on top of the upper cell layer 5 to be examined. In certain cell types 5 that have to be anchored on both sides, it is possible to additionally apply a third matrix layer above the cell layer 5 to be examined. This is indicated in the figure by the dotted line ~. In this case as well, the layer of culture or nutritive medium 6 represents the upper border above tha third matrix layer 7. A third matrix layer 7 will generally be envisaged if, for example, the upper cells 5 to be examined are also liver cells.
Using the illustrated method, it is possible to co-cultivate liver cells 3 with any second cell type 5, while at the same time maintaining a hepatocellular phenotype that is stable over a prolonged period.
Since the oxygen supply of the liver cells 3 is ensured by mem~rane oxygenation, any other cell type can be cultivated in the conventional way on the foundation of the second upper collagen layer 4. The cell type 5 to be examined is supplied from above via the culture or nutritive medium 6.
The method illustrated in the example can be used, for example, for the toxicity testing of pharmaceuticals, since cells from target organs of potential toxicity can be directly combined with a human metabolic system. This offers the possi-amended sheet bility of a screening method, i.e. a test series of severalsubstances.
Human-specific metabolic products that cannot be detected in animal experiments, as well as the widest variety of other cell systems can also be studied using this method. Further-more, this offers the possibility of a precise localisation of toxic effects according to cell systems.
As required, the method can also be integrated into a dynamic culture medium cycle.
.
Instead of applying the second cell type 5, i.e. the cell type in itself to be examined by the influence of the metabolites, to the side of the matrix layer 4 pointing in the direction of the culture medium above the liver cells 3, this second cell type 5 can also be applied to a separate support (not shown).
This support is then rinsed together with the cells arranged thereupon with the metabolite-rich medium from the liver cell system. It is then possible to approximate the second support with the second cell type 5 in such a way that a gap with culture medium is located between the two systems.
Instead of a gas-permeable membrane 1, a simple gas-imperme-able plate can also be used as a support. However, for an ade~uate oxygen supply it would then be necessary to analyse the cells in a special incubator in which the partial oxygen pressure can be increased in accordance with the cell density located thereupon so that the liver cells can be adequately supplied with oxygen. In practice, however, a gas-permeable membrane will generally be used for reasons of simplicity and easier execution.
.
amended sheet -
The cases mentioned under points 1 to 3 particularly involve substances that are not converted into their "active form"
until after metabol~sm. What is known about testing these substances is that they can either be administered to animals, or applied to particular cell types in an enzyme mixture after metabolism, or examined in a conventional monoIayer co-culti-vation system between liver cells and another cell type.
However, the disadvantage of these known methods is, among other things, that there are extreme differences in the pattern of metabolite production, not only between different animal species, but especially between animals and humans as well. This means that all substances, the active or toxic form of which can only occur after being metabolised by a human , ~ . . .
metabolism, cannot be usefully determined in animal experi-ments.
,, . .
~- ~ For this reason, attempts have already been made to study -- various cell types using co-cultivation methods with liver - cells (see e.g. WO 84/04325 and "Annals of the New York Academy of Sciences" 349, 1980, pages 264-272). However, all , ; ` .
amended sheet , of these known co-cultivation methods have the inherent proh lem that the function of the hepatocytes, i.e. the liver cells, starts to deteriorate immediately after preparation of the culture, since basic hepatocyte-specific and cytobiologi-cal requirements were not and could not be taken into account in the method applied. Longer-term studies which use such systems do not therefore make any sense. However, such studies would be very important, since it is a well known fact that the properties of carcinogenic substances only manifest them-selves after a longer period.
In the known co-cultivation methods, the cells were mixed in a two-dimensional layer, which meant that there was no longer an easy way to separate them selectively. In other words, the test system is not reusable.
Particularly due to the unstable function experienced with the known test systems and the fact that their reactions do not reflect in-vivo conditions, the application of substances to the respective cell types in an enzyme mixture following meta-bolism has already been proposed for studying such effects.
The disadvantage of such a test system, however, is that again onl~ a certain percentage of the metabolites that occur in vivo can be detected, since complete metabolism is bound to an intact celLular system. This means that substances that may ~e .
therapeutically effective or toxic remain undetected, since there is no way that they can even form. However, the main disadvantage is that potential carcinogens or cytostatics can-not be detected in this way.
An_investigation of interactions between different cell types, in-pa-rticular between hepatocytes and a further cell type in co-culture, e~uivalent to case 4, would only make sense if the amended sheet hepatocytes were able to maintain stable function in the culture. However, this is not the case in the known methods.
