Preparation of liquid-core microcapsules for cell cultures

A method for preparing liquid-core microcapsules for cell cultures, using a hardening solution containing CaCl.sub.2 and polyethyleneimine to harden gel-core beads before coating them with polylysine solution.

BACKGROUND OF THE INVENTION 
The present invention relates to a method for preparing liquid-core 
microcapsules for cell cultures, and in particular to a method for 
preparing liquid-core microcapsules for cell cultures, which uses a 
hardening solution containing CaCl.sub.2 and polyethyleneimine to harden 
gel-core beads before coating them with polylysine solution. 
When culturing high-density animal cells, the use of liquid-core 
microcapsules has many advantages, for example, low cost, ease of 
culturing and high suitability for culturing various animal cells. Due to 
the strict requirements on the growing conditions for animal cell 
cultures, when the microencapsulation technique is used for culturing 
animal cells, the materials selected for the microcapsules and the method 
of encapsulation are usually more stringent than the fixing conditions of 
the cells. 
T. Yoshioka et al disclose a process for producing liquid-core 
microcapsules, which involves adding a polyelectrolyte compound dropwisely 
to another polyelectrolyte compound of opposite charge (Biotechnol. 
Bioeng. Vol. 35, pp 66, 1990). 
M. C. Bane et al disclose microcapsules made of a new synthetic polymer 
(Biotechnol. Bioeng. Vol. 9, pp 468, 1991). S. Iijima et al disclose a 
method for producing gel-core microcapsules by using interfacial 
polymerization wherein the gel-core contains alginate and polyurea (Appl. 
Microbiol. Biotechnol. Vol. 28, pp 572, 1988). 
In the high-density culturing of animal cells, using liquid-core 
microcapsules can provide larger growing space for the animal cells 
encapsulated therein as compared to gel-core microcapsules, and thereby 
provide higher production. However, as some liquid-core microcapsules are 
fragile, when the culturing is conducted in a large scale culture device, 
they are easily ruptured due to mechanical collisions caused by necessary 
stirring and aeration, causing leakage of the cells and accordingly 
affecting the whole manufacturing process thereof. E. R. Mckillip et al 
have reported the problems encountered when cells are mass-cultured by 
using liquid-core microcapsules (Bio./Technol., Vol. 9, pp 805, 1991). 
Heretofore liquid-core microcapsules are produced by mixing a 1-3% alginate 
solution as core material with cultured cells to form a mixture, dropping 
the mixture into a 1-3% CaCl.sub.2 solution (hardening solution) to gel 
the mixture to form gel-core beads, placing the gel-core beads in a 
solution containing 0.1% of polylysine or polyethyleneimine to coat the 
beads and form a membrane thereon, followed by dissolving the calcium ion 
in the gel cores with sodium citrate. The resulting liquid-core 
microcapsules, when used in culture of animal cells, are able to produce 
10.sup.8 cells per ml. However, they have the following disadvantages: 
(1) The lifetime of the liquid-core microcapsules is only 2 weeks when they 
are used for animal cell culture due to their weak mechanical strength. 
(2) In accordance with the conventional processes, the operating time for 
each step influences the quality of the final microcapsules greatly. For 
example, assuming the coating time of polylysine is set at 3 minutes, 
increasing or reducing the coating time will result in the instability of 
the resulting liquid-core microcapsules. Therefore, according to the 
above-mentioned conventional manufacturing process, when mass production 
is desired, it is difficult to obtain liquid-core microcapsules having 
controlled quality. 
(3) If cells capable of producing high molecular weight proteins 
(MW.gtoreq.160 Kd), for example, monoclonal antibodies, are encapsulated 
in liquid-core microcapsules prepared by the above conventional method, as 
the produced protein can not permeate the microcapsule membrane and 
therefore can not be released thereform, the continuous production of the 
cells will be hindered. 
Gel-core microcapsules made of alginate and polyurea as proposed by S. 
Iijima et al can overcome a part of the above disadvantages, however, they 
have the disadvantage that the cell density is low, and can not reach the 
value of 10.sup.8 cells per ml. 
