EP0815211A1

EP0815211A1 - New defined enzyme mixtures for obtaining cells and treating wounds

Info

Publication number: EP0815211A1
Application number: EP96907436A
Authority: EP
Inventors: Claus Otto Markert; Hans Thom; Jürgen Weymann; Wolfgang Zahn
Original assignee: Knoll GmbH
Current assignee: Abbott GmbH and Co KG
Priority date: 1995-03-16
Filing date: 1996-03-12
Publication date: 1998-01-07
Also published as: WO1996028543A1; CA2213723A1; EA000583B1; US6146626A; MX9706725A; IL117479A0; NO974260D0; JPH11501517A; IL117479A; HUP9901188A2; CZ280397A3; NO974260L; AU5106696A; BR9607143A; EA199700241A1; AU702514B2; PL322271A1

Abstract

The invention concerns the use of defined mixtures of purified enzymes obtained from Clostridium histolyticum and intended for use in the reproducible, standardised extraction of cells or tissue fragments from human or animal tissues. The invention also concerns the enzymes in question and mixtures thereof, and the direct or indirect medical use of these enzymes, either on their own or as components in mixtures, e.g. for treating wounds.

Description

New, defined enzyme mixtures for cell extraction and wound treatment

description

The present invention relates to the use of defined mixtures of purified enzymes from Clostridium histolyticum for the reproducible, standardized extraction of cells or tissue fragments from human or animal tissue and the use of these purified enzymes for wound treatment.

Methods for isolating cells from tissues should be reproducible and guarantee the least possible damage to the cells obtained. For this purpose, preparations from Clostridium histolyticum containing collagenase, which are composed in an undefined manner, have generally been used so far, with which these goals cannot be achieved with certainty. The use of other enzyme preparations, or non-enzymatic methods, is not common or only provides poor isolation results (1), (9).

The normally used and recommended collagenase-containing preparations (2) are obtained from culture filtrates of the bacterial strain Clostridium histolyticum and, in addition to various collagenases and proteases (3a), (3b), also contain cleavage products of these enzymes formed by proteolysis, as well as others, some of which are harmful acting and unknown components.

With a single enzyme from Clostridium histolyticum, it is currently not possible to obtain viable cells in good yield. Rather, different enzymes from this bacterium have to work together in order to achieve effective tissue breakdown. However, it has not yet been known which ratio of enzymes is required. So far, defined mixtures of enzymes from Clostridium histolyticum have not been available to the user.

Based on the problems presented, various working groups have attempted to use purified enzymes for the isolation of cells from tissues. However, uniform enzymes and therefore no clearly defined hybrids were never used.

By Suggs et al. (4) Human vein endothelial cells were purified using a mixture consisting of a

Collagenase fraction and purified trypsin from bovine pancreas, isolated. The collagenase fraction used was enriched However, 2 was not defined in terms of its content of various enzymes and also contained, for example, small amounts of clostripain. The success of the isolation was not significantly different from that which was obtained when using undefined compositions containing collagenase. Satisfactory tissue disintegration could not be achieved with individual components of the mixture. However, the use of trypsin for cell isolation is not without problems, since this proteolytic enzyme attacks membrane proteins. For example, insulin binding to liver membranes and adipocytes is negatively influenced (5).

Hefley (6), (7) used mixtures of purified collagenase fractions to isolate bone cells from the skull cap of mice (6). Earlier (7) the author described that a mixture of a purified collagenase fraction can be used together with a neutral protease to successfully isolate these cells. In both studies, however, eluates from column fractionations were used, whose content of various collagenases, which differ in their substrate specificity, and of other components was not known.

By Wolters et al. (8) Mixtures of neutral protease and "collagenase type VII * from Sigma were used to isolate islet cells from rat pancreas, but it is not known what types and amounts of different collagenases they contained Content of clostripain and "unspecific protease *. The neutral protease was used in a highly purified form. Mixtures of the two components mentioned gave good yields on viable islet cells.

The invention relates to the individual enzymes collagenase HP, collagenase AZ and elastase, in high purity and mixtures thereof.

Collagenase HP has a specific activity of at least 20 U / mg, preferably at least 50 U / mg in the Graßmann and Nordwig (11) test with the synthetic hexapeptide Z-Gly-Pro-Gly-Gly-Pro-Ala as substrate. For pharmaceutical purposes, it preferably has a specific activity of 100 U / mg or more.

Collagenase AZ has a specific activity of at least 10 U / mg, preferably at least 30 U / mg in the test according to Mandl et al. (12) using azocoll as substrate. For phar a- for therapeutic purposes, it preferably has a specific activity of 50 U / mg or more.

Elastase has a specific activity of at least 2 U / mg, preferably at least 5 U / mg in the test with elastin from the neck band of the cattle as a substrate. For pharmaceutical purposes, it preferably has a specific activity of 12 U / mg or more.

