EP1468285A1

EP1468285A1 - Method for detecting the biocompatibility of polymers

Info

Publication number: EP1468285A1
Application number: EP03702524A
Authority: EP
Inventors: Ulrich Zimmermann
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-01-24
Filing date: 2003-01-24
Publication date: 2004-10-20
Also published as: DE10202710A1; WO2003062820A1

Abstract

Disclosed is a method for detecting the biocompatibility of polymers, according to which the apoptosis rate and mitogenicity of cells are measured in the presence of the polymers.

Description

Method for demonstrating the biocompatibility of polymers

The invention relates to a method for detecting the biocompatibility of polymers. This method is particularly applicable in transplant medicine.

Polymers, especially biopolymers, have become increasingly important in transplantation medicine in recent years. It is known to encapsulate grafts for their immune isolation. Spherical alginate capsules have proven to be advantageous for encapsulating allogeneic or xenogeneic, endocrine tissue (see, for example, T. Zerkon et al. In "Acta Diabetol.", Vol. 29, 1992, pp. 41-52, P. DE Vos et al. in "Biomaterials", vol. 18, 1997, pp. 273-278, C Hasse et al. in "Exp. Clin. Endocrinol. Diabetes", vol. 105, 1997, pp. 52-56 and in " J. Microencapsulation ", Vol. 14, 1997, pp. 617-626).

However, this presupposes that the alginate encapsulation material is absolutely biocompatible in order to avoid rejection reactions. Such reactions include, for example, the typical immune rejection reactions that lead to fibrotic overgrowth of the graft.

The use of unpurified or insufficiently purified alginate materials in the form of alginate capsules leads to differently strong fibrotic reactions in the animal model. The reason for this is, among other things, contamination of alginate batches with mitogenic substances (Zimmermann et al. In "Electrophoresis", Vol. 13, 269-274). DE-OS 198 36 960 describes a process for the production of highly purified alginates. The mitogenicity test (Zimmermann et al. In "Biotechnology", vol. 10, pp. 547-571) showed that there are no detectable mitogenic impurities in the product purified by this method. The traveled The product was tested for tissue tolerance in animal experiments. It has been shown that with these alginates a significantly higher tissue tolerance can be achieved compared to other purified alginates, but depending on the starting material, different, albeit minor, fibrosis symptoms can occur. This means that an itogenicity test alone cannot predict the results in animal experiments, but that other effects that are not due to mitogenic properties are also responsible for this reaction. It is therefore desirable to improve this mitogenicity test, especially with a view to completely avoiding animal testing.

So far it has been indispensable, finally due to legal regulations, that the materials intended for transplantation and implantation are tested for their biocompatibility in animal experiments. It is known that even minor qualitative or quantitative changes in the alginate composition when used in encapsulation technology lead to incompatibilities. As a result, practically every batch has to be tested in animal experiments. However, this inevitably produces a large number of animals that must be sacrificed in animal experiments. In addition, not every animal model is suitable for predicting behavior in the human organism. Special, expensive and difficult to maintain animal models with an overbred immune system are required for this. In addition, animal experiments are highly variable, which means that a batch to be tested must be examined in a large number of animals in order to obtain reliable information about it. To date, there is no alternative to animal testing. However, the goal of science and industry is to replace time-consuming and costly animal experiments. However, this presupposes that special sensitive detection methods are available that can be carried out with as little effort as possible.

It is therefore an object of the present invention to provide a way of simply testing substances and materials for their biocompatibility in order to replace animal experiments and thus to contribute to animal protection.

This object is achieved with the method according to the invention.

The present invention relates to a method for detecting the biocompatibility of polymers by measuring the apoptosis rate of cells and detecting the mitogenicity of the cells in the presence of the polymers.

The method according to the invention is particularly suitable for distinguishing between non-immunologically active polymers (biocompatible) and immunologically active polymers. This means that a simple, combined detection method without animal testing can be used to determine whether or not the polymer to be tested for biocompatibility would lead to rejection reactions. It is therefore no longer necessary to carry out animal experiments and, in the interests of animal protection, no more animals are being killed for biomedical compatibility studies to the previous extent.

