CA1230566A - Method of encapsulating bio material in bead polymers - Google Patents
Method of encapsulating bio material in bead polymersInfo
- Publication number
- CA1230566A CA1230566A CA000440377A CA440377A CA1230566A CA 1230566 A CA1230566 A CA 1230566A CA 000440377 A CA000440377 A CA 000440377A CA 440377 A CA440377 A CA 440377A CA 1230566 A CA1230566 A CA 1230566A
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- Prior art keywords
- cells
- fungi
- algae
- bacteria
- agarose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Spherical polymer particles may be produced by dispers-ing water-soluble monomers or polymers in a hydrophobic solvent, such as toluene or chloroform. These solvents, however, have a dewatering effect on sensitive biomaterials if these are simulta-neously enclosed in the polymer particles. The present invention shows that it is possible to enclose sensitive biomaterials selected from plant cells, animal cells, bacteria, algae and fungi, in spherical polymer particles with retained viability and growth capacity if a suitable combination of polymer and dispers-ing medium is used. The polymer is selected from agar, agarose and fibrinogen. The dispersing medium is selected from paraffin oil, soybean oil, liquid silicone, phthalic acid dibutyl ester and tri-n-butyl phosphate.
Spherical polymer particles may be produced by dispers-ing water-soluble monomers or polymers in a hydrophobic solvent, such as toluene or chloroform. These solvents, however, have a dewatering effect on sensitive biomaterials if these are simulta-neously enclosed in the polymer particles. The present invention shows that it is possible to enclose sensitive biomaterials selected from plant cells, animal cells, bacteria, algae and fungi, in spherical polymer particles with retained viability and growth capacity if a suitable combination of polymer and dispers-ing medium is used. The polymer is selected from agar, agarose and fibrinogen. The dispersing medium is selected from paraffin oil, soybean oil, liquid silicone, phthalic acid dibutyl ester and tri-n-butyl phosphate.
Description
~.~30~6 The present invention relates to a method of immobiliz-ing bio-material selected from animal cells, plant cells, bacte-ria, algas or fungi by encapsulation in polymer beads.
In recent years there has been a considerable interest in immobilizing bio-material. The most usual polymers used for encapsulating bio-material are alginate, polyacrylamide, car-rageenan, agar or agarose. Of these alginate and carrageenan are the only ones which can be manufactured simply in spherical form with encapsulated material. This is done by ionotropic gelling, i.e. the alginate is dropped down into calcium solution and the carrageenan in a potassium solution. However, relatively large beads (2-3 mm diameter) are usually obtained in this way, and beads that are stable only in the presence of ions (calcium and potassium ions, respectively). In the use of agar, agarose, col-lagen, polyacrylamide, gelatine or fibrinogen, the biomaterial is usually mixed with polymer or polymer solution which is then caused to gel. In order to obtain a suitable size, this gel is fragmented and possibly further cross-linked to attain higher stability. In the preparations produced in this way there is leakage of the encapsulated material in the fragmentation, and due to their heterogeneity they are found to have poor flow in column processes.
Spherical polyacrylamide particles with encapsulated enzymes can be produced by a bead polymerization process, where the monomer solution together with enzyme and catalysts are dis-persed in a hydrophobic phase (see Swedish patent 7204481-1).
The hydrophobic phase comprises an organic solvent (exemplified by toluene plus chloroform) and an emulsifier. Since both these solvents and emulsifiers have a denaturing effect, this method is suitable only for relatively insensitive material.
There has therefore been a great need for a more gentle dispersion medium for the production of spherical polymer par-ticles.
. ~
i" ~ -- 1 --~ 6 According to the present invention there is provided a method of immobili~ing viable animal cells, plant cells, bacte-ria, algae, or fungi with retained abillty of growth by encapsu-lation in polymer beads, which comprises: (a) adding said viable animal cells, plant cells, bacteria, algae or fungi to an aqueous solution of agar, agarose or fibrinogen; (b) dispersing said aqueous solution in a non-toxic water-lnsoluble dispersion medium selected from the group consisting of soybean oil, tri-n-butylphosphate, liquid silicone, paraffin oil and phthalic acid dibutylester; and (c) allowing said agar, agarose or fibrinogen to gel to form polymer beads encapsulating said viable animal cells, plant cells, bacteria, algae or fungi either by cooling or by enzymatic action under conditions such that the growth ability of said cells is unaffected.
