Process for determining the tolerance, or toxicity of gaseous, liquid and/or viscous substances for the human or animal organism

The invention relates to a novel process for determining the tolerance, especially the toxicity of gaseous, liquid and/or viscous substances for the human or animal organism. A tissue sample, obtained without proteolytic disintegration, is placed in a chamber which has at least two compartments which are separated from each other by the tissue sample. A nutrient solution is supplied to both sides of the tissue and the tissue is exposed to a test substance on at least one side. The tissue is evaluated to determine the toxicity of the substance.

BACKGROUND OF THE INVENTION 
In the medical and pharmaceutical fields, as well as in other areas, such 
as nutrition, etc., it is often necessary to determine the toxicity of 
certain gaseous, liquid and/or viscous substances. 
The conventional processes at the present time call first of all, for cell 
culture tests. Subsequently animal tests are added. This is especially the 
case when in the cell culture tests, no toxicity has been ascertained. 
Then, if necessary, clinical studies on humans are undertaken. In 
particular, the prior art can be described as follows: 
Model systems currently used to determine acute toxicity of liquid or 
particular substances are based on the use of cell cultures. Adherently 
growing cells and non-adherently growing cells are exposed for different 
time intervals to the substance to be tested, which is either offered with 
the nutrient solution or is to be colonized as a culture substrate by the 
cells. 
Application of gaseous or liquid test substances is done in this system 
jointly with the test substance. Specific polar application of the test 
substance to the basal or apical aspect of epithelial cell monolayer is 
not done. The cells are either completely flushed by the medium and test 
substance, or exposed to both components jointly in the area of the apical 
cell membrane. 
It is characteristic of these systems that they work only with a certain 
cell type. Often they are cells capable of unlimited division, such as 
cells with tumor characteristics. 
Both epithelial cell lines and also cell lines with connective tissue 
characteristics are used for these tests. They differ in their properties 
however significantly from differentiated epithelial or connective tissue 
cells in human or animal organs, since tumor cells are dedifferentiated 
and can execute the typical cellular functions of differentiated cells 
only to a limited degree. According to the knowledge underlying the 
invention, this is one of the reasons for the lack of applicability of 
results obtained in cell culture experiments to the situation in the 
organism. Chronic toxic effects on cells are unsatisfactorily studied 
using cell culture systems. 
If test substances in the cell culture systems have been found to be 
nontoxic, they must be studied for their action in an animal organism in 
different ways. Still, results of animal tests can only be applied to 
humans to a limited degree. There are major differences in the metabolic 
event between the human and animal organism. On the one hand, substances 
toxic to certain species are tolerated by humans without extensive adverse 
side effects (example: aspirin). On the other hand, there are a large 
number of test substances which are recognized as toxic to humans only in 
clinical studies. 
One disadvantage of animal tests is that they are timeconsuming and often 
indicate little with reference to the human organism. Likewise, clinical 
tests on humans are expensive and complex and often cannot be done. 
In particular, a process is disclosed for the in-vitro testing of effects 
on biological structures in German patent No. DE 42 29 013 A1. The process 
is an analytic technique, and a tolerance, toxicity, side effect and 
efficacy test. In this process, the respective sample is placed in a 
chamber in which it is completely flushed by the test substance so that 
disadvantages arise. These are the same disadvantages that were indicated 
above in the discussion of cell culture tests. 
Further, a system or process is known for measuring transepithelial 
resistances on tissue samples with a small diameter as disclosed in German 
Patent No. DE 40 20 013 A1. The patent uses a perfusion chamber for 
holding the respective tissue sample. In this system, small organisms or 
organs are studied with respect to their transport properties (transport 
systems of the membrane for ions) with special application areas of 
ecotoxicology, pharmacology and transport physiology in biology and 
medicine. The use of this known process is limited exclusively to 
epithelial cells. Transepithelial resistance is determined. 