SUMMARY OF THE INVENTION 
It is therefore an object of this invention to provide a method for 
preparing liquid-core microcapsules obviating the above-mentioned 
disadvantages. 
To attain the above object, it has now been found that dropping a solution 
containing CaCl.sub.2 and polyethyleneimine as hardening agent into a 
mixture of cultured cells and core material including alginate and 
optionally carboxymethyl cellulose to form gel-core beads, followed by 
coating them with polylysine solution can produce liquid-core 
microcapsules having superior mechanical stability that are capable of 
producing high density cells and allowing the produced cells to be 
released, and can make more flexible the operating time for each step of 
manufacturing liquid-core microcapsules, for example coating with 
polylysine. 
More specifically, the method for the preparation of liquid-core 
microcapsules according to the invention includes adding a core material 
solution dropwisely to cultured cells in a culture medium to form a 
mixture, wherein the core material solution is a solution of alginate; 
adding the mixture to a hardening solution containing CaCl.sub.2 and 
polyethyleneimine to form gel-core beads; coating the gel-core beads with 
polylysine solution; and dissolving the coated gel-core beads with citrate 
salts to form the liquid-core microcapsules. The microcapsules prepared by 
this method, for the ease of description, hereinafter are refered to as 
Type A microcapsules. 
Alternatively, liqid-core microcapsules can be produced by adding a core 
material solution to cultured cells in a culture medium to form a mixture, 
wherein the core material solution is a solution of alginate and 
carboxylmethyl cellulose; adding the mixture to a hardening solution 
containing CaCl.sub.2 and polyethyleneimine to form gel-core beads; and 
coating the gel-core beads with polylysine solution to form the 
liquid-core microcapsules. The microcapsules prepared by this method are 
hereinafter referred as Type B microcapsules.

DETAILED DESCRIPTION OF THE INVENTION 
According to the invention, core material suitable for use in the method 
includes alginate or carboxylmethyl cellulose, because they are 
polyelectrolyte compounds and thus have good physiological compatability 
with animal cells, are nontoxic and are able to form stable nonsoluble 
microcapsules under mild conditions. The core material should be in the 
form of a solution when used. According to the present invention, in the 
prepartion of Type A microcapsules, only alginate is used as the core 
material, and the concentration should be in the range of 0.2 to 1.0 
percent by weight, preferably 0.9 percent by weight. In the preparation of 
Type B microcapsules, a mixture of alginate and carboxulmethyl cellulose 
is used as the core material. The core material solution is added to the 
cultured cells contained in culture medium to form a mixture. The amount 
of the core material usually is 5 to 10 times that of the total amount of 
cultured cells and culture medium. 
The hardening solution of the invention contains CaCl.sub.2 and 
polyethyleneimine (PEI). The concentration of CaCl.sub.2 should be in the 
range of from 1 to 3, preferably 1.5 percent by weight and the 
concentration of PEI should be in the range of from 0.1 to 1.5, preferably 
0.5 percent by weight. The core material mixture is dropped into the 
hardening solution to form gel-core beads. 
To reinforce the mechanical stability of the formed gel-core beads, 
according to the method of the invention, coating with polylysine is 
necessary. The coating is conducted by immersing the gel-core beads into 
polylysine solution. The concentration of the polylysine should be in the 
range of from 0.05 to 1.0 percent by weight. 
According to the method of the invention, if only alginate is used as the 
core material, that is, in the case of preparing Type A microcapsules, the 
resulting gel-core beads should further be treated with citrate salts, for 
example sodium citrate, to dissolve the gel-cores to form the liquid-core 
microcapsules. However, if a mixture of alginate and polyethyleneimine is 
used as core material, as in the Type B microcapsules, it is not necessary 
to use citrate salts to dissolve the gel-cores because the content of the 
alginate is very low, for example 0.2 percent by weight, and therefore 
will be easily dissolved by phosphate which are usually included in 
culture medium for the cultured cells, to form liquid-core microcapsules. 
The invention is more specifically described by the following illustrative 
examples. 