The invention further relates to the use of a mixture of collagenase HP and elastase, optionally with the addition of collagenase AZ and / or clostripain, for the isolation of cells or tissue fragments from human or animal tissue.

The invention further relates to the direct or indirect use of these enzymes, alone or as a component of mixtures, for medical applications, e.g. in wound treatment.

For use in tissue disintegration, the mixtures can, for example, be packaged in vials in lyophilized form in such a way that they are sufficient for disintegration from an individual rat liver (approx. 9-11 g wet weight of the organ).

Suitable mixtures are those which consist of at least two of the purified enzymes collagenase HP (50-300 U; preferably 70-170 U) and elastase (5-70 U, preferably 10-25 U) and optionally an addition of collagenase AZ (1-20, preferably 2-8 U) and / or clostripain (10-280 U, preferably 20-50 U) contain. The numbers given indicate the amounts in one unit of the mixture - for example in a vial - available.

The purity criterion for the enzymes used is their respective specific activity and their uniformity detection in the electrophoretic methods commonly used for this purpose (SDS gel electrophoresis, isoelectric focusing and electrophoresis on agarose gel). The specific activity of the purified enzymes is up to 100 times higher than that of the starting material.

Purified enzymes with the following specific activities are used for the preparation of the mixtures: collagenase HP with a specific activity of at least 20 U / mg, elastase with at least 2 U / mg, collagenase AZ with at least 10 U / mg and clostripain with at least 10 U / mg. 4

The mixtures are distinguished by the fact that, owing to their defined composition of synergistic collagenolytic, as well as elastinolytic and proteolytic enzymes, they are particularly suitable for a gentle and effective isolation of cells or tissue fragments from animal and human tissues.

A major advantage is that a time-consuming and costly inspection of batches is no longer necessary, since the production of purified enzymes is based on known properties. This eliminates the workload that is necessary for the functional characterization of the cells, or at least it is reduced. For these reasons, the number of animal experiments can be reduced in many areas.

The use of less well-purified enzymes, or the use of enzymes with a lower specific activity, generally leads to poorer cell yields in the abovementioned applications, and is furthermore of the usual problems of the lack of reproducibility of the isolation success, a lack of batch constancy and the presence of unknown, possibly negative components.

Many of the experimental results described in the literature are difficult to interpret because of the degradation of the enzymes by accompanying proteases that occurs during production, purification or characterization. The clarification of the contribution of the individual enzymes to the success of the experiment in cell isolation was also made more difficult by the fact that the fragments formed from the collagenases, the elastase and the proteases partially retain their enzymatic activity. To what extent the substrate specificities change has not yet been clarified. Even in many commercial preparations containing collagenase, sometimes only degradation products of the original collagenases are contained. According to the prior art, it was therefore not possible to standardize collagenase-containing preparations clearly either for the preparation manufacturers or for the users of cell isolation methods.

Because of their broad tissue-degrading activity, all human and animal tissues and cells, preferably tissue fragments and / or cells, are suitable for the use of the mixtures of purified enzymes according to the invention:

the biliary tract, the blood system, glands, the vascular system, the brain, the skin, the heart, the intes inu, Langerhans' islets, the liver, the lungs, the stomach, the spleen, muscles, the Umbilical cord, nerves, the kidney, the pancreas, the spinal cord, the thyroid, the terminal ileum, tumor tissue, the uterus, the digestive tract and the tongue.

The cells or tissue fragments isolated with the aid of the mixtures described are particularly suitable for use in cell and tissue transplantation, as well as in gene therapy (e.g. islets of Langerhans, islet cells, hepatocytes, tumor cells, adipocytes), in immunotherapy or in Wound treatment.

Of particular importance is the use of the hybrids according to the invention in the isolation of hepatocytes, Langerhans's islet cells, endothelial cells, epithelial cells, adipocytes, oocytes and tumor cells. In these areas of application, standardization and improvement of the methodology are particularly advantageous.

For example, a well-defined mixture of 2 collagenases with different substrate specificity and one elastase has proven to be ideally suited for the isolation of hepatocytes from rat liver (application examples A, D, E) and human liver (application example F) from bile duct epithelial cells Rat livers (application example G), endothelial cells from human umbilical cords (application examples H), and for the isolation of tumor cells from human tumors (application example I) and islet cells from porcine pancreas (application example J).

Compared to the best undefined composite collagenase-containing enzyme preparations, better or at least equally good isolation success is achieved.

The mixtures according to the invention can be used as a substitute for all commonly used preparations containing collagenase, since they have the components necessary for tissue disintegration. The disadvantages of the previously used collagen-containing preparations are, inter alia, avoided due to the constant composition of the mixtures.

