Note: Descriptions are shown in the official language in which they were submitted.
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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.
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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.
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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
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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
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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
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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