Further, a device is known for treatment of tissue samples held in vitro 
and drawn up on glass slides as described in German Patent No. DE 36 35 
013 C2. The device has a chamber for holding the preparation. This chamber 
can be filled with the respective treatment reagent, can be sealed tight 
with a cover and can be provided with feed and drain openings for the 
reagents. It is basically a single-chamber system. The respective sample 
itself is located on the impermeable glass slide and is wetted on only one 
side by the respective test liquid. In this system, cells of the sample 
can only execute typical cellular functions of differentiated cells to a 
very limited degree, or not at all. The results obtained are not 
applicable or only conditionally applicable or only conditionally 
applicable to the actual situation in a specific organism. 
Further, a device is known for determining and measuring membrane-permeable 
substances (see U.S. Pat. No. 5,525,475). This device does not determine 
tolerance, for example, the toxicity of gaseous, liquid or viscous 
substances for the human or animal mechanism. 
The device calls for two chambers which are separated from one another by a 
semipermeable membrane. The substance to be tested is placed in one of the 
chambers. The diffusion of the substance is measured in a second chamber. 
Cultivation of cells or tissues is not provided in either of these 
chambers. The purpose of this system is the determination of the 
diffusibility of a test substance, not the determination of its biological 
action, especially not the determination of tolerance for human or animal 
organisms. 
An object of the invention is to devise a process which avoids these 
defects and enables the determination of the tolerance or toxicity of 
gaseous, liquid or viscous substances in the laboratory for a human or 
animal mechanism. 
SUMMARY OF THE INVENTION 
The process embodying the present invention calls for organs or organ parts 
which have been obtained without proteolytic disintegration from a human 
or animal body to be cultivated in a gradient perfusion chamber in the 
presence of a substance to be tested. The gradient chamber has an upper 
and a lower compartment which are separated from one another by the organ 
or tissue sample. One compartment of the gradient perfusion chamber is to 
be flushed with a nutrient solution while the organ or tissue sample is 
exposed to the substance to be tested, preferably via the other 
compartment. 
The test results are evaluated using antibodies or antigens for the 
detection of whether certain cells in the tested organ or tissue sample 
are still present after the test (cultivation) or have died during 
treatment. Such detection can be accomplished by microscopic examinations, 
or other means. 
Organ or tissue samples are not cell cultures of simple structure, but 
interactive tissue culture systems with the following advantages: 
1. Interactive tissue culture systems are composed of cells in the 
organ-typical differentiation state. Depending on the tissue type, there 
is contact inhibition for some of the cells. These cells do not 
proliferate. They are embedded in a specific extracellular matrix and form 
structured cell associations with organ-typical function. 
2. Interactive tissue culture systems consisting of cell associations which 
are composed of identical and different cells in an organ-specific matrix. 
Thus intercellular interactions can be acquired in the action of toxic 
substances between the different cell components. 
3. Tests of toxic substances in interactive tissue culture systems can 
contribute to reduction of test numbers. There are two important 
advantages of the invention, specifically: 
Substances which did not exhibit toxic effects in the cell culture system, 
since they become evident only by intercellular interactions, can be 
precluded early from other tests by using cell culture systems. The 
interactive cell culture model as claimed in the invention enables use of 
human tissue material. Thus the process, as described in the invention, is 
better suited than the existing processes for applying data acquired in 
the culture experiment to the situation in the human organism. This 
contributes to a reduction in the number of experiments. 
The interactive tissue culture system as described in the invention also 
satisfies the following requirements: 
prevention of necrosis formation in the cultivated tissue 
abandonment of undefined medium additives 
preservation of an organ-specific degree of cell differentiation 
acquisition of organ-typical tissue composition 
possibility of specific basal or apical application of test substance. 
Organ or tissue parts are obtained in the process, embodied by the 
invention without proteolytic preparation while acquiring the 
organ-typical tissue composition and while obtaining a organ-typical 
degree of cell differentiation. The tissue or organ samples can be 
obtained as tissue sections, for example, vibratome sections which can be 
obtained from all organs, as teased-out preparations, such as tissue 
explants from the kidneys, or mucosa explants of the stomach. Further, 
relatively thin tissue parts, for example, the retina, cornea, skin, etc., 
can also be used as tissue samples. 