An important area of application for the mixtures mentioned is the reproducible, standardized extraction of hepatocytes from the liver according to the recognized method of Berry and Friend (9), (10), which previously worked with undefined composite collagen-containing enzyme preparations, and the use of the mixtures mentioned according to the prior art 6 better extraction of islet cells in intact form from pancreatic tissue.

Another main area of application is wound treatment. For this, the enzymes are used in the highest possible purity.

I. Preparation and characterization of the enzymes from Clostridium histolyticum

1. Collagenase HP

a. Manufacturing

Fractional protein precipitation with ammonium sulfate

All enzyme purification operations were carried out at 4-8 ° C.

200 g of raw collagenase were dissolved in 3 L of water and 680 g of powdered ammonium sulfate were initially introduced into the solution obtained. The precipitated protein precipitate AI was separated by centrifugation; a further 420 g of ammonium sulfate were added to the supernatant. The protein fraction A2 precipitated was spun off, the precipitate was dissolved in 1 L of water and dialyzed against water. The mixture was then concentrated to about 100 mL via an ultrafiltration membrane (cutoff limit 10,000 Da). The concentrate was then lyophilized. The powdered protein fraction A2 obtained in this way contained collagenase HP and collagenase AZ as main components.

Chromatographic separation of HP and collagenase AZ using metal chelate affinity chromatography

Carried out the chromatographic separation of collagenase HP and collagenase AZ of Zn ²⁺ -beladener Chelating Sepharose 6B. For this purpose, this material was packed into a column (5 x 100 cm) at a layer height of 70 cm and equilibrated with starting buffer (500 mM sodium acetate + 20 mM calcium acetate, pH 8.0). Then about 1.2 g of the protein fraction A2, dissolved in 15 ml of starting buffer, the pH of which had been corrected to 8.4 with TRIS [tris (hydroxymethyl) aminomethane], were applied to the column and various constituents were added to the column the protein fraction A2 washed from the column. In the subsequent elution with buffer A (500 mM sodium acetate + 20 mM calcium acetate, corrected to pH 6.3 with acetic acid), first collagenase AZ and then collagenase HP were collected in separate fractions. The two separate enzyme fractions of collagenase HP and collagenase AZ were concentrated to 50-100 ml with the aid of an ultrafiltration membrane (cutoff limit 10,000 Da), then dialyzed against water and again through an ultrafiltration membrane (cutoff limit 10,000 Da) to approx. 10 liters concentrated.

Fine cleaning of collagenase HP

The fine purification of collagenase HP was carried out on the anion exchanger Mono Q (HR 10/10, Pharmacia) using the FPLC device from Pharmacia. For this purpose, a mixture of 1 ml of buffer B and 2 ml of the fraction from the previous chromatography step containing collagenase HP was applied to the column, which had been equilibrated with buffer B (20 mM TRIS / HCl pH 7.5).

After washing with buffer B, the collagenase HP was eluted from the column with buffer C (20 mM TRIS / HCl + 200 mM NaCl, pH 7.5) in purified form.

b. characteristics

The collagenase HP got its name * HP "because of its property to break down the hexapeptide Z-Gly-Pro-Gly-Gly-Pro-Ala particularly well. It is therefore suitable for a specific detection of the activity. Collagenase HP is characterized in that it can only convert denatured collagens such as gelatin or azocoll to a small extent, but it attacks bovine tendon collagen and cleaves the synthetic peptides mentioned in Example 2b between glycine and glycine or between any amino acid and glycine with high conversion.

It should be emphasized that collagenase HP together with collagenase AZ (vice-versa, i.e. also any mixture of at least collagenase HP and AZ) has an over-additive effect on the degradation of native collagen. This synergistic effect in vitro is also important when used for tissue integration (e.g. application example G).

The specific activity of collagenase HP is a maximum of 146 U / mg in the test with the synthetic substrate Z-Gly-Pro-Gly-Gly-Pro-Ala (11). This corresponds to an approximately 100-fold increase in their specific activity compared to the starting material. The collagenase HP purified in this way forms only a single band both in SDS gel electrophoresis and in isoelectric focusing and electrophoresis on agarose gel.

Its molecular weight determined by SDS electrophoresis is 106,000 Da. Their isoelectric point is at pH 5.8-6.0.

2. Collagenase AZ

a. Manufacturing

All enzyme purification operations were carried out at 4-8 ° C. The first two steps in the purification of collagenase AZ (fractional protein precipitation and metal chelate affinity chromatography) are described under 1.

Fine cleaning of collagenase AZ

The fine purification of collagenase AZ was carried out on the anion exchanger Mono Q (HR 10/10, Pharmacia) and with the aid of the FPLC device from Pharmacia. For this purpose, a mixture of 1 ml of buffer D and 1 ml of the fraction containing collagenase AZ (from the metal chelate described above) was added to the column, which had been equilibrated with buffer D (20 mM calcium acetate, pH 7.2). Affinity chromatography).