Example 1 
Solutions containing respectively the core materials of the invention as 
set forth in Table 1 were dropped into hardening solutions having the 
compositions as set forth in Table 1. Gel-core beads were formed and 
tested for mechanical stability (mechanical strength). The test was 
conducted by placing the beads in a 50 mM citrate solution and observing. 
The gel-core beads are deemed stable if no rupture was observed within 10 
minutes. The test results are summarized in Table 1. 
The same procedures as Example 1 were repeated except the core materials 
used were alginate and dextran sulfate having concentrations as set forth 
in Table 1. The test results are also summarized in Table 1. 
TABLE 1 
__________________________________________________________________________ 
Hardening Solution 
CaCl.sub.2 2.5% CaCl.sub.2 1.5% 
DEAE- DEAE- 
Core dextran 
PEI PLL dextran 
PEI PLL 
Material 
1.0% 
0.5% 
1.0% 
0.5% 
0.05% 
1.0% 
0.5% 
1.0% 
0.5% 
0.05% 
__________________________________________________________________________ 
alginate 
1.0% - - + + - - - + + - 
0.7% - - + + - - - + + - 
0.5% - - + + - - - + + - 
alginate/ 
CMC 
0.5%/1.0% 
- - + + - - - + + - 
0.5%/0.5% 
- - + + - - - + + - 
alginate/ 
dextran 
sulfate 
0.5%/1.0% 
- - + + - - - + + - 
0.5%/0.5% 
- - + + - - - + + - 
__________________________________________________________________________ 
+: stable in 50 mM citrate solution for at least 10 min 
-: ruptured within 10 min in 50 mm citrate solution. 
As can be seen from the results of Table 1, including polyethyleneimine in 
addition to CaCl.sub.2 in the hardening solution can increase the 
mechanical stability of the gel-core beads. 
Example 2 
Solutions containing the core materials of the invention as set forth in 
Table 2 were dropped into a hardening solution containing 1.5 percent by 
weight of CaCl.sub.2 and 0.5 percent by weight of polyethyleneimine. 
Gel-core beads were formed. The formed gel-core beads were then coated 
with polylysine, polyethyleneimine and DEAE-dextran at concentrations 
indicated in Table 2, and tested for the mechanical stability by the same 
method as in Example 1. The test results are summarized in Table 2. 
The same procedures as Example 2 were repeated except the core materials 
used were alginate and dextran sulfate having concentrations as set forth 
in Table 2. The test results are also summarized in Table 2. 
TABLE 2 
______________________________________ 
Coating Material 
Core polyethyl- DEAE- 
Material polylsine eneimine dextran 
(wt %) 0.05% 0.1% 0.1% 
______________________________________ 
alginate 
0.7% + - - 
0.5% + - - 
alginate/CMC 
0.5%/1.0% + + - 
0.5%/0.5% + + - 
alginate/DS 
0.5%/1.0% + - - 
0.5%/0.5% + - - 
______________________________________ 
+: stable in citrate solution for at least 10 min 
-: ruptured within 10 min in citrate solution 
As can be seen from the results of Table 2, adding polyethyleneimine into 
hardening solution followed by coating with a layer of polylysine can 
enhance the mechanical strength of the formed gel-core beads. 
Example 3 
Solutions containing the core materials as set forth in Table 3 were 
dropped into hardening solutions having the compositions as set forth in 
Table 3. Gel-core beads were formed and then coated with 0.05 percent by 
weight of polylysine solution and washed with 20 mM, pH 7 sodium citrate 
solution for 10 minutes and tested for mechanical stability (mechanical 
strength). The test was conducted by placing the beads in a 20 mM 
phosphate solution and shaking it with a shaker at a rate of 200 rpm with 
an amplitude of 8 cm for 24 hours or 48 hours. The test results are 
summarized in Table 3. 