Only by using the gradient perfusion chamber is it possible to preserve the 
organ structure and the function which are linked to a functioning 
vascular system, such that disintegration of the tissue is prevented. In a 
normal culture dish with a nutrient solution, the process as described in 
the invention, cannot be carried out, since sufficient supply of 
nutrients, and especially oxygen, is not ensured. Only use of the unique 
gradient perfusion chamber prevents metabolites, which are naturally 
removed by the vascular system which supplies the organ, from accumulating 
in the tissue. Cultivation of a tissue sample in a conventional culture 
disk results in tissue necroses, destroying the original tissue structure. 
An assessment of whether this occurrence is the consequence of the action 
of toxic substance, or whether it is the consequence of culture conditions 
is no longer possible. These defects are avoided in the invention. 
In the process embodying the invention, the location of the application can 
also be of decisive importance for the action of the substance to be 
tested. Epithelial cell associations are for example organized to be flat. 
The cells with the apical membrane border a lumen which can contain very 
different substances depending on the organ (for example, bladder--urine, 
intestine--digestive pulp, etc). The cell basal membrane conversely is in 
contact with the blood-like tissue fluid. Depending on the location of the 
application (basal or apical), test substances can have very different 
effects on the cells, at the same time substance gradients in tissue parts 
can greatly influence the intercellular interactions. 
The invention focuses on the fact that toxic actions are triggered not only 
via direct contact of the test substance with a certain cell type, but are 
mainly conveyed via interactions between the different cells of an organ. 
In the process embodying the present invention these interactions are 
fully considered by the use of organ or tissue samples which represent an 
interactive tissue system. 
Organs have a complex structure and they are composed of more than one cell 
type. The cells are present in different stages of differentiation. In 
addition to so-called reserve cells, which are necessary for replacement 
of dead tissue, terminally differentiated cells occur which no longer 
profile, but perform a specific function. With reference to the 
proliferation capacity of the differentiated cells, contact inhibition 
prevails. As long as the tissue association is undamaged, the specific 
extracellular matrix is intact and the cells are linked to one another, 
and they remain in their high differentiation state. Reserve cells are 
only activated to close wounds or for regeneration. The percentage of 
reserve cells capable of proliferation and the lifetime of the terminally 
differentiated cells are organ-specific. While the epithelial cells of the 
skin are replaced within a short time, for the epithelial cells of the 
kidneys have a lifetime of several years. If tissues are cultivated in the 
presence of mitogenic nutrient media, this acts to stimulate the reserve 
cells. Cell proliferation is excited, but not their differentiation. This 
changes the organ-typical ratio of proliferating to differentiated cells, 
and the typical organ function is limited. To approach organ-typical 
conditions as closely as possible in the tissue culture, as embodied in 
the invention, tissue material and a nutrient medium must be used which 
has with a cellular composition which corresponds to the ratios in the 
organ with reference to the degree of differentiation. 
Organ function is determined both by its individual components and also by 
the interaction between the different cell types. Not only cells of the 
same type are in contact with one another, but also the cells of different 
tissue components interact with one another. In addition, not only the 
cellular components, but also the organ-typical extracellular matrix are 
of decisive importance for organ-typical interactions. Only by the 
interaction of the various tissue components does organ function become 
possible. This complexity, which can be of decisive importance for 
determination of the action of the substance, is not achieved by simple 
cell culture systems nor by co-culture systems. 
Therefore, the invention takes into account the circumstance that in the 
organism a substance acts not only on a single cell type, but 
fundamentally on all cells of the body which come into contact with the 
substance or its metabolic products. For the toxicity of a substance 
therefore not only its acute action on an individual cell is decisive. 
Rather, there are intercellular interactions between the different cell 
types which, when influenced by the test substance, are responsible for 
its action in the organism. 
In the process embodied in the invention, through the use of animal tissue 
or samples of human tissue test conditions can be established which enable 
direct applicability of the culture or test results to the conditions in 
an animal and human organism.