After washing with buffer D, collagenase AZ was eluted from the column with buffer E (20 mM calcium acetate, pH 5.0).

b. characteristics

The collagenase AZ received its designation * AZ "due to its property of degrading the substrate azocolla particularly well. It is characterized in that it converts denatured collagens such as gelatin or azocolla, but also bovine tendon collagen well. However, it is capable of small synthetic peptides such as 2 -Furanacryloyl-Leu-Gly-Pro-Ala, 4-phenyl-azobenzyloxy-carbonyl-Pro Leu-Gly-Pro-Arg and Z-Gly-Pro-Gly-Gly-Pro-Ala.

The specific activity of collagenase AZ is a maximum of 82 U / mg in that of Mandl et al. (12) developed test using Azokoll as substrate. This corresponds to an approximately 80-fold increase in their specific activity compared to the starting material. The collagenase AZ purified in this way forms only a single band both in SDS gel electrophoresis and in isoelectric focusing and electrophoresis on agarose gel. Its molecular weight determined by SDS gel electrophoresis is 111,000 Da. Their isoelectric point is at pH 5.9-6.1.

3. Elastase

a. Manufacturing

All enzyme purification operations were carried out at 4-8 ° C.

The first cleaning step of elastase was carried out by protein precipitation with ammonium sulfate, as described under l.a. described.

The protein precipitate AI is dissolved in 1 L of water, concentrated to about 170 mL via an ultrafiltration membrane (cutoff limit 10,000 Da) and dialyzed against a 0.1 mM calcium acetate solution. The enzyme solution was then again concentrated over an ultrafiltration membrane (cut-off limit 10,000 Da) to approximately 50 mL and then lyophilized.

The fine purification was carried out by gel chromatography on SEPHADEX G100 or G200 (Pharmacia).

b. characteristics

The elastase is characterized in that it can break down elastin with high conversion. Their specific activity (see 3.c.) was 18 U / mg. This corresponds to an approximately 80-fold increase in their specific activity compared to the starting material. The elastase purified in this way forms only a single band in SDS gel electrophoresis as well as in isoelectric focusing and electrophoresis on agarose gel.

The molecular weight of the elastase determined by SDS electrophoresis is 35,000 Da. c. Activity determination

The enzyme activity was determined with the aid of the substrate elastin.

20 mg of finely powdered elastin from the neck of the cattle (Sigma) were pre-shaken in 0.4 mL buffer (50 mM TRIS / HCl + 10 M calcium acetate, pH 7.2) in a water bath at 37 ° C with shaking. incubated. The reaction was then started by adding elastase, dissolved in 0.1 ml of buffer, and shaken for a further 10 min at 37 ° C. (lifting height 25 mm, frequency 150 min − ¹ ). Then 3.5 ml of ice-cold water were added and the unreacted elastin was immediately removed via a filter. The absorbance E of the filtrate was measured spectrophotometrically at a wavelength of 280 nm. The extinction value E _B »obtained in a similar test was used as the blank value, in which the elastase solution was only added to the mixture after the elastin had been removed. The absorbance E _{B of} this filtrate was then also determined at 280 nm.

The elastase activity is expressed in units [U]. The enzyme activity which dissolves 1 mg elastin per minute under the given test conditions is defined as 1 U. The amount of elastin dissolved in the filtrate of the test batch was determined by measuring the absorbance at 280 nm.

The specific activity was calculated using the following formula:

° Δ E x F x test volume x dilution factor

U / mg = min x mg elastase in the test mixture

ΔE = E - EB 5 F = 1.376

The factor F is determined by completely dissolving 10 mg elastin of a certain batch with the aid of elastase and measuring the corresponding extinction difference ΔE. The multiplication of this factor F by the extinction difference ΔE gives the dissolved milligram amount of elastin per mL test batch.

5 4. Clostripain

a. Manufacturing

Clostripain was isolated by the method described by Ullmann and Jakubke (14).

Their specific enzyme activity was determined with the aid of the synthetic substrate α-N-benzoyl-L-arginine-ethyl ester (= BAEE) after activating the for 3 hours beforehand

Clostripain solution with 2 mM 1,4-dithioerythritol solution (15). It was around 83 U / mg.

b. characteristics

The thiol protease clostripain is characterized in that it cleaves specifically behind the amino acid L-arginine in polypeptide chains and in synthetically produced substrates.

Its molecular weight determined by SDS electrophoresis is 55,000 Da.

II. Examples of use

The following examples A-J exemplify the special one

Suitability of some mixtures of the purified enzymes for the isolation of cells and tissue fragments from animal and human tissue. Examples K and L show an example of the suitability of the purified enzymes for use in wound treatment.

