CH668984A5 - METHOD FOR OBTAINING CELL INGREDIENTS. - Google Patents

Description

DESCRIPTION The present invention relates to an improved method for obtaining cell constituents from cells of any origin while maintaining the intact cell structure.

Since the beginning of the fermentative processing of microorganisms as well as plant and animal cells, there has been an interest in the release of synthetic products (e.g. primary or secondary metabolites) from the producing cell. With the development of genetic engineering processes for manipulating microorganisms as well as animal and plant cells, there is an increasing need for refined and gentle isolation methods.

A number of economically interesting and important cell constituents are not able to pass through the cell membranes and are normally not released into the surrounding nutrient medium, but remain within the producer cell. This applies primarily to macromolecular compounds such as polypeptides, proteins, polysaccharides and nucleic acids. But also low molecular synthesis products, e.g. Numerous metabolites of the secondary metabolism of microorganisms, fungi and plants are partly deposited in the producing tissues and cells and are therefore not readily available.

In the plant cell, the cell vacuole primarily serves as a reservoir and storage for a wide variety of metabolic products. Examples are the dyes dissolved in the cell vacuole, such as anthocyanins and anthoxanthines. In addition, there are also active substances that have or could have gained great importance in human and veterinary medicine or naturopathy, e.g. various glycosides (digitalis glycoside), alkaloids (morphine) and many others.

To isolate these cell constituents, a cell disruption first had to take place, which is usually only possible with a great deal of time and effort and generally leads to cell destruction.

Since in many cases biological products are sensitive compounds, the structural and functional integrity of which can only be guaranteed within a very limited milieu, processing can only be carried out using appropriately refined methods.

As a rule, two different procedures have been used so far.

On the one hand, it is possible to break up the cells with the help of mechanical manipulations, e.g. by grinding in stirred ball mills or colloid mills, by applying pressure and subsequent relaxation (homogenizer) and by ultrasound treatment. Another possibility is the use of thermal, chemical or enzymatic methods. This includes, for example, drying and lyophilizing the cells and treating the cells with surface-active compounds (detergents) or with organic solvents.

However, these known procedures have decisive disadvantages, since they are associated with time-consuming and labor-intensive processing steps and generally end with the destruction of the cells.

In the event of mechanical destruction of the cell structure, the cell wall debris and other cellular components must first be removed from the culture solution before further processing and isolation of the desired cell contents can take place.

Ultrasound treatment often leads to unwanted and disadvantageous heating and the undesirable formation of free radicals, which can lead to uncontrollable reactions. In addition, there are high energy and cost expenses.

When using detergents, on the other hand, undesirable denaturation of proteins can occur. In addition, the added chemicals usually have to be removed from the fermentation broth before further processing can take place.

The use of enzymes is generally expensive and is therefore usually limited to the laboratory scale. In this case, too, there may be undesirable interactions that make further processing more difficult.

All of these disadvantages can surprisingly be overcome by the simple measures of the present invention, in particular by the fact that when the method according to the invention is used, the cell structure is preserved to the same extent

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What remains is that no cell debris or other cell components that interfere with processing get into the culture medium.

More recently, methods have been developed which make it possible to introduce macromolecules into microorganism cells as well as into animal and plant cells or protoplasts without permanently impairing their cell structure and thus the viability of the cells.

It was possible to increase the membrane permeability of protoplasts by treatment with electrical impulses and thus to allow macromolecules (e.g. DNA in the form of plasmids) to pass from the culture medium into the protoplasts (ShilILto, RP et al., (1985). Bio / Technology 3, 12, pp. 1099-1103).

Surprisingly, it has now been possible to develop a process which, in reverse of the principles mentioned above, is now outstandingly suitable for removing cell contents from cells while maintaining the intact cell structure.

The present invention is thus a method for obtaining cell contents from cells of any origin, while maintaining the intact cell structure, which is characterized in that said cells are subjected to high-intensity electrical voltage pulses in a suitable incubation medium and which in the ingredients released from the incubation medium are isolated.

The cells are expediently first cultivated in a suitable nutrient medium for a suitable period of time, then concentrated and resuspended in the same or any other suitable medium. The cell suspension is placed in an electroporator chamber between two electrodes. By discharging a capacitor over the cell suspension, the cells are briefly exposed to very high electrical field strengths, which leads to a release of contents from the cell interior into the surrounding nutrient medium. The cell contents are isolated from the culture medium using methods known per se, such as e.g. by extraction, by column chromatography or similar purification processes.