DETAILED DESCRIPTION OF THE INVENTION 
Perfusion chamber 1 shown in FIG. 1, includes two housing parts 2 and 3 
which form a housing interior closed to the outside when the housing is in 
the closed position. Between two housing parts 2 and 3 there is a holder 
4, formed by a retaining ring, on which the tissue or organ sample 5 is 
inserted and fixed. In this form globularization of an organ sample 5 is 
prevented. Holder 4, and organ or tissue sample 5, divide the interior of 
chamber 1 into two component spaces 6 and 7, each of which have an inlet 8 
for supplying a nutrient solution or substance to be tested and an outlet 
9, for removing the nutrient solution with the metabolic products, or the 
test substance. For example, the figure has the upper component space 6 is 
filled with the test substance. For this purpose, inlet 8 of this 
component space, is connected via a hose line 10 with a charge, not shown, 
for the test substance. The nutrient solution flows through lower 
component space 7. Inlet 8 there is connected via a hose line 11 and a 
multiple hose pump 12 with a reserve 13 for the nutrient solution. Outlet 
9 of lower component space 7 is connected via a hose line, not shown, to a 
receiving tank for collecting used nutrient solution. In each test, it is 
preferred that several tissue samples 5 are cultivated at the same time 
and are partially exposed to the test substance. 
Because a medium flows continuously especially through lower component 
space 7, metabolites and paracrine factors are continuously removed. 
Furthermore, optimum oxygen supply of respective tissue sample 5 is 
ensured. In a culture medium (nutrient solution) additives are abandoned 
which influence the test result, especially fetal sera (fetal calf serum), 
tissue extracts, etc. which are needed in nutrient solutions for cultures 
in culture dishes and which consist of a plurality of protein, growth 
factors, and other components which are not precisely defined and which 
adversely affect the test result, or which could lead to uncontrollable 
interference with the respective test substance. Use of perfusion culture 
techniques also minimize necroses. 
Use of the perfusion chamber makes it easily possible to allow the 
respective test substance to act selectively basally or apically on tissue 
sample 5. Gaseous, liquid, or viscous substances are possible as the test 
substance. 
As an example, the detection of the toxic action of vitreous body 
replacements in a renal explant culture is reproduced in the following for 
the process as embodied in the invention. 
EXAMPLE 
Vitreous body replacements, as the name indicates, are used for replacement 
of damaged or injured vitreous body in ophthalmology. In this example, two 
of these replacements (the substances perfluorophenathrene and 
perfluorodecaline) were studied. The substances have been tested in a cell 
culture system. This test yielded no indications of toxic effects of the 
vitreous body replacements. A test of the substance perfluorophenathrene 
in a rabbit eye however resulted in extensive changes of the blood 
vascular system in the area of the retina and major damage to the eye. The 
substance perfluorodecaline showed good tolerance in an animal test. No 
damage to the retina or other parts of the eye were observed. 
An interactive tissue culture system was established for testing of 
toxicity. The culture system should provide information on whether the 
action of the substance perfluorophenathrene which changes the vessels can 
be established in vitro. Explants of neonatal rabbit kidneys were chosen 
as the test tissue. Antibodies for detection of vessels in the rabbit 
tissues are available in laboratory. In addition, the vascular system in 
this tissue has an extraordinarily high degree of spacial order. The 
preservation of the structure of the vascular system after application of 
the test substance was the parameter for assessment of the toxic effect in 
this test. 
Material and methods: 
Tissue preparation: One to three day old rabbits were sacrificed by 
cervical dislocation. Organ explants were prepared immediately after 
removal of the kidneys. 
The kidneys of neonatal rabbits are not yet completely developed. In the 
outermost region of the organ directly under the organ capsule, all 
nephron developments stages are found in addition to the embryonal tissue. 
Microsurgical organ explants were prepared from this region. To do this 
the kidneys were cut in half lengthwise. With fine tweezers the thin organ 
capsule was removed. With the organ capsule, the renal tissue still in 
development was detached. Proteases were not used in this preparation 
method, i.e. the organ-typical extracellular matrix and the intercellular 
connections remained intact. The organ explants were then stretched onto 
the retaining ring system. 