The invention submitted is therefore based on the task of creating a method for co-cultivating a cell type in which at least an approximation to in-vivo conditions can be achieved over a longer period in order to investigate this cell type.
In the sense of this invention, this task is solved by culti-vating liver cells on a support above which a matrix layer is arranged and above which a second cell type to be examined is applied.
i In the sense of this invention, the problems outlined above are solved by a kind of "sandwich culture technique" for hepatocytes by cultivating the second cell type to be examined above this structure in a three-dimensional culture system.
One of the advantages of the type of co-cultivation method embodied in this invention is that a polarised anchorage of - the hepatocytes is thus maintained.
Since this kind of cell stacking or cell density wauld normal-ly very rapidly lead to an oxygen deficiency for the liver cells in the lower layer, a very advantageous and not obvious further development of the -invention is proposed in order to supply oxygen to the liver cells via the support in tha form of membrane oxygenation.
, In this way, the -support is appropriately constructed to achieve so-called membrane oxygenation, thus pro~iding the er cells with a~ sufficient oxygen supply.
In practice, three techniques are combined, making it possible amended sheet PCT/EP93/017~j6 to exploit the functional advantages of a sandwich hepatocyte culture technique for the production of hepatocellular pro-ducts and their immediate action on any chosen co-cultivated cell type above them, and vice versa.
The method used in the sense of this invention thus comprises three/ to some extent known, techniques, namely membrane oxy-genation via a support, a sandwich technique for cultivating the liver cells and a co-cultivation method which is new in this form.
This method eliminates oxygenation problems, provides a stable liver cell culture and can be used to simultaneously co-culti-vate a cell type with which one can investigate effects that, for example, are produced by the liver cells and their metabo-lic capacity.
In contrast to known test systems, especially of known co~cul-tivation methods, the invention presented provides a function-ally stable test system in which metabolites are produced over a period of several weeks and their effects studied. The same applies to cellular interactions. In this way, medium to long-term toxicity studies are-now possible for the first time. ~~ ~
Since tha arrangement of the liver cells and the second cell type to be investigated one above the~:other can lead to their separation, e.g. by a matrix layer, both cell types can be selectively separated from each other again as required. This can be done, for example, by adding -an enzyme to the culti-vated cell type located uppermost.- Trypsin has proved to be a suitable enzyme for separating the cells.
The longer-term functional stability of the liver cells and amended sheet -~1~978~
the capacity of a cell type to be separated while maintaining the liver cells immobilised in the sandwich system also makes the test system reusable.
For example, another cell type which is to be tested can be re-cultivated after the system has been washed. This is of particular importance because human cells, such as human liver cells, are not limitlessly obtainable.
Another very im~ortant advantage of the invented method is also the feasibility of using human hepatocytes which achieve a very high degree of relevance with regard to their results in humans, which is after all the object of all human pharma-ceutical and human toxicological studies.
There is also a higher level of relevance compared with methods that use enzyme mixtures, since the invented method provides an intact cellular system and thus all metabolites can be tested as a whole. Known co-cultivation methods only have very limited relevance for medium and longer-term studies in this case, since the liver cells in such known systems very rapidly lose metabolic enzymes.
In the following, one~example will be presented in more-detail to illustrate the principle of the invented method.
The figure shows a method for co-cultivating a celi type-with the cell types arranged one above the other.
I
!~ ' - For co-cultivation, an at least gas-permeable me~brane 1 is first coated with a matrix 2 for use as a support. As the gas-permeable membrane 1, the support used can ~e- made of a material with appropriately large pores or in which holes have been drilled and through which oxygen is passed or diffused.
amended sheet -213978~
l?CT/EP93/01756 For example, sintered metal discs are suited for this purpos~
into which oxygen deriving from an oxygen source is passed into channels or drilled holes in the interior of the dlsc, from where the oxygen is conveyed via appropriately drilled holes or pores to the surface of membrane 1. Porous plastics of cellulose are also suitable as membrane 1.
A layer containing protein, for example, such as collagen, can be used as matrix 2. If necessary, further components such as glycosaminoglycans or glycoproteins can be admixed as required. Such components exert inductive functions on hepa-tocytes. Hepatocytes, i.e. liver cells 3, are polarised and embedded in or above matrix 2.
V-79 cells of lymphocytes can be used as a celI type for co-cultivation with the hepatocytes. These cells are particularly advantageous for muta~enicity tests.
.
Matrix 2 also serves to improve the adhesion of the liver cells 3. If necessary, the liver cells 3 can also be applied directly to membrane 1 without the intermediately positioned matrix 2 if this membrane promotes the adhesion of the liver cells 3, as for example polyurethane foil, polypropylene or- -organic membranes, such as human or animal connective tissue. ~~
,:: , -The Iiver cells 3 can be isolated using the standard method of collagenase digestion of the connective tissue structure of a liver and then washed.