The invention is not restricted to these application examples.

Example A

Isolation of hepatocytes in the liver with mixtures of three enzymes

Hepatocytes were isolated using the standard Berry and Friend method (9) and the modification by Seglen (16). Wistar rats (200-280 g) were anesthetized by ip injection of nembutal (35 mg pentobarbital / kg body weight). After opening the abdominal cavity, the vena portae was cannulated, and the following carbogen-gassed solutions with a pH value of 7.4 ± were subjected to constant hydrostatic pressure (12 cm water column, variable flow rate) 0.05 infused at a temperature of 36-36.8 ° C after opening of the inferior vena cava:

Perfusion:

1. 5 min approx. 100 mL cell buffer without Ca ²⁺

2.5 minutes approx. 100 mL cell buffer without Ca ²⁺ with EGTA (0.42mM)

3. 8 min approx. 200 mL cell buffer without Ca ²⁺

4. 5-30 min approx. 100-600 mL cell buffer in which the lyophilized mixtures of the purified enzymes had been dissolved.

Cell buffer:

120.0 mM NaCl 1.29 mM CaCl ₂

5.50 mM D (+) - glucose 1.19 mM KH ₂ P0 ₄ 4.81 mM KC1 1.20 mM MgS0 15.0 mM NaHC0 ₃ 10.0 mM HEPES

Carbogen: gas mixture of 95% 0 ₂ and 5% C0 ₂ (v / v)

The enzyme mixture was a lyophilizate of 130 U collagenase HP, 5 U collagenase AZ and 21 U elastase, which was dissolved in 87.5 mL cell buffer.

Perfusion was stopped when the liver tissue softened. After the glisson's capsule had been detached, the hepatocytes were shaken out of the liver tissue and filtered through a sieve (100 μm mesh size). After the filtration, the cells were shaken under a carbogen atmosphere in a water bath for 20 min at 37 ° C. Intact hepatocytes were enriched by three 2-minute centrifugation processes in cell buffer at 50 times the gravitational acceleration. The supernatant, which predominantly contains dead cells, was discarded after each centrifugation. The proportion of vital hepatocytes, hepatocyte-cell pairs or multicellular aggregates from hepatocytes was determined microscopically after resuspension of the cell precipitate obtained in a Burker count (staining with 0.08% trypan blue, for 2 minutes). Result

In a test series (n = 4), cell suspensions were obtained using the enzyme mixture described, which contained an average of 88.7% vital cells with a yield of 360 × 10 ⁶ cells per liver.

Comparative experiments with a collagenase-containing preparation of an undefined composition with particularly high specific collagenolytic activity led to severe cell damage and only to 72.5% vital cells (n = 4).

Example B

Isolation of hepatocytes from the liver with a mixture of two enzymes

The procedure was analogous to Example A, but using the following enzyme mixture: 60 U collagenase HP and 3 U elastase. Cell suspensions were obtained, which were average

90.5% vital cells with a yield of 263 × 10 ⁶ cells (n = 2).

Example C

Isolation of hepatocytes from the liver with a hybrid of four enzymes

The procedure was analogous to Example A, but using the following enzyme mixture: 30 U collagenase HP, 5 U collagenase AZ, 3 U elastase and 16 U clostripain.

Cell suspensions were obtained which contained an average of 83% vital cells with a yield of 232 x 10 ⁶ cells (n = 2).

Example D

Comprehensive validation of the isolation success of hepatocytes from the rat liver with a mixture of three enzymes in 4 different laboratories in comparison to collagenase-containing preparations of an undefined composition

Small, but possibly important for the isolation success of hepatocytes, differences are known in the method of cell isolation from laboratory to laboratory. In order to determine the effectiveness of the mixture mentioned in Example A, regardless of the method used To check the cell isolation, hepatocyte isolations were carried out in large numbers in 4 different laboratories.

The enzyme mixture used was a lyophilisate of 130 U collagenase HP, 5 U collagenase AZ and 21 U elastase, which was in 87.5 ml cell buffer (analogous to example A).

The success of the isolation of hepatocytes was measured using the following five parameters: 1. microscopic determination of the proportion of vital hepatocytes (in% of the total number of hepatocytes in the cell suspension obtained by means of a trypan blue exclusion test), 2. determination of the total number of isolated hepatocytes, 3. determination measurement of the total number of hepatocytes isolated per gram of animal weight, 4. determination of the proportion of single cells (in% based on all forms of aggression in the cell suspension obtained, ie compared to two and multiple aggregates of hepatocytes), and 5. recording of the success rate Tissue disintegration necessary perfusion time with the collagenase solution.

In these tests, the mixtures of the enzymes according to the invention showed results which were much more reproducible than conventional preparations.