Culture of the kidney explants: By using a retaining ring system it was 
possible to use tissue explants which had been obtained by proteolytic 
disintegration for the tests. The tissue fragment was prepared in the 
corresponding size and fixed in the retaining ring. Proteolytic 
disintegration of the tissue, as can be necessary for culture in the 
conventional culture dish applications, was not carried out. The tissue 
fragment stretched onto the retaining ring was brought into contact with 
the tissue top or bottom with the test substance. The tissue top was 
defined as the side covered by the organ capsule. The tissue bottom was 
the tissue side exposed by the preparation. The tissue explants prepared 
in this way were cultivated with continuous throughflow of the medium. Use 
of perfusion culture technique in this case enabled complete abandonment 
of additives of fetal calf serum, adult serum or tissue extracts. The 
medium consists of a commercially available basic medium (Iscove's 
Modified Dulbecco's Medium, IMDM) to which the hormones aldosterone 
(10.sup.-7 M) and 1.25 dihydroxyvitamin D.sub.3 (10.sup.-9 M) have been 
added. In the presence of these hormones the structure of the renal tissue 
is completely preserved. Necroses were not observed even after culture 
times of more than 10 days. 
For the perfusion culture, stretched explant 5 (organ samples) were 
inserted into the gradient chamber so that one upper and one lower 
compartment 6 or 7 are formed which are separated from one another by 
tissue 5. Upper compartment 6 was filled with the given test substance and 
no flow takes place through it in the experiment since the test substance 
was of viscous consistency. Throughflow of the test substance however 
would be possible in the application of liquid or gaseous substances. 
Lower compartment 7 was filled with nutrient solution which was 
continuously pumped through the chamber compartment. Storage bottle 13 of 
the medium was connected via a hose connection 11 to the inlet 8 of lower 
chamber 7. Chamber outflow 9 was connected to the waste bottle. Storage 
bottle 13 was kept at 4.degree. C. during the entire culture time. Supply 
hose 11 was inserted into 12 which ensured constant flushing of chamber 
with medium at 1 ml/h. This perfusion rate was found to be very suitable 
for the culture of renal tissue. Fundamentally other flow rates are 
possible. 
The test substance (substance perfluorophenathrene, substance 
perfluorodecaline) was added to the upper chamber compartment 6 so that 
the tissue fragment 5 was reached on one side by the nutrient solution, 
while opposite tissue side was in direct contact with the test substance. 
The control consisted in that upper chamber compartment 6 was not filled 
with test substance, but with basic medium. 
Another experiment was designed such that in perfusion chamber 1, the test 
substance was placed in the lower chamber compartment 7, while the 
nutrient solution was pumped through the upper compartment 6. A number of 
perfusion chambers were placed on a heat plate with a temperature of 
37.degree. C. and the tissues were cultivated for 24 hours with continuous 
throughflow of medium. 
After culture, the explants were flushed briefly in a buffer solution and 
frozen in liquid nitrogen. 
Optical microscopic evaluation process: Two parameters were used as the 
criteria for assessment of the toxic effect of the respective test 
substance: 
1. The different cell types of an organ are characterized by the expression 
of typical molecules which can be detected using specific antibodies. 
Endothelial cells in the rabbit kidneys likewise bear these molecules. 
To detect endothelial cells, in our experiments antibody EC 1 was used. The 
EC 1 antigen is expressed solely by endothelial cells. If the endothelial 
cells die, the EC 1 antigen can no longer be detected in the tissue. 
2. The developing vascular network of the kidneys is characterized by an 
extremely high degree of spacial order. This characteristic structure is 
observed only in a distinct differentiation state of the tissue. It is an 
expression of the coordinated development of different organ components of 
the kidneys. Preservation of the three-dimensional structure of the 
vascular network in the explant after application of the test substance 
was another criterion for assessment of the toxicity of the substance. 
To detect endothelial antigens in the histological preparation, indirect 
immune peroxidase marking was used. In this process, murine monoclonal 
antibody EC 1 was used as a primary antibody for detection of endothelial 
cells. The antibody bonding was then detected in a second step by marked 
species-specific secondary antibodies. 
The specificity of the antibody reaction was safeguarded by different 
controls. On the one hand control sections which have been treated not 
with the primary antibody but with all other fixing agents, buffers and 
antibody conjugates used were carried along. In addition, in the 
experiments sections with irrelevant primary antibodies of the same class 
or subclass of the specific antibody were incubated. Murine preimmune sera 
were used for control. 