The present invention also provides immobillzed viable animal cells, plant cells, bacteria, algae, or fungi with a retained ability of growth, and being encapsulated in polymer beads of agar, agarose or fibrinogen.
Accordlng to the present invention sensitive bio-mate-rial can be encapsulated with full viability and with retained growth ability if the organic solvent is exchanged for any of the following dispersion media: soybean oil, tri-n-butylphosphate, liquid silicone, paraffin oil or phthalic acid dibutyl ester.
Certain of these dispersion media have been used before in the preparation of spherical polymer particles. In the U.S. patent 4,169,804 there is described a method of producing magnetic microspheres by dispersing an albumin solution in vegetable oil with subsequent heating thereof to 140C or cross-linking the albumin polymer with aldehydes. This technique cannot be used for encapsulating sensitive bio-material, since either the high temperature or the cross-linking aldehyde then result in inacti-vation.
A method is described in "Biotechnology Letters: vol.
~230S~6 3, pages 65-70, 1981, of producing spherical polyurethane parti-cles by dispersing the monomers in paraffin oil. By including bacteria in the monomer mixture there is obtained a ca~alytically active preparation (although whether the bacteria retain their propagating ability is doubtful, furthermore there is great risk that they are linked covalently to the carrier). On the other hand, by combining these gentle dispersion media with a suitable immobilization-method carrier there may be obtained, as with the preparations described here, 100% viability and retained growth ability. The denaturing action of different dispersion media on agar-encapsulated plant cells is compared in Table l. It will be seen from the Table that these dispersion media give a retained viability compared with the usual standard method for the produc-tion of spherical polymer particles (swedish patent 7204481-l).
In Table 2, this relationship is also shown to apply to yeast cells encapsulated in polyacrylamide.
In accordance with a preferred embodiment of the pre-sent invention said gelation is conducted so as to obtain beads having a granular size of 0.05-3mm. More preferably said gela-tion is conducted so as to obtain beads having a granular size of 0.1-l.Omm. Suitably said retained growth ability is such that after the encapsulation of said viable animal cells, plant cells, bacteria, algae or fungi, a relative respiration in the range of 82-100% is exhibited.
There are two principally different types of animal cells, suspension cells and surface-dependent cells. The sur-face-dependent cells must be attached to a surface in order to survlve and grow. In this invention the fibres formed by colla-gen or fibrin are used as the necessary surface, this resulting in the surface-dependent anlmal cells being able to survive a~d grow ln an encapsulated condition. Surface dependent cells can-not grow encapsulated in agarose, but by mixing agarose with fib-rin or collagen this polymer can also be used for encapsulatingsurface-dependent cells. Animal suspension cells do no have this o 123~i6 requirement and may be encapsulated in agarose, collagen or fib-rin with retained ability to grow. Since the potential field of use of animal cells is very large (the production of vaccines, proteins or hormones, transforming of precursors, etc.) it is important to develop systems where they can be used on a large scale.
Encapsulated animal cells have great advantages from the aspect of production technique, compared with .
- 3a -"
- 123(~ 6 free cells. For example, they can easily be used in column processes, and there are also advantages in other types of reaction, since they are much easier to adapt to c~tLnuous production.
The size of the bead formed with encapsulated bio material can be varied within wide limits, depend-ing on the force with which the polymer solution is dispersed.
Example 1. Agar, agarose Agar or agarose is dissolved in water (5.6 %w/v) by heating. The polymer solution (8 ml) is brought to a temperature of 50C and mixed with plant cells (2 g) subsequent to which dispersion in soybean oil (40 ml) takes place. When suitably large beads have been obtained the mixture is cooled to 5C and the beads washed over to water.
Example 2. Carrageenan The carrageenan is dissolved in 0.9 ~ NaCl (3.1 % w/v) by heating. The beads are manufactured according to example 1.