Example E

Isolation of hepatocytes from rat liver with a mixture of 3 enzymes: maintenance of cell function

In order to prove not only the suitability of the mixtures according to the invention with regard to the simple isolation success of hepatocytes, but also with special consideration of the cell function, comparative cell isolations were made using a collagenase-containing preparation of an undefined composition which is particularly suitable for this purpose performed on rat livers.

Hepatocytes were isolated using the standard Berry and Friend method (9) and the modification by Seglen (16).

The enzyme mixture used was a lyophilizate of 130 U.

Collagenase HP, 5 U collagenase AZ and 21 U elastase, which was dissolved in 87.5 mL cell buffer to isolate hepatocytes from rat livers (analogously to Example A). The simple isolation success of rat hepatocytes was determined according to the following parameters: proportion of vital cells after the trypan blue exclusion test, cell yield per g of liver, proportion of single cells (in%).

The quality of the cell suspension obtained was further characterized with regard to the function of the hepatocytes (21, 22) by the following parameters: ATP content, energy charge (EC), lidocaine metabolism to monoethylglycine xylidide (MEGX), and absorption of cholyItaurin ((3α, 7α, 12α-trihydroxy-5ß-cholan-24-oyD-2-aminoethanesulfonic acid) at a substrate concentration of 21 μ-M.

For all parameters examined, there were no significant differences between the two collagenase-containing preparations.

It could thus be shown that the hepatocytes isolated with an enzyme mixture according to the invention not only after assessment by the simple isolation success, but also after evaluation of various cell functions, e.g. certain functions of differentiated mass transport, or certain metabolic performances of cytochrome P450-dependent enzymes of the hepatocytes, are completely intact.

Example F

Isolation of hepatocytes from human liver with a mixture of 3 enzymes

Hepatocytes were isolated by the Berry and Friend method (9) and the modifications by Seglen (16) using a Biopsy perfusion technique (21).

The enzyme mixture used was a lyophilizate of 130 U collagenase HP, 5 U collagenase AZ and 21 U elastase, which was dissolved in 50 mL cell buffer.

The success of the isolation of human hepatocytes was determined according to the following parameters: proportion of vital cells after the trypan blue exclusion test and cell yield per g of liver.

It was found that hepatocytes from Hu an liver can also be isolated successfully and with constant results (proportion of vital cells 85-90%) with the mixtures according to the invention. Example G

Isolation of biliary epithelial cells from the rat liver

In a series of experiments (n = 4), biliary epithelial cells from the rat liver were isolated according to the method described in reference (18). In a first step of tissue integration, hepatocytes were first removed enzymatically from the tissue, and in a second step biliary epithelial cells were isolated from the remaining tissue with trypsin.

The enzyme mixture for removing the hepatocytes was a lyophilizate of 130 U collagenase HP, 5 U collagenase AZ and 21 U elastase, which was dissolved in 87.5 mL cell buffer.

The proportion of vital epithelial cells was over 95% in all preparations, the cell yield was 4-6 x IO ⁶ cells per liver (n = 4). The use of the enzyme mixture described made it extremely easy to isolate biliary epithelial cells from the remaining vascular system using trypsin, since the residual tissue obtained after the first step was practically free of hepatocytes.

The problem with the methods used up to now for obtaining biliary epithelial cells was that the hepatocytes remaining in the cell suspension, as well as von Kupffer cells and other cell types, were generally difficult to separate from the epithelial cells to be isolated.

By using the mixture of pure enzymes described, a clear time and cost saving can be achieved, since compared to the previously used method there is a clearly improved, practically complete disintegration of the liver tissue. It is thus possible to remove the contaminating hepatocytes, which are usually present in large numbers.

Example H

Isolation of endothelial cells from human umbilical cord with a mixture of 3 enzymes

Human umbilical vein endothelial cells were isolated using the method of Jaffe (17). For this purpose, the umbilical cords (20 - 30 cm long) were halved, filled in pairs with the respective enzyme solution and incubated for 15 min at 37 ° C and 5% CO ₂ in the incubator. The detached cells were removed and kept in primary culture for evaluation. The yield of living and Divisible cells were determined after washing the non-adherent cells after four days in culture.

The enzyme mixture was a lyophilizate of 130 U collagenase HP, 5 U collagenase AZ and 21 U elastase, which was dissolved in 87.5 mL cell isolation buffer.

The day after the cells are sown, the non-adherent cells (erythrocytes, macrophages, etc.) are washed out. The medium is changed on the fourth day. Confluence is usually reached no later than the eighth day after sowing (> 10 ⁵ cells / cm ² ). The confluent cell lawn consists of over 95% endothelial cells, such as FACS studies with antibodies against factor VIII-related antigen (Von Willebrand factor) or two endothelium-specific surface antigens (EN-4, PAL-E) or with a fluorescent ligand of the scavenger receptor (dil-Ac-LDL).