The method according to the invention can also be used as a continuous method by moving the cell-containing suspension continuously and at a constant speed between the capacitor plates and briefly exposing the cells to high electric field strengths.

The method according to the invention is suitable for the production of synthesis products from cells of any origin, which can be stably maintained and multiplied in culture. The method is not limited to specific cells and can be applied equally to cells of microorganisms such as bacteria, yeast and mycelium-forming fungi, as well as cells or protoplasts of human, animal and vegetable origin.

Said cells can also be in immobilized form.

A preferred embodiment of the method according to the invention relates to the isolation of cell contents from plant cells. Plant cells are cultivated e.g. in a nutrient medium suitable for this purpose, e.g. in a medium according to Murashige and Skoog (Murashige T. and Skoog F., Physiol. PI. 15, pp. 473-497, 1962), which can be modified in a corresponding manner depending on the desired metabolite.

The plant cells are grown in one of the said media and then expediently separated from large cell aggregates via a noble metal sieve. This is followed by centrifugation and resuspension of the cells in an identical or any culture medium suitable for the cultivation of plant cells or in a culture medium modified by the addition of a suitable cell stabilizing agent. The cell suspension thus obtained is placed in a previously e.g. electroporator chamber sterilized with ethanol, where the cells are then briefly exposed to very high electrical field strengths by discharging a capacitor over the cell suspension. As a rule, there is a three to ten discharge with a repetition frequency between 0.1 and 20 seconds, the field strengths achieved being between approximately 0.1 kV-cm-1 and approximately 15 kV-cm-1. The field strengths required for the method according to the invention are dependent on the cell size of the cells used, the size of the cells and the field strength being negatively correlated. The pulse width is advantageously in the range from 1 nanosecond to 1000 microseconds.

This treatment of the cells with high voltage pulses leads to the release of cell contents, e.g. of stains or active ingredients dissolved in the cell vacuole into the culture medium surrounding the cells without destroying the cell structure. It has been shown that the method according to the invention can also be applied to immobilized cells which e.g. are present in culture media solidified with agarose or alginate or other crosslinking agents.

However, cells immobilized in another way can also be used in the method according to the invention.

The extraordinarily high proportion of substance released from the cell by the method according to the invention can be demonstrated by control tests, the control of the extraction of the cell contents using conventional methods, e.g. by extraction.

The viability of the cells after the application of electroporation can e.g. be checked based on the growth of the treated cells in fresh MS medium.

The release of cell contents from the treated cells and their viability generally correlate with one another. High electrical field strengths between e.g. 8 kV-cm-1 and 15 kV-crrr1 lead to a quantitative release of the ingredients from the cell (Fig. 1), but the vitality of the treated cells is severely impaired (Fig. 2). In contrast, high survival rates of the treated cells are usually associated with lower yields of cell contents. One way of compensation is to add stabilizing agents to the incubation medium, which means that the vitality of the cells is maintained even at relatively high voltage values and the resulting high field strength values.

In contrast to the field strength values, the influence of the capacities used on the release of the cell contents over a wide range (10 nF- 40 nF) seems to be small (Fig. 3).

A connection between the vitality of the treated cells and the capacities used can only be determined at low field strengths (1200 V-cm-1), while at high values (4800 V-cm-1) there is no correlation between the two parameters (Fig. 4 ).

As the explanations given above show, when using the method according to the invention, it is possible, depending on the problem at hand, to select the experimental parameters in such a way that a desired result is obtained, i.e. too high yields of synthesized ingredients with simultaneously reduced vitality of the treated cells or too high survival rates with a somewhat lower yield of ingredients.

The following examples serve to illustrate the present invention and have no limiting character.

The following abbreviations are used in the following examples and illustrations:

1 kV 1000 volts

2.4 D (2,4-dichlorophenoxy) acetic acid

FG fresh weight

Ms-Medium Murashige and skoog's Medium

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Rpm revolutions per minute w / v weight / volume (volumetric weight)

w / w weight / weight s second h hour m minute

example 1

Release of berberine from cells of a cell line from Thalictrum rugosum

Berberine is a characteristic ingredient of the Berberida-ceae and Ranunculaceae. It is an intensely yellow colored compound, which chemically belongs to the benzyl-isoquinoline alkaloids and is present in solution in the vacuole.