The figures serve to explain the invention. Show it: 1 shows a graphical representation for the detection of mitogenicity and FIGS. 2A and 2B recordings of histological sections through the marginal region of a rat kidney

With the method according to the invention, preference is given to biopolymers and synthetic polymers which are suitable for biological purposes, such as transplantations, before their use, e.g. in clinical studies, tested for biocompatibility in vitro. The biopolymers include, for example, polysaccharides and their derivatives. Alginates are very particularly preferred.

According to the invention, the biocompatibility of the polymers is demonstrated on a laboratory scale by measuring the apoptosis rate of cells and demonstrating the mitogenicity of the cells in the presence of the polymers. Eukaryotic cells, for example, can be used for the detection. Suitably, Jurkat T lymphocytes are used in the method according to the invention for measuring the apoptosis rate.

The phenomenon of apoptosis has been studied intensively for many years. Apoptosis, the "programmed cell death", is a natural mechanism for the self-destruction of cells. It works equally well in humans, animals and plants. This suicide program is genetically designed for the organisms and is vital. For example, apoptosis is also very important for the body's defenses. It causes infected or damaged cells to commit suicide, protecting the body. The cells can recognize themselves when they no longer function properly. The basic idea of the present invention is to evaluate the apoptosis rate of cells and the detection of mitogenicity as an indication of whether the polymer to be tested in animal experiments for biocompatibility has immunogenic effects. Apoptosis and mitogenicity are determined in vitro, ie cells are cultivated in the presence of the polymers and the apoptosis rate is determined by measuring the apoptotic cells and the mitogenicity is determined by the stimulation rate of certain cells. If the samples to be examined show an increased mitogenicity and / or an increased apoptosis rate compared to a control sample (cultivation of the cells without the polymer to be tested), it can be assumed that the material to be tested is not biocompatible. Only when both detection methods have no elevated values compared to the control group can it be assumed that the polymer to be tested is biocompatible.

Mitogenicity can be determined using the following procedure. Living cells, such as human lymphocytes (cell lines) or mouse lymphocytes, are kept in culture with the polymers for a few days. If mouse lymphocytes are used, it is necessary to sacrifice animals in this case too, but a spleen preparation is sufficient to carry out several hundred mitogenicity tests. This is in no comparison to the biocompatibility studies in the animal model, in which several animals are required per batch. If the polymers have an immunogenic effect, the cells enlarge and the number of cells increases (in order to clarify this effect, additional stimulants, for example LPS or PHA, can be added to the culture). The cell size and the cell number can be determined with the help of the CASY1 Cell Analyzer (Model TTC, Sharpness Technology, Reutlingen, Germany), which works according to the Coulter principle. Such a This device enables fast, precise detection (size, size distribution and number of cells) of several thousand cells per measurement. No significant cell enlargement and increase in cell number after incubation can be measured with mitogen-free polymers, whereas with mitogenic polymers a significant cell enlargement and an increased cell number can be demonstrated.

FIG. 1 shows the result of this process for different alginate batches. It can be seen that commercially available alginates (top peaks at about 9.5 μm in diameter), in contrast to the control group (bottom peak at about 9.5 μm in diameter), which was not cultivated with the alginate to be tested, significantly increased the number of cells in the Effect area of stimulated cells. This means that they contain a high proportion of mitogenic substances and are therefore not biocompatible. The fact that commercial alginates cause fibrotic reactions in the animal model has been described in numerous publications (Klöck et al. In "Biomaterials", Vol. 18, 1997, pp. 707-713; Klöck et al. In "Applied Microbiology and Biotechnology" , Vol. 40, 1994, pp. 638-643; Zimmermann et al. In "Bio Techniques", Vol. 29, 2000, pp. 564-581). In contrast, alginates A and B, which are highly purified products, show no significant increased mitogenicity compared to the control.

The apoptotic cell rate is measured on a laboratory scale. First, suitable cells, such as human Jurkat T lymphocytes, are grown. The harvested cells are then cultivated in the presence of the polymers. If necessary, a check for cell vitality and cell cycle can then be carried out. Finally, the cells incubated in the presence of the polymers are subjected to apoptosis after a determined period, usually after ten and fifty hours, preferably after 16 and 40 hours.