The yield (n = 2) achieved with the above-mentioned mixture of purified enzymes exceeded that which had been obtained with enzyme preparations containing collagenase and having an undefined composition. The cell density after 4 days was 1.8 x IO ⁴ per cm ² . If you use previously known methods, you get a cell density that is significantly lower. The confluence of the monolayer was reached earlier (already after 5-6 days) when using the above mixture.

In the enzymatic isolation of the endothelial cells with the various enzyme preparations, the umbilical vein was never ruptured. The extracted cells were well separated. No cytotoxic effects were observed.

After one hour, endothelial cells were attached.

The prepared cells were functionally checked in the first passage (endothelin production, release of LDH). All values were within the normal range. The functions of the cells were therefore indistinguishable from results from control experiments in which cells with preparations containing collagenase containing an undefined composition were isolated.

The superiority of the mixture of purified enzymes used thus results in the higher possible cell yield, a faster formation of a confluent monolayer, and an optimal integrity of the isolated cells (cell structure and function). Comparably good results were achieved in experiments in which other mixtures, for example consisting of collagenase HP and elastase, and mixtures of collagenase HP, collagenase AZ, elastase and clostripain were used instead of the enzyme mixture mentioned above.

Example I

Isolation of tumor cells from human tumors with a mixture of 3 enzymes

Tumor cells were isolated from human tumors according to protocol (23).

The enzyme mixture used was a lyophilizate of 130 U

Collagenase HP, 5 U collagenase AZ and 21 U elastase, which was dissolved in 28.4 L cell isolation buffer (Ringer's lactate / PBS).

The success of the isolation was determined on the basis of the proportion of vital cells after the trypan blue exclusion test and counting of the number of lymphocytes. Directly comparative cell isolations were carried out using the enzyme mixture according to the invention and a preparation of an undefined composition containing collagenase, which is very suitable for this purpose, in 4 different tumor tissues.

Result

The enzyme mixture according to the invention can be used very well to isolate tumor cells from human tumors.

In the context of the experimental fluctuations, the isolation success corresponds to the isolation success which is obtained with selected, undefined composition preparations containing collagenase which are particularly suitable for tumor cell isolation.

Since, according to the results presented here, a wide variety of connective tissue structures in tumor tissue in humans can always be successfully disintegrated with the enzyme mixture according to the invention, or with improvement in the prior art (success in isolation, quality of the isolated cells, and retention of the methodological variables) Suitability of the enzyme mixtures according to the invention for disintegration from other animal and human tissues can also be assumed. Example J

Isolation of islet cells from pig pancreas with a mixture of 3 enzymes

For the isolation of islet cells from pig pancreas, the well-known preparative method according to Ricordi was introduced with the introduction of some essential modifications (24) and others. applied to better control the insulation success. The collagenase solution acts on the pancreatic tissue for a long time after infusion through the pancreatic duct under particularly well-standardized conditions. The islands which have already been detached are continuously discharged into a reservoir at a lower temperature, and after the disintegration has ended, the islands which have already been partially freed from their matrix in the pancreatic tissue are completely detached and isolated using light mechanical treatment.

The isolation of islet cells from the pancreas places extremely high demands on a preparation containing collagenase, since the foundations for successful isolation of intact islets from the pancreas are not sufficiently known.

In the following, the suitability of a defined enzyme mixture according to the invention is demonstrated by way of example. In this type of tissue, too, the suitability for successful tissue integration with the aim of isolating large, native cell aggregates (Langerhans's Island) is transferable to the use of pancreata also from other species (eg human pancreas). This perspective is supported by experiences that generally consider the isolation of islets from porcine pancreas to be more difficult than from pancreas of other species. It is known, for example, (25) that islets from porcine pancreas are very easily decomposed into smaller, undesirable fragments by commonly used preparations of an undefined composition containing collagenase. This is explained by the special properties of the cells in island aggregates, which have many protease-sensitive cell-cell contacts, both between endocrine and exocrine cells, and also between endocrine cells and the islets. The aim of an improved isolation method can therefore only be to obtain more intact islet cell aggregates, fragmentations to aggregates <100 μ should only occur to a smaller extent. The enzyme mixture used in this application example to isolate islands from porcine pancreas was a lyophilisate of 130 U collagenase HP, 5 U collagenase AZ and 21 U elastase, which was dissolved in 10 mL isolation buffer. Deoxyribonuclease (0.4 mg / 10 ml, 440 Kunitz units / mg, SIGMA) was routinely added to this solution.

The success of the isolation can be determined on the basis of the size distribution of the intact cell aggregates obtained and other customary parameters.