Example 3. Chitosan Chitosan is dissolved in 0.1 M HAc/0.1 M NaAc.
The polymer solution (8 ml) is mixed with yeast cells (2 ml) or enzymes (peroxidase 10 mg/ml, 2 ml) after which it is dispersed in soybean oil (40 ml), and formaldehyde (37 ~ w/v, 2.2 ml) is now added, after which stirring is carried out for 30 minutes. The cross-linked beads are washed over to water.
Example 4. Polyacrylamide Acrylamide (17.6 g) and bisacrylamide (1.2 g) are dissolved in tris~buffer (100 ml, 0,05 M,pH7). The monomer solution (8 ml) is mixed with yeast cells or enzymes (peroxidase, 10 mg/ml, 2 ml) and ammonium persulphate (0,4 g/ml, 20 ~l) and dispersed in soybean 35 oil (40 ml). TEMED (100 41) is added when a suitable bead size has been reached. The polymerized beads are washed over to water.
Example 5. Gelatine Gelatine (15 ~ w/v) is dissolved by heating in water. The polymer solution (8 ml) is brought to a temperature 37C and mixed with yeast cells (2 ml), subsequent to which it is aispersed in soybean oil (40 ml). After cooling to 15C, the beads are washed over to water.
Example 6. Gelatine capsules Gelatine (15 % w/v) is dissolved in a phosphate buffer (0.1 M pH 8). The polymer solution (8 ml) is brought to a temperature 37C and mixed with cells (plant cells
In recent years there has been a considerable interest in immobilizing bio-material. The most usual polymers used for encapsulating bio-material are alginate, polyacrylamide, car-rageenan, agar or agarose. Of these alginate and carrageenan are the only ones which can be manufactured simply in spherical form with encapsulated material. This is done by ionotropic gelling, i.e. the alginate is dropped down into calcium solution and the carrageenan in a potassium solution. However, relatively large beads (2-3 mm diameter) are usually obtained in this way, and beads that are stable only in the presence of ions (calcium and potassium ions, respectively). In the use of agar, agarose, col-lagen, polyacrylamide, gelatine or fibrinogen, the biomaterial is usually mixed with polymer or polymer solution which is then caused to gel. In order to obtain a suitable size, this gel is fragmented and possibly further cross-linked to attain higher stability. In the preparations produced in this way there is leakage of the encapsulated material in the fragmentation, and due to their heterogeneity they are found to have poor flow in column processes.
Spherical polyacrylamide particles with encapsulated enzymes can be produced by a bead polymerization process, where the monomer solution together with enzyme and catalysts are dis-persed in a hydrophobic phase (see Swedish patent 7204481-1).
The hydrophobic phase comprises an organic solvent (exemplified by toluene plus chloroform) and an emulsifier. Since both these solvents and emulsifiers have a denaturing effect, this method is suitable only for relatively insensitive material.
There has therefore been a great need for a more gentle dispersion medium for the production of spherical polymer par-ticles.
. ~
i" ~ -- 1 --~ 6 According to the present invention there is provided a method of immobili~ing viable animal cells, plant cells, bacte-ria, algae, or fungi with retained abillty of growth by encapsu-lation in polymer beads, which comprises: (a) adding said viable animal cells, plant cells, bacteria, algae or fungi to an aqueous solution of agar, agarose or fibrinogen; (b) dispersing said aqueous solution in a non-toxic water-lnsoluble dispersion medium selected from the group consisting of soybean oil, tri-n-butylphosphate, liquid silicone, paraffin oil and phthalic acid dibutylester; and (c) allowing said agar, agarose or fibrinogen to gel to form polymer beads encapsulating said viable animal cells, plant cells, bacteria, algae or fungi either by cooling or by enzymatic action under conditions such that the growth ability of said cells is unaffected.
The present invention also provides immobillzed viable animal cells, plant cells, bacteria, algae, or fungi with a retained ability of growth, and being encapsulated in polymer beads of agar, agarose or fibrinogen.
Accordlng to the present invention sensitive bio-mate-rial can be encapsulated with full viability and with retained growth ability if the organic solvent is exchanged for any of the following dispersion media: soybean oil, tri-n-butylphosphate, liquid silicone, paraffin oil or phthalic acid dibutyl ester.