TABLE 3 
______________________________________ 
Hardening Solution 
CaCl.sub.2 (wt %) 
Core 2.5% 1.5% 1.0% 
Alginate 
polyethyleneimine, PEI (wt %) 
(wt %) 0 0.5 1.0 0 0.5 1.0 0 0.5 1.0 
______________________________________ 
0.9% 24 hr 0 98 99 0 98 98 0 96 93 
48 hr 0 93 90 0 97 91 0 95 93 
0.7% 24 hr 0 97 99 0 98 99 0 94 94 
48 hr 0 92 96 0 93 96 0 91 90 
0.5% 24 hr 0 96 100 0 100 100 0 90 100 
48 hr 0 88 100 0 97 99 0 85 99 
______________________________________ 
*the numeral indicates the percent of gelcore beads remaining unruptured 
after the shaking test 
As can be seen from Table 3 that all the gel-core beads coated with a 
hardening solution without polyethyleneimine ruptured and 90 percent of 
the gel-core beads coated with hardening solution containing both 
CaCl.sub.2 and polyethyleneimine remained unruptured. 
The same gel-core beads were prepared by following the same procedures as 
Example 3 except that no coating with polylysine were applied to the 
formed gel-core beads. The resulting gel-core beads were also tested by 
the same method. All gel-core beads without the polylysine coating 
ruptured. 
Example 4 
Gel-core beads were prepared by following the procedures of Example 3 
except that the concentration of polyethyleneimine, the hardening time and 
the coating time used are set forth in Table 4. The prepared gel-core 
beads were tested for their mechanical stability by the same method as 
Example 3 for 48 hours. The test results are summarized in Table 4. 
TABLE 4 
______________________________________ 
polyethyl- 
eneimine hardening time 
coating time (minutes) 
(wt %) (minutes) 6 10 15 
______________________________________ 
0.1% 6 0 0 15 
10 0 98 41 
15 31 75 80 
0.5% 6 0 0 17 
10 50 97 87 
15 55 55 94 
1.0% 6 0 0 10 
10 47 98 90 
35 75 70 96 
______________________________________ 
*the numeral indicates the percent of gelcore beads remaining unruptured 
after the shaking test 
As can be seen from Table 4 that if both the hardening time and the coating 
time exceed 10 minutes, most of the prepared gel-core beads remain 
unruptured. 
Example 5 
Gel-core beads were prepared by using 0.2 wt % alginate, 1.2 wt % 
carboxylmethyl cellulose as core material, 1.5 wt % CaCl.sub.2 and 
polyethyleneimine as hardening solution and 0.05 wt % polylysine solution 
as coating solution. The concentration of polyethyleneimine, the hardening 
time and the coating time are set forth in Table 5. The prepared gel-core 
beads were tested for their mechanical stability by the same method as 
Example 3 for 48 hours. The test results are summarized in Table 5. 
TABLE 5 
______________________________________ 
polyethyl- 
eneimine hardening time 
coating time (minutes) 
(wt %) (minutes) 6 10 15 
______________________________________ 
0.1% 6 0 0 0 
10 0 70 99 
15 75 95 100 
0.5% 6 0 0 0 
10 7 87 100 
15 55 95 100 
1.0% 6 0 0 0 
10 0 78 91 
15 75 100 100 
______________________________________ 
*the numeral indicates the percent of gelcore beads remaining unruptured 
after the shaking test. 
As can be seen from Table 5 that if the hardening time and the coating time 
exceed 10 minutes, most of the prepared gel-core beads remain unruptured. 
Also, as can be seen from Table 4 and Table 5, if the hardening time and 
the coating time are within 10 to 15 minutes, the quality of the resulting 
gel-core beads remains mostly unaffected, indicating the operating of the 
present method is more flexible than conventional methods. 
Example 6 
Murein hybridoma CT04 obtained from Veterans General Hospital, Taipei, 
Taiwan was cultured in a culture medium containing F12:DMEMG(1:1), 10 wt % 
FCS, penicillin G(100 ng/ml) and streptomycin (100 ng/ml) in T flask. The 
cultured Murein hybridoma CT04 was then centrifugated with 800.times.g for 
5 minutes, recovered and suspended in an Erlenmeyer flask. A Murein 
hybridoma CT04 solution with a concentration of 5.times.10 cells/ml was 
obtained. 
The prepared Murein hybridoma CT04 solution was then encapsulated with 
various microcapsules. The preparation of these microcapsules are 
summarized in Table 6 below. 