The measurement of the apoptotic cells can be used by any known method for the determination of apoptotic cells. The measurement based on a fluorescent dye reaction has proven to be particularly suitable.

The fluorescence detection reaction takes advantage of the mitochondral transmembrane potential of the cells. The destruction of the mitochondral transmembrane potential is known to be one of the earliest intracellular events that follows after apoptosis induction. The fluorescence detection method is based on the fact that a distinction is made between healthy and apoptotic cells by detecting the changes in the mitochondrial transmembrane potential. In healthy cells, the fluorescent dye accumulates and aggregates in the mitochondria, resulting in a broad red fluorescence. In apoptotic cells, on the other hand, the fluorescent dye cannot aggregate in the mitochondria, which is due to the changed mitochondral transmembrane potential. Therefore, it remains in the cytoplasm in its monomeric form and fluoresces green.

The fluorescence signals can easily be detected by fluorescence microscopy using an appropriate bandpass filter or in a flow cytometer. Virtually any fluorescent dye suitable for detection can be used as the fluorescent dye. An example of such a dye is 5, 5 ', 6, 6' tetrachloro-1, 1 ', tetraethylbenzimidazolylcarbocyanine iodide. The method according to the invention is meaningful and indicates the incompatibility of the tested material even at low apoptosis rates. The apoptosis rate of the cells that are cultivated with the polymer to be tested is compared with the apoptosis rate of the control cells (same cell lot) that are not cultivated with the polymer to be tested. Normally it can be assumed that an apoptosis rate that is greater than 2% higher is a critical value for the cells cultivated with the polymer in comparison with the cells cultivated without the polymer.

The following table shows a summary of the results with different alginate batches, which were both pre-tested with the method according to the invention and later examined in animal experiments. The animal experiments were carried out as described in DE-OS 198 36 960. Camptothecin, which is an apoptosis-inducing detergent, was used as a positive control.

Camptothecin therefore causes a significantly increased apoptosis rate compared to the control group. The batches of commercial alginates with high mitogenicity and low biocompatibility (swo) show that no mitogenic impurities are found in the substances to be tested with this method; here the apoptosis rates are even slightly below those of the control. Reference is made to the following Table 1. Table 1

In the case of mitogen-free alginates, batch A shows a low apoptosis rate (less than 2% increased compared to the control) in the area of the control and batch B shows a significantly increased rate

Apoptosis rate (more than 2% increased compared to the control). This means that only Charge A is biocompatible and would not cause fibrosis in the animal model.

The statements of this method according to the invention were checked in a sensitive animal model (spontaneously diabetic BB / OK rat). The animal experiments were carried out as described in DE-OS 198 36 960. The results of fibrosis formation are summarized in Table 2 below. This shows the variability of animal experiments. Several animals must be examined in order to obtain reliable, average statements. Table 2

(- = no fibrous, + very light fibrous; +++++ very strong fibrous)

In the fibrosis examinations, the alginates of batch B were found to be very immunogenic on average, since strong fibrosis sometimes appeared in the animals tested. In contrast, only a slight fibrosis occurred in the batch A alginates used.

Figure 2 shows an example of the histological sections on which these data are based. Alginate capsules cross-linked with Ba ^{2+ were} implanted under the kidney capsule of the spontaneously diabetic, immune-sensitive BB / OK rats. After three weeks, the kidneys were explained and histological examinations were performed. FIG. 2A shows a histological section through the edge region of a rat kidney, in which the capsules from the alginate of batch A were implanted. Since the cut was drained for fixation, the alginate is only visible in fragments. However, the space that the capsules have occupied is clearly visible. There is no or only a very slight formation of fibrosis around this room. FIG. 2B (same section preparation as in FIG. 2A) shows a histological section through the edge region of a rat kidney, in which the capsule from the alginate of batch B was implanted. A strong fibrosis is clearly visible around the capsule. Similar and sometimes stronger fibrotic reactions can be observed when looking at implants of capsules from commercial or mitogen-containing alginates (Klöck et al. in "Biomaterials" Vol. 18, 1997, pp. 707-713; Klöck et al. in "Applied Microbiology and Biotechnology", Vol. 40, 1994, S 638-643; Zimmermann et al. In BioTechniques ", Vol. 29, 2000, pp. 564-581).