Certain of these dispersion media have been used before in the preparation of spherical polymer particles. In the U.S. patent 4,169,804 there is described a method of producing magnetic microspheres by dispersing an albumin solution in vegetable oil with subsequent heating thereof to 140C or cross-linking the albumin polymer with aldehydes. This technique cannot be used for encapsulating sensitive bio-material, since either the high temperature or the cross-linking aldehyde then result in inacti-vation.
A method is described in "Biotechnology Letters: vol.
~230S~6 3, pages 65-70, 1981, of producing spherical polyurethane parti-cles by dispersing the monomers in paraffin oil. By including bacteria in the monomer mixture there is obtained a ca~alytically active preparation (although whether the bacteria retain their propagating ability is doubtful, furthermore there is great risk that they are linked covalently to the carrier). On the other hand, by combining these gentle dispersion media with a suitable immobilization-method carrier there may be obtained, as with the preparations described here, 100% viability and retained growth ability. The denaturing action of different dispersion media on agar-encapsulated plant cells is compared in Table l. It will be seen from the Table that these dispersion media give a retained viability compared with the usual standard method for the produc-tion of spherical polymer particles (swedish patent 7204481-l).
In Table 2, this relationship is also shown to apply to yeast cells encapsulated in polyacrylamide.
In accordance with a preferred embodiment of the pre-sent invention said gelation is conducted so as to obtain beads having a granular size of 0.05-3mm. More preferably said gela-tion is conducted so as to obtain beads having a granular size of 0.1-l.Omm. Suitably said retained growth ability is such that after the encapsulation of said viable animal cells, plant cells, bacteria, algae or fungi, a relative respiration in the range of 82-100% is exhibited.
There are two principally different types of animal cells, suspension cells and surface-dependent cells. The sur-face-dependent cells must be attached to a surface in order to survlve and grow. In this invention the fibres formed by colla-gen or fibrin are used as the necessary surface, this resulting in the surface-dependent anlmal cells being able to survive a~d grow ln an encapsulated condition. Surface dependent cells can-not grow encapsulated in agarose, but by mixing agarose with fib-rin or collagen this polymer can also be used for encapsulatingsurface-dependent cells. Animal suspension cells do no have this o 123~i6 requirement and may be encapsulated in agarose, collagen or fib-rin with retained ability to grow. Since the potential field of use of animal cells is very large (the production of vaccines, proteins or hormones, transforming of precursors, etc.) it is important to develop systems where they can be used on a large scale.
Encapsulated animal cells have great advantages from the aspect of production technique, compared with .
- 3a -"
- 123(~ 6 free cells. For example, they can easily be used in column processes, and there are also advantages in other types of reaction, since they are much easier to adapt to c~tLnuous production.
The size of the bead formed with encapsulated bio material can be varied within wide limits, depend-ing on the force with which the polymer solution is dispersed.
Example 1. Agar, agarose Agar or agarose is dissolved in water (5.6 %w/v) by heating. The polymer solution (8 ml) is brought to a temperature of 50C and mixed with plant cells (2 g) subsequent to which dispersion in soybean oil (40 ml) takes place. When suitably large beads have been obtained the mixture is cooled to 5C and the beads washed over to water.
Example 2. Carrageenan The carrageenan is dissolved in 0.9 ~ NaCl (3.1 % w/v) by heating. The beads are manufactured according to example 1.
Example 3. Chitosan Chitosan is dissolved in 0.1 M HAc/0.1 M NaAc.
The polymer solution (8 ml) is mixed with yeast cells (2 ml) or enzymes (peroxidase 10 mg/ml, 2 ml) after which it is dispersed in soybean oil (40 ml), and formaldehyde (37 ~ w/v, 2.2 ml) is now added, after which stirring is carried out for 30 minutes. The cross-linked beads are washed over to water.