Cells from a cell line from Thalictrum rugosum are grown over a period of two weeks in a medium according to Murashige and Skoog (MS), to which 2.4 D had previously been added at a concentration of 2 μl, at a temperature of 26 ° C. The cells are cultivated in a liquid shaking culture (120 rpm) in the dark. After the incubation period has elapsed, the cell suspension is filtered through a stainless steel sieve with a mesh size of 500 μm in order to separate any large cell aggregates that may be present.

The cells are then centrifuged off and resuspended in fresh MS medium. In order to achieve a uniform cell density of the cell suspensions intended for the electroporation experiments, the centrifuged cells are diluted in a ratio (w / v) of 1:10 (fresh weight in 9 ml of medium).

The electroporation experiments are carried out in an electroporator chamber by Dialog © "Porator" (Dialog GmbH, Harff-strasse 34,4000 Düsseldorf, Germany-West).

These are cylindrical chambers with a volume of 0.35 ml and 1 ml. At the chamber ends are stainless steel electrodes with an electrode spacing of 1 cm.

Before the cell suspension is introduced, the electroporator chamber is sterilized by washing with 70% and 100% ethanol and then dried over a sterile air flow from a blower with laminar air flow.

0.30 ml or 1 ml of the cell suspension mentioned above are then transferred to the electroporator chamber and here 3 to 10 times an electrical pulse in the nano- to microsecond range from 0.3 kV to 15 kV, which corresponds to an electrical field strength of 0.3 kV-cm_I to 15 kV-cnr1 , exposed. The interval between the individual pulses is 10 s. Capacitors with different capacitances are used to generate the electrical field pulses, which leads to different exponential decay constants of the induced pulses, which are between 5 and 20 microseconds.

The release of the vacuole dye berberine from cells of Thalictrum rugosum into the culture medium is determined using thin-layer chromatography or spectroscopic methods.

The amount of substance released from the cells is shown in the figures in relative sizes compared to control experiments. The controls are cells that are grown under the same conditions, but whose dye release is achieved using conventional methods such as e.g. by extraction with methanol.

Example 2

Release of betanin from cells of a Chenopodium rubrum cell line

Betanine is an intensely red-colored compound that can be found, for example, as a cell substance in red beets. This dye, which can be regarded chemically as ß-D-glucopyranoside, is also present in solution in the vacuole.

The cultivation of the cells of Chenopodium rubrum takes place over a period of 3 weeks in a MS medium enriched with 2.4 D (2 (iM) as a liquid shaking culture (80 rpm) while observing a day / night rhythm (16 h / 8 h The incubation temperature in this case is also 26 ° C.

The electroporation experiments are carried out analogously to the information given above for Thalictrum rugosum.

The amount of betanin released into the culture medium is determined after the cells have been separated from the cell suspension by centrifugation based on the absorption of the culture solution at 540 nm.

Example 3

Release of berberine from immobilized cells of Thalictrum rugosum

The cells of a Thalictrum rugosum cell line are cultivated in MS medium with the addition of 2.4 D over a period of 2 weeks in a liquid shaking culture (120 rpm) at a temperature of 26 ° C. in the dark, as described in Example 1.

The cell suspension is then first filtered to separate any cell agglomerates that may be present, then the cells are centrifuged off and resuspended in a w / v ratio of 1:10 in fresh MS medium (1 g cells (FG) / 9 ml MS medium).

To produce immobilized cells, agarose (e.g. Sigma Type VII) is added to the culture medium up to a concentration of 4% (w / w) at a temperature of 37 ° C.

This batch is then mixed with constant stirring using a magnetic stirrer with 40 ml soybean oil of the same temperature. After droplets with a diameter of 0.5 mm - 1 mm are formed, the mixture is cooled to 10 ° C. In this way, agarose beads are formed in which the cells are enclosed. These agarose beads are collected and freed of any oil residues by washing several times with MS medium.

The cells immobilized in the manner described above are then used in an analogous manner to the suspended, free cells for the electroporation experiments. In this case, the immobilized cells in the 1 ml electroporation chamber are subjected to voltage pulses with an exponential decay constant of 10 microseconds.