About 30 minutes to one hour after the liver ~ells 3 have been - -~ . .
applied to the first matrix layer 2, the application of a ``-second matrix layer 4 above the liver cells 3 can be started `~ using collagen as a basal adhesion structure. The second-~-~
matrix layer 4 serves to anchor the liver cells 3 along with ; ,,. ~, .
~: .
1~ amended sheet ,~
~, their upper cell membranes to this structure. As is the case for the first matrix layer 2, if necessary, further substances can be admixed to the second matrix layer 4.
After adhesion of the second, upper matrix layer 4, any second cell type or even several cell types 5 can be applied on top of this.
Finally, culture of nutritive medium 6 is applied in a layer on top of the upper cell layer 5 to be examined. In certain cell types 5 that have to be anchored on both sides, it is possible to additionally apply a third matrix layer above the cell layer 5 to be examined. This is indicated in the figure by the dotted line ~. In this case as well, the layer of culture or nutritive medium 6 represents the upper border above tha third matrix layer 7. A third matrix layer 7 will generally be envisaged if, for example, the upper cells 5 to be examined are also liver cells.
Using the illustrated method, it is possible to co-cultivate liver cells 3 with any second cell type 5, while at the same time maintaining a hepatocellular phenotype that is stable over a prolonged period.
Since the oxygen supply of the liver cells 3 is ensured by mem~rane oxygenation, any other cell type can be cultivated in the conventional way on the foundation of the second upper collagen layer 4. The cell type 5 to be examined is supplied from above via the culture or nutritive medium 6.
The method illustrated in the example can be used, for example, for the toxicity testing of pharmaceuticals, since cells from target organs of potential toxicity can be directly combined with a human metabolic system. This offers the possi-amended sheet bility of a screening method, i.e. a test series of severalsubstances.
Human-specific metabolic products that cannot be detected in animal experiments, as well as the widest variety of other cell systems can also be studied using this method. Further-more, this offers the possibility of a precise localisation of toxic effects according to cell systems.
As required, the method can also be integrated into a dynamic culture medium cycle.
.
Instead of applying the second cell type 5, i.e. the cell type in itself to be examined by the influence of the metabolites, to the side of the matrix layer 4 pointing in the direction of the culture medium above the liver cells 3, this second cell type 5 can also be applied to a separate support (not shown).
This support is then rinsed together with the cells arranged thereupon with the metabolite-rich medium from the liver cell system. It is then possible to approximate the second support with the second cell type 5 in such a way that a gap with culture medium is located between the two systems.
Instead of a gas-permeable membrane 1, a simple gas-imperme-able plate can also be used as a support. However, for an ade~uate oxygen supply it would then be necessary to analyse the cells in a special incubator in which the partial oxygen pressure can be increased in accordance with the cell density located thereupon so that the liver cells can be adequately supplied with oxygen. In practice, however, a gas-permeable membrane will generally be used for reasons of simplicity and easier execution.
.
amended sheet -
Claims (8)
1. Method for co-cultivating a cell type with liver cells, c h a r a c t e r i s e d b y a cultivation of liver cells (3) on a support (1) above which a matrix layer (4) is arranged and above which a second cell type to be examined (5) is applied.
2. Method in accordance with claim 1, c h a r a c t e r i s e d b y t h e f a c t that oxygen is supplied to the liver cells (3) in the form of membrane oxygenation via the support (1).
3. Method in accordance with claim 1 or 2, c h a r a c t e r i s e d b y t h e f a c t that a first matrix layer (2) is arranged between the liver cells (3) and the support (1) to anchor the liver cells (3).
4. Method in accordance with any of the claims 1 to 3, c h a r a c t e r i s e d b y t h e f a c t that a culture or nutritive medium (6) is arranged above the second cells (5) that are arranged above the liver cells (3).
5. Method in accordance with claim 4, c h a r a c t e r i s e d b y t h e f a c t that a third matrix layer (7) is arranged for the second cell type (5), above which the culture or nutritive medium (6) is placed.
6. Method in accordance with any of the claims 1 to 5, c h a r a c t e r i s e d b y t h e f a c t that the second cell type (5) is arranged on its own second support, a gap with culture or nutritive medium (6) being adjusted between the two cell types (3,5).
7. Method in accordance with claim 1, c h a r a c t e r i s e d b y t h e f a c t that it is conducted in an incubator with increased partial oxygen pressure.