Example 4. Polyacrylamide Acrylamide (17.6 g) and bisacrylamide (1.2 g) are dissolved in tris~buffer (100 ml, 0,05 M,pH7). The monomer solution (8 ml) is mixed with yeast cells or enzymes (peroxidase, 10 mg/ml, 2 ml) and ammonium persulphate (0,4 g/ml, 20 ~l) and dispersed in soybean 35 oil (40 ml). TEMED (100 41) is added when a suitable bead size has been reached. The polymerized beads are washed over to water.
Example 5. Gelatine Gelatine (15 ~ w/v) is dissolved by heating in water. The polymer solution (8 ml) is brought to a temperature 37C and mixed with yeast cells (2 ml), subsequent to which it is aispersed in soybean oil (40 ml). After cooling to 15C, the beads are washed over to water.
Example 6. Gelatine capsules Gelatine (15 % w/v) is dissolved in a phosphate buffer (0.1 M pH 8). The polymer solution (8 ml) is brought to a temperature 37C and mixed with cells (plant cells
2 g), subsequent to which it is dispersed in soybean oil (40 ml) containing a water-soluble cross-linking agent (toluene diisocyanate, 2.5 % w/v), the beads being washed over to water after 30 minutes. In heating to 37C, gelatine which has not been cross-linked goes into solution and leaves the shell intact. Plant cells immobilized in this way are unaffected by the cross-linking agent and are viable to 95 % with respect to respiration.
Example 7. Fibrinogen The fibrinogen solution (1 ml, 2 % w/v) is mixed with the fibrinogen buffer (1 ml) and fetal calf serum (0.3 ml). Animal cells (0.7 ml) and thrombin (1.5 U) are then added. The mixture is dispersed in paraffin oil (40 ml), and after 15 minutes the beads are washed over to a cultivating medium and the encapsulated cells are cultivated at 37C.
Example 8.
Agarose is dissolved in PBS (5 %, w/v) by heating. The polymer solution (5 ml) is brought to 37C and mixed with animal cells (5 ml), after which the mixture is dispersed in paraffin oil (40 ml).
After cooling, the beads are washed over to a cultivat-ing medium and the encapsulated cells cultivated at 37C
- ~L2305~6 Example 9.
Collagen is dissolved in diluted hydrochloric acid. The polymer solution (cooled to 5C, 2 ml~ is mixed with 10 times concentrated medium and sodium hydroxide to obtain a neutral pH, and also with animal cells (2 ml). The mixture is dispersed in paraffin oil (40 ml), The mixture is gelled by heating to 37C, the beads washed over to a medium and the encapsulated cells are cultivated at 37C.
Table 1 The action of the hydrophobic phase on the respiration of plant cells encapsulated in agar Hydrophobic phaseRelative respiration (~) Soybean oil 100 15 Tri-n-butyl phosphate100 Paraffin oil 91 Liquid silicone 100 Phthalic acid dibutylester 82 Toluene: chloroform, (73:27 v/v), 20 Arlacel 83 (2 ~ w¦~v)0 Table 2 The action of the hydrophobic phase on polyacryl-amide-encapsulated yeast cells.
Hydrophobic phase Relative respiration (~) 25 Soybean oil 100 Toluene: chloroform, (73:27v/v) Arlacel 83 (2 % w/v) 7
Example 7. Fibrinogen The fibrinogen solution (1 ml, 2 % w/v) is mixed with the fibrinogen buffer (1 ml) and fetal calf serum (0.3 ml). Animal cells (0.7 ml) and thrombin (1.5 U) are then added. The mixture is dispersed in paraffin oil (40 ml), and after 15 minutes the beads are washed over to a cultivating medium and the encapsulated cells are cultivated at 37C.
Example 8.
Agarose is dissolved in PBS (5 %, w/v) by heating. The polymer solution (5 ml) is brought to 37C and mixed with animal cells (5 ml), after which the mixture is dispersed in paraffin oil (40 ml).
After cooling, the beads are washed over to a cultivat-ing medium and the encapsulated cells cultivated at 37C
- ~L2305~6 Example 9.
Collagen is dissolved in diluted hydrochloric acid. The polymer solution (cooled to 5C, 2 ml~ is mixed with 10 times concentrated medium and sodium hydroxide to obtain a neutral pH, and also with animal cells (2 ml). The mixture is dispersed in paraffin oil (40 ml), The mixture is gelled by heating to 37C, the beads washed over to a medium and the encapsulated cells are cultivated at 37C.