As a particular advantage of using the enzyme mixture according to the invention, it can be stated that proportionally more free islands over 100 μm can be obtained than with conventional preparations.

Example K

Experimental studies on wound treatment in vivo

In wound healing, collagenases are responsible for the effective removal of destroyed tissue, the recruitment of cells in the wound area and the reshaping of the extracellular matrix.

In an experimental in vivo model for wound cleaning according to Webster (19), the tissue-degrading property of the purified enzymes on rats was checked.

Depending on the amount of the purified enzymes applied to the third degree burn, an accelerated breakdown of the denatured tissue was observed within the first 16 hours. The enzymes were applied individually or in combination with other enzymes. The best results were obtained when applying a mixture of collagenase HP (3-48 U / cm ² , preferably 16 U / cm ² ), collagenase AZ (0.2-3 U / cm ² , preferably 1 U / cm ² ) and elastase (1-12 U / cm ² , preferably 3 U / cm ² ). The scab was almost completely dissolved in 8 out of 15 animals, and partial dissolution was achieved in 6 out of 15 animals. In these cases, the necrotic tissue was extremely easy to remove mechanically, in contrast to the control. After a shorter application time of 4 hours, softening of the necrotic material was found with the same amounts used, and in all cases the scab was easily removed (in contrast to the control).

In addition to this mixture, other mixtures could also be used successfully, for example those composed of collagenase HP (3-48 U / cm ² ) and elastase (1-12 U / cm ² ).

Example L

Experimental studies on wound treatment in vitro

The results obtained on the degrading effect of the necrotic tissue (Example K) were characterized further in an in vitro model. In a shaken buffer solution, the release of 4-hydroxyproline (20) was determined after 2, 4, 6 and 24 hours by the action of collagenase HP, collagenase AZ and / or elastase on the burn scab mentioned in Example K.

Such activities on purified enzymes were used which were identical to the relative activate in an effective comparison amount of a preparation containing collagenase. This experimental simplification disregards the respective contribution of the other components in the collagenase-containing preparation to the release of hydroxyproline.

When the purified enzymes were used, a release of comparable amounts of hydroxyproline was found in all cases, depending on the time, as when using a corresponding amount of collagenase-containing preparation.

According to these results, the purified enzymes and enzyme mixtures according to the invention can also be used in therapeutic areas, such as in wound treatment or for the treatment of keloids or fibrils. They can be applied externally or injected. literature

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Claims

claims

1. Enzymes available from Clostridium histolyticum, which are either

a) a collagenase with a specific activity of at least 20 U / mg in the Nordwig and Strauch test deals with the synthetic hexapeptide Z-Gly-Pro-Gly-Gly-Pro-Ala as the substrate, the denatured collagens such as gelatin and Azokoll can only implement to a small extent, but attacks bovine tendon collagen, whose molecular weight at 106,000 Da determined by SDS gel electrophoresis. and which has an isoelectric point at pH 5.8 to 6.0, or around

b) a collagenase with a specific activity of at least 10 U / mg in the test according to Mandl et al is using Azokoll as a substrate, the denatured collagens, such as gelatin or azocoll, but also bovine tendon collagen, small synthetic pro ¬ teins such as 2-furan-acryloyl-Leu-Gly-Pro-Ala, 4-phenyl-azobenzoloxycarbonyl-Pro-Leu-Gly-Pro-Arg and Z-Gly-Pro-Gly-Gly-Pro-Ala, however, cannot attack , whose molecular weight determined by SDS gel electrophoresis at 111,000 Da. lies and which has an isoelectric point at pH 5.9 to 6.1 or around

c) Elastase with a specific activity of at least 2 U / mg in the test with elastin from the neck band of the cattle acts as a substrate, whose molecular weight determined by SDS electrophoresis at 35,000 Da. lies

or mixtures thereof, the enzyme according to a) having a specific activity of at least 20 U / mg, the enzyme according to b) an activity of at least 10 U / mg and the enzyme according to d) a specific activity of at least 2 U / mg ¬ points.

2. Enzyme mixture according to claim 1, characterized in that it additionally contains the thiol protease clostripain. contains.

3. Use of a mixture according to claim 1 of enzyme according to a) and enzyme according to c) for the isolation of cells or tissue fragments from human or animal tissue.

4. Use of a mixture according to claim 1 of enzymes according to a), b) and c) for the isolation of cells or tissue fragments from human or animal tissue.

5. Use of a mixture according to claim 2 for the isolation of cells or tissue fragments from human or animal tissue.

6. Use of the enzymes according to claim 1 either alone or in a mixture or according to claim 2 in wound treatment or in diseases which are associated with an altered collagen metabolism.

7. Collagenase HP in pure form.

8. Collagenase AZ in pure form.

9. Elastase in its pure form.