8. Use of a method in accordance with claims 1 to 7 for toxicity testing of substances to be examined.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4222345.8 | 1992-07-08 | ||
| DE4222345A DE4222345A1 (en) | 1992-07-08 | 1992-07-08 | Process for growing a cell type in a co-culture process with liver cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2139782A1 true CA2139782A1 (en) | 1994-01-20 |
Family
ID=6462712
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002139782A Abandoned CA2139782A1 (en) | 1992-07-08 | 1993-07-07 | Process for co-cultivating a type of cells with liver cells |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0651788B1 (en) |
| JP (1) | JPH09502081A (en) |
| KR (1) | KR950702238A (en) |
| AT (1) | ATE190649T1 (en) |
| AU (1) | AU685056B2 (en) |
| CA (1) | CA2139782A1 (en) |
| DE (2) | DE4222345A1 (en) |
| DK (1) | DK0651788T3 (en) |
| ES (1) | ES2145051T3 (en) |
| PT (1) | PT651788E (en) |
| WO (1) | WO1994001535A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4336399A1 (en) * | 1993-10-26 | 1995-04-27 | Augustinus Dr Med Bader | Process for improving the matrix conditions of bipolar adhered hepatocytes and for producing a suitably configured cell culture kit |
| EP1046911A1 (en) * | 1999-04-23 | 2000-10-25 | Universiteit van Amsterdam | Methods for identification of proteinaceous substances capable of inducing cellular reactions |
| DE10023505A1 (en) * | 2000-05-13 | 2001-11-22 | Fraunhofer Ges Forschung | Reactor module for use in artificial organs contains ceramic hollow fibers on which cells are immobilized |
| JP2003052364A (en) * | 2001-08-17 | 2003-02-25 | Fuji Photo Film Co Ltd | Method for producing cell cultural system |
| AU2006315163A1 (en) * | 2005-11-16 | 2007-05-24 | University Of North Carolina At Chapel Hill | Extracellular matrix components for expansion or differentiation of hepatic progenitors |
| WO2023132371A1 (en) * | 2022-01-07 | 2023-07-13 | インテグリカルチャー株式会社 | Method for culturing liver-derived cells and culture system including liver-derived cells |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2545100B1 (en) * | 1983-04-29 | 1985-12-20 | Inst Nat Sante Rech Med | PROCESS FOR OBTAINING CULTURES OF HUMAN HEPATOCYTES, THE CULTURES OBTAINED AND THEIR BIOLOGICAL AND BIOCHEMICAL APPLICATIONS |
| DE4206585C2 (en) * | 1992-03-03 | 1994-11-24 | Augustinus Dr Med Bader | Device for mass culture of cells |
-
1992
- 1992-07-08 DE DE4222345A patent/DE4222345A1/en not_active Withdrawn
-
1993
- 1993-07-07 ES ES93915821T patent/ES2145051T3/en not_active Expired - Lifetime
- 1993-07-07 DK DK93915821T patent/DK0651788T3/en active
- 1993-07-07 EP EP93915821A patent/EP0651788B1/en not_active Expired - Lifetime
- 1993-07-07 AU AU45651/93A patent/AU685056B2/en not_active Ceased
- 1993-07-07 PT PT93915821T patent/PT651788E/en unknown
- 1993-07-07 AT AT93915821T patent/ATE190649T1/en not_active IP Right Cessation
- 1993-07-07 JP JP6502947A patent/JPH09502081A/en active Pending
- 1993-07-07 DE DE59309981T patent/DE59309981D1/en not_active Expired - Fee Related
- 1993-07-07 WO PCT/EP1993/001756 patent/WO1994001535A1/en active IP Right Grant
- 1993-07-07 CA CA002139782A patent/CA2139782A1/en not_active Abandoned
-
1994
- 1994-12-31 KR KR1019940704873A patent/KR950702238A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| KR950702238A (en) | 1995-06-19 |
| JPH09502081A (en) | 1997-03-04 |
| EP0651788A1 (en) | 1995-05-10 |
| ATE190649T1 (en) | 2000-04-15 |
| PT651788E (en) | 2000-08-31 |
| WO1994001535A1 (en) | 1994-01-20 |
| DE4222345A1 (en) | 1994-01-13 |
| AU685056B2 (en) | 1998-01-15 |
| EP0651788B1 (en) | 2000-03-15 |
| ES2145051T3 (en) | 2000-07-01 |
| AU4565193A (en) | 1994-01-31 |
| DK0651788T3 (en) | 2000-08-28 |
| DE59309981D1 (en) | 2000-04-20 |
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| EEER | Examination request | ||
| FZDE | Discontinued |