Table 1 The action of the hydrophobic phase on the respiration of plant cells encapsulated in agar Hydrophobic phaseRelative respiration (~) Soybean oil 100 15 Tri-n-butyl phosphate100 Paraffin oil 91 Liquid silicone 100 Phthalic acid dibutylester 82 Toluene: chloroform, (73:27 v/v), 20 Arlacel 83 (2 ~ w¦~v)0 Table 2 The action of the hydrophobic phase on polyacryl-amide-encapsulated yeast cells.
Hydrophobic phase Relative respiration (~) 25 Soybean oil 100 Toluene: chloroform, (73:27v/v) Arlacel 83 (2 % w/v) 7
Claims (9)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of immobilizing viable animal cells, plant cells, bacteria, algae, or fungi with retained ability of growth by encapsulation in polymer beads, which comprises: (a) adding said viable animal cells, plant cells, bacteria, algae or fungi to an aqueous solution of agar, agarose or fibrinogen; (b) dis-persing said aqueous solution in a non-toxic water-insoluble dis-persion medium selected from the group consisting of soybean oil, tri-n-butylphosphate, liquid silicone, paraffin oil and phthalic acid dibutylester; and (c) allowing said agar, agarose or fib-rinogen to gel to form polymer beads encapsulating said viable animal cells, plant cells, bacteria, algae or fungi either by cooling or by enzymatic action under conditions such that the growth ability of said cells is unaffected.
2. The method as claimed in claim 1, wherein said gelation is conducted so as to obtain beads having a granular size of 0.05-3mm.
3. The method as claimed in claim 2, wherein said gelation is conducted so as to obtain beads having a granular size of 0.1-1.0mm.
4. The method as claimed in claim 1, wherein said retained growth ability is such that after the encapsulation of said viable animal cells, plant cells, bacteria, algae or fungi, a relative respiration in the range of 82-100% is exhibited.
5. Immobilized viable animal cells, plant cells, bac-teria, algae, or fungi with a retained ability of growth, and being encapsulated in polymer beads of agar, agarose or fibrino-gen.
6. The immobilized cells of claim 5, having a granular size of 0.05-3mm.
7. Immobilized viable animal cells, plant cells, bac-teria, algae, or fungi with a retained ability of growth, and being encapsulated in polymer beads of agar, agarose or fibrino-gen, and being produced by: (a) adding said viable animal cells, plant cells, bacteria, algae or fungi to an aqueous solution of agar, agarose or fibrinogen; (b) dispersing said aqueous solution in a non-toxic water-soluble dispersion medium selected from the group consisting of soybean oil, tri-n-butylphosphate, liquid silicone, paraffin oil and phthalic acid dibutylester; and (c) allowing said agar, agarose or fibrinogen to gel to form the polymer beads encapsulating said viable animal cells, plant cells, bacteria, algae or fungi such that the growth ability of said cells is unaffected.
8. The immobilized cells as claimed in claim 5, wherein the retained growth ability of said cells is such that after the encapsulation of said viable animal cells, plant cells, bacteria, algae or fungi, a relative respiration in the range of 82-100% is exhibited.
9. The immobilized cells as claimed in claim 7, wherein the retained growth ability of said cells is such that after the encapsulation of said viable animal cells, plant cells, bacteria, algae or fungi, an relative respiration in the range of 82-100% is exhibited.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000440377A CA1230566A (en) | 1983-11-03 | 1983-11-03 | Method of encapsulating bio material in bead polymers |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000440377A CA1230566A (en) | 1983-11-03 | 1983-11-03 | Method of encapsulating bio material in bead polymers |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1230566A true CA1230566A (en) | 1987-12-22 |
Family
ID=4126441
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000440377A Expired CA1230566A (en) | 1983-11-03 | 1983-11-03 | Method of encapsulating bio material in bead polymers |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1230566A (en) |
-
1983
- 1983-11-03 CA CA000440377A patent/CA1230566A/en not_active Expired
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