The results for application of the test substances in the lower chamber 
compartment are reproduced. Renal explants which had been cultivated with 
the substance perfluorophenathrene for 24 hours, like the control 
explants, showed optimum preservation of the spacial organization of the 
vascular network. In contrast, the vascular network which was exposed to 
the substance perfluorophenathrene for 24 hours was largely decomposed. 
Individual endothelial cells however were detectable, i.e. expression of 
the EC 1 antigen was not influenced by the incubation of the tissue with 
the substance perfluorophenathrene. The destructive action of substance 
perfluorophenathrene on the structural preservation of the vascular 
network was however unambiguous. In addition, the entire tissue structure 
showed structural changes. The results of the tests are summarized in the 
following table. 
______________________________________ 
Detection of the Toxic Effect of Vitreous Body 
Replacements in the Renal Explant Culture 
Perfluorophenathrene 
Perfluorodecaline 
Control 
______________________________________ 
Expression 
+ + + 
of EC 1 
antigen 
Vascular - + + 
structure 
Tissue changed + + 
structure 
______________________________________ 
Legend: - antigen/structure no longer detectable, + antigen/structure 
clearly detectable 
The test of the vitreous body replacements perfluorophenathrene and 
perfluorodecaline on renal explants, in vitro, convincingly confirmed the 
results which were obtained after application of these substances to the 
rabbit eye. While perfluorodecaline did not cause damage to the retina or 
eye, the vascular system of the retina after application of 
perfluorophenathrene showed significant changes. Also the vascular system 
in the renal explants was already decomposed after 24 hours of incubation 
with perfluorophenathrene. Both the control explants and the explants 
incubated with perfluorodecaline after 24 hours of culture exhibited 
excellent preservation of the vascular system. This result clearly shows 
that interactive tissue culture systems can be successfully used for 
detection of toxic effects. 
In the testing of the substance perfluorophenathrene on tumor cells there 
were no toxic effects of the substance. The importance of the cellular 
interaction for the shaping of the toxic effect of a substance is 
impressively highlighted by this discrepancy. Cell culture systems are 
outstandingly suited for detection of acute toxic effects. They allow 
prompt testing of the direct toxic effect. If the toxicity of a substance 
is however based on cellular interactions, other systems must be used for 
analysis. 
The culture conditions described here by way of example can be varied as 
required. Variation of the flow rates of the medium, culture time 
intervals of varied length, and a different medium composition depending 
on the tissue requirement are possible and can be easily adapted. In 
addition to the teased-out preparations described here, tissue sections or 
organ parts can also be cultured. 
Likewise other methods can be used for evaluation. In addition to 
techniques using transmission and raster electron microscopes, in-situ 
hybridization and biochemical and histochemical detection are conceivable. 
One important simplification of the evaluation can be achieved by 
measurement of molecules in the culture supernatant. For example, 
detection of the increase of apoptotic cells after application of test 
substances could be done via studying of the culture supernatant using 
apoptosis markers and suitable analysis systems. 
For the tests described herein, explants of neonatal rabbit kidneys were 
used. Fundamentally any tissue can be cultivated with this system. In our 
laboratory, initial experiments with tissues from the rabbit stomach 
showed that this tissue can also be cultivated with good success over long 
time intervals. The use of a gradient perfusion chamber described herein, 
for specific apical or basal application of toxic substances, is also 
possible for other tissues (for example, stomach, skin, cornea, retina). 
For the experiments described herein, a culture duration of 24 hours was 
chosen. Shorter or longer culture intervals are possible with this system. 
It was assumed above that the tissue sample is frozen prior to evaluation 
to improve storage. It is also possible to immediately evaluate the tissue 
sample after its cultivation. 
For the evaluation process other techniques are also possible, for example, 
automated test systems, for example, the test system known to one skilled 
in the art under ELISA. Instead of the aforementioned EC 1 antigen, other 
markers can also be used for evaluation, for detection of proliferating 
and/or apoptotic cells. 
Reference Number List 
1 perfusion chamber 
2,3 housing part 
4 holder 
5 tissue or organ sample 
6,7 component space 
8 inlet 
9 outlet 
10,11 hose line 
12 hose pump 
13,14 reserve tank