The investigations shown prove that the method according to the invention reliably predicted the animal experiments. Only the alginates of batch A showed both a low mitogenicity and a low apoptosis rate in the test systems. Therefore a high biocompatibility of these batches can be concluded. This claim is supported by animal experiments.

The method according to the invention is an ideal exclusion method. It is used to check the biocompatibility of polymers and biomaterials without animal testing and to provide or exclude these materials for their use in transplant medicine. This procedure can be carried out quickly and inexpensively on the laboratory measuring stick. It can be easily standardized. These materials can be alginate materials, for example, which are usually used to encase the graft in a capsule. Furthermore, other polymer materials can also be tested in advance for this purpose.

In the sense of the method according to the invention, there is no restriction with regard to the order of the two verifications. For cost reasons, it makes sense to carry out the mitogenicity test first. If the polymers already show increased mitogenicity, then the more expensive apoptosis test could be dispensed with. In principle, it would also be possible to carry out both tests with the same cells. However, there are demonstrably clearer results in the mitogenicity test when mouse lymphocytes are used.

The following examples serve to explain the method according to the invention.

Examples

example 1

Cell and culture conditions

Human Jurkat T lymphocytes (ACC 282, DSMZ, Heidelberg) were cultured in T25 or T75 culture bottles (TPP, Switzerland) in RPMI 1640 complete growth edium (CGM) with phenol red (5 mg / ml). The medium was supplemented with fetal calf serum (FCS, 10%), 2mM L-glutamine, 100 U / ml penicillin streptomycin (all PAA, Linz, Austria). The cultivation was carried out at 37 ° C, enriched with 5% CO ₂ . The suspension cells were regularly passaged into new T75 culture bottles with fresh CGM every 2 to 3 days.

Example 2

cell stimulation

Jurkat cells were harvested in the exponential growth phase and centrifuged (10 min, 180 xg). A living cell number of 0.17 x 10 ⁶ / ml was then set (living cell number at the beginning of the exponential growth phase). For alginate stimulation, alginate-containing CGM- Solutions (final concentration 0.1%) prepared (alginate stock solutions: 0.5% in 0.9% NaCl solution). 12 ml of cell suspension were prepared per batch and divided into three 4 ml wells on a 12-hole test plate (TPP) (n = 3). The negative control was recorded in normal CGM. Camptothecin (0.75 μg / ml cell suspension; stock solution 5 mg / ml in 1 N NaOH, stored at 4 ° C), a topoisomerase I inhibitor and thus detergent that triggers apoptosis was added to the positive control (Amstad et al., 2000; Bortner et al., 1995). The cells stimulated in this way were then incubated at 37 ° C., enriched with 5% CO ₂ .

Example 3

Cell vitality and cell cycle

Before the start of the experiment (day 0), after 16 h (day 1) and 40 h (day 2), the number of cells and vitality were determined using the trypan blue stain (biochrom, Berlin, Germany) with a Neubauer chamber. In addition, a cell cycle determination was carried out before starting the experiments (day 0) and in the case of the negative control on days 1 and 2. 500 μl of the non-stimulated cell suspension with 0.2% saponin (stock solution 5% in aqua dest) (Sigma, Deisenhofen, Germany) for permeabilization of the membrane and 25 μg / ml propidium iodide for staining the DNA (PJ, Stock solution 1 mg / ml in aqua dest., Stored at 4 ° C) (Sigma) added. After a one minute incubation, the measurement was carried out at flow cytometer (488 nm argon laser, Coulter EPICS ^® ^® XL, Coulter, Krefeld, Germany). The DNA content was determined by measuring the PJ emission (10,000 events / sample) (Djuzenova et al., 1994). The data were analyzed using the System II ™ Version 3.0 software programs (Coulter) or WinMDI (Windows Multiple Cocument Interface Version 2.8).

The intactness and vitality of the cells were checked before and during the experiment by the staining methods.