Example 4

Release of berberins from immobilized cells of Thalictrum rugosum

The cultivation of the cells of Thalictrum rugosum is carried out in the same way as in Example 1.

In this case, alginate is used to produce the immobilized cells. The filtered and centrifuged cells are resuspended in alginate-containing Ms medium (e.g. 2% w / w, Portan HF) at room temperature in a ratio of 1:10 (lg cells (FG) / 9 ml MS medium).

The cell suspension is then added dropwise in MS medium containing 50 mM CaCk. Beads form which are stirred for a further 30 minutes before being washed and separated several times with fresh MS medium containing 5 mM CaCk.

In this case too, the electroporation experiments are carried out in the same way as described above in Example 4 for the cells in suspension culture.

Test results

The results of the tests carried out prove that, if suitable parameters are selected, a quantitative release of cell contents from the vacuoles is achieved (Fig. 1 and 3),

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regardless of whether the cells used are in suspension or in the form of immobilized cells. (Fig. 5).

There is a clear dependence of the release of the vacuole dyes on the electric field strength. In a range between 1 kV ■ cm-1 and 4 kV-cm-1, a strong increase in the release can be observed, which occurs quantitatively at values of 8 kV ■ cm-1 and 15 kV-cm-1 (Chenopodium rubrum) . (Fig. 1).

The capacitance of the capacitor used, on the other hand, is apparently of little importance for the amount of substance released.

The fluctuations in the release rates across the used capacity range (10 nF - 40 nF) are only slight (Fig. 3). It turns out that in this case too, high field strengths (4800 V-cm-1) lead to high release rates (up to 100%), whereas with low electrical field strengths (1200 V-cm-1) the values are correspondingly lower (approx. 25%).

The number of voltage pulses emitted apparently plays only a minor role in the release of the dyes. As Figure 6 shows with the example of the Berberin release, the curves for 3 or 10 impulses 5 run practically parallel.

In contrast to the release of dye from the vacuole, the viability of the cells after electroporation decreases with increasing field strength.

As shown by the example of Thalictrum rugosum (Fig. 2), a strong decrease in viable cells can be observed in a range between 600 V • cm-1 and 2400 V • cm "" '.

The capacity used in this case also has an influence on the viability of the cells, primarily in the lower field strength range (1200 V-cm-1. This decreases significantly at values between -15 see 10 nF-40 nF it is practically unchanged at an electric field strength of 4800 V • cm _1 (Fig. 4).

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3 sheets of drawings

Claims (15)

668 984 1. A method for obtaining cell contents from cells of any origin, while maintaining the intact cell structure, characterized in that said cells are subjected to high-voltage electrical voltage pulses in a suitable incubation medium and the ingredients released into the incubation medium are isolated. 2. The method according to claim 1, characterized in that said cells are those that can be stably maintained or grown in culture. 2nd PATENT CLAIMS 3. The method according to any one of claims 1 or 2, characterized in that said cells are microorganism cells. 4. The method according to claim 3, characterized in that said microorganisms are bacteria, yeast or mycelium-forming fungi. 5. The method according to any one of claims 1 or 2, characterized in that said cells are those of animal or human origin. 6. The method according to any one of claims 1 or 2, characterized in that said cells are of plant origin. 7. The method according to claim 6, characterized in that said cells are cell lines of Thalictrum rugosum. 8. The method according to claim 6, characterized in that said cells are cell lines of Chenopodium rubrum. 9. The method according to any one of claims 1 or 2, characterized in that cells of any origin in a suitable incubation medium are subjected to electrical voltage pulses in such a way that said cells are subjected to electrical field strengths between 0.1 kV-cm-1 and 15 kV-cm_1 are. 10. The method according to claim 9, characterized in that the cells are incubated in a liquid nutrient medium. 11. The method according to claim 9, characterized in that said cells are immobilized cells. 12. The method according spoke 11, characterized in that the cells are immobilized with agarose or alginate. 13. The method according to claim 9, characterized in that that said cells are subjected to 1 to 10 electrical voltage pulses with a repetition frequency between 0.1 and 20 seconds. 14. The method according to claim 9, characterized in that the pulse width is 1 nanosecond to 1000 microseconds. 15. The method according to any one of claims 1 to 14, characterized in that it is berberine and / or betanine in the cell constituent obtained.