Example 4

Fluorescence labeling and measurement of apoptotic cells

Cells preincubated with alginate (0.1% in CGM), the positive (without alginate in CGM with camptothecin) and negative control (without alginate in CGM) were examined for apoptosis on days 1 and 2. 2 ml of cell suspension were removed from each sample and centrifuged (10 min, 180 xg). After discarding the supernatant, the cells were treated with a lipophilic, cationic fluorochrome (5, 5 ', 6, 6' tetrachloro-1, 1 ', 3, 3' - tetraethylbenzimidazolylcarbocyanine iodide [JC-1]; (staining solution: 0, 5 μl / ml in incubation buffer) (test kit MitoCapture Mitochondrial Apoptosis Detection Kit, BioCat, Heidelberg, Germany) for 20 min at 37 ° C, 5% C0 ₂ (1 x 10 ⁶ cells / ml staining solution) Incubation was centrifuged again, the supernatant discarded, the cell pellets taken up in 1 ml PBS (PAA) and resuspended, and the fluorescence-labeled cells were measured immediately afterwards on a flow cytometer (Coulter) (10,000 events / sample) Fluorescence shift from red to green to (Kroemer and Reed, 2000, Pigue et al., 2000; Samali et al., 1999) This effect was based on the change in the (trans) membrane potential of mitochondria (ΔΨ _m , i-tochondrial membrane potential MMP) (Kroemer et al. , 1998; Bernardi et al. , 1999; Petit et al., 2001). In intact cells, the accumulation and aggregation of the fluorochrome led to the Mitochondria show a broad red fluorescence (absorbance 488 nm, emission 590 ± 42 nm, similar to PJ). Apoptotic cells no longer had this property due to a decreasing ΔΨ _m , so that monomer could no longer aggregate and therefore emitted in the green area (530 ± 30 nm, similar to FITC). The fluorescence for apoptotic cells was therefore measured via the green (FL1, such as FITC), for healthy cells via the red fluorescence channel (FL3, such as PJ). A distinction was also made using fluorescence microscopy.

Example 5

Mitogenitätsbestimmung

A mouse lymphocyte suspension (1 ^x 10 ⁶ cells / ml in complete growth medium, the same concentration is selected when using human lymphocytes) was added to the alginate solution to be tested (final concentration: 0.05%) and 3 days at 5% CO ₂ and 37 ° C cultivated. An aliquot (100 μl) of the well resuspended lymphocyte suspension, which had been centrifuged off and taken up in inositol (or PBS), was washed twice in isoosmolar inositol solution (290 mOsm / kg) and taken up in 10 ml PBS (phosphate-buffered saline solution) and immediately in CASY 1 cell Analyzer (Model TTC, Sch Sharpness Technology, Reutlingen, Germany) measured. The proportion of large, stimulated cells in the total population can be measured from the corresponding histograms.

Claims

1. Method for detecting the biocompatibility of polymers by measuring the apoptosis rate of cells and detecting the mitogenicity of the cells in the presence of the polymers.

2. The method according to claim 1, characterized in that the biocompatibility of biopolymers and synthetic polymers is detected.

3. The method according to claim 2, characterized in that the biopolymers include polysaccharides and their derivatives.

4. The method according to claim 3, characterized in that the polysaccharides are alginates.

5. The method according to at least one of the preceding claims, characterized in that eukaryotic cells are used as cells.

6. The method according to claim 5, characterized in that eukaryotic cells are human Jurkat T lymphocytes.

7. The method according to at least one of the preceding claims, characterized in that the apoptosis rate is determined by measuring the apoptotic cells.

8. The method according to claim 7, characterized in that the measurement is carried out with a fluorescent dye reaction.

9. The method according to claim 8, characterized in that 5, 5 '6, 6' tetrachloro-1, V, 3, 3 '- tetraethylbenzimidazolylcarbocyanine iodide is used as the fluorescent dye.

10. The method according to at least one of the preceding claims, characterized in that the apoptosis rate of the cells is at least 2% higher than that of the control group not cultured with the polymer to be tested.

11. The method according to at least one of claims 1 to 10, characterized in that the detection of the mitogenicity of cells is carried out by measuring the stimulation rate.

12. The method according to claim 11, characterized in that the stimulation of the cells used by mitogenic substances takes place by detecting the increased metabolic activity.

13. The method according to claim 12, characterized in that the stimulation of the cells used by mitogenic substances by detecting cell enlargement and increasing the number of cells.

14. The method according to at least one of the preceding claims for the biocompatibility detection of polymer materials in transplantation medicine.