TABLE 6 
______________________________________ 
Core hardening polylysine 
Citrate 
Material 
Solution coating Treatment 
______________________________________ 
Alginate 0.9% 1.5% 
Gel-beads 
alginate CaCl.sub.2 
0.05% no 
Polylysine- 
0.9% 1.5% 
coated liquid- 
alginate CaCl.sub.2 
0.05% yes 
core capsules 
PEI- 0.9% 1.5% 
reinforced 
alginate CaCl.sub.2 + 
0.05% no 
gel-beads 0.5% PEI 
A Type 0.9% 1.5% 
microcapsules 
alginate CaCl.sub.2 + 
0.05% yes 
0.5% PEI 
B Type 0.2% 1.5% 
microcapsules 
alginate + 
CaCl.sub.2 + 
0.05% no 
1.2% CMC 0.5% PEI 
______________________________________ 
*the concentration indicated in the table is wt % 
In the preparation of the above microcapsules, the amount of the core 
material solution added is 9 times that of the Murein hybridoma CT04 
solution. The volume ratio of core material solution to the hardening 
solution is 1:20, and the volume ratio of the formed gel-core beads to the 
polylysine solution is also 1:20. The hardening time and the coating time 
in all the above prepareations are both 6 to 15 minutes. 
The encapsulated Murein hybridoma CT04 cells were then placed in T-flasks 
and incubated in a state of 20 microcapsules per ml of culture medium. The 
cell concentrations after incubating for 12 days are shown in FIG. 1. 
As can be seen from FIG. 1, the viable cell concentrations cultivated by 
using Type A and Type B microcapsules of the invention are higher than 
that of conventional microcapsules. 
Example 7 
Gel beads, Type A and Type B microcapsules were prepared by following the 
procedures of Example 6 except that concentrations of polyethyleneimine 
(PEI) in the hardening solutions were used as indicated in Table 7 below. 
TABLE 7 
______________________________________ 
PEI in hardening 
Cell Concentration 
solution (10.sup.6 cells/ml-capsules) 
solution (wt %) 
7th day 10th day 12th day 
______________________________________ 
gel-core beads 
0% 5 5 16 
0.1% 5 4 10 
0.5% 10 32 42 
1.0% 9 17 33 
A Type 
0.1% 10 49 73 
0.5% 16 28 47 
1.0% 10 21 68 
B Type 
0.5% 20 49 70 200* 
______________________________________ 
*the result of the 15th day 
As can be seen from Table 7, by using the microcapsules of the invention, 
the cell concentrations can reach 2.times.10.sup.8 per ml which is far 
larger than the cell concentration contained by using conventional 
microcapsules. 
Example 8 
Type A microcapsules were prepared by adding a mixture containing 1 wt % 
alginate and the above prepared Murein hybridoma CT04 solutions to a 
hardening solution containing 1.5 wt % of CaCl.sub.2 and 0.1 wt % of 
polyethylene imine, coating the formed gel-core beads with 0.05 wt % 
polylysine solution and followed by washing with water and sodium citrate 
solution. 
The encapsulated Murein hybridoma CT04 cells were then placed in a T-flask 
and cultivated in a state of 20 microcapsules per ml of culture medium for 
10 days. The rate of glucose uptake, lactate prodution and IgG production 
of Murein hybridoma CT04 cells after the cultivation are shown in Table 8 
below. The amount of glucose uptake, lactate production and IgG production 
from the encapsulated cells is shown in FIG. 4. 
The rate of glucose uptake, lactate production and IgG production from the 
cells encapsulated by alginate gel-beads and polylysine coated liquid-core 
capsules of Table 6 were measured and also summarized in Table 8. The 
amount of the glucose uptake, lactate production and IgG production of 
alginate gel-beads and polylysine coated liquid-core microcapsules are 
respectively depicted in FIG. 2 and FIG. 3. 
Example 9 
Type B microcapsules were prepared by adding a mixture containing 1.2 wt of 
carboxylmethyl cellulose and 0.2 wt % pf alginate and the above prepared 
Murein hybridoma CT04 solutions to a hardening solution containing 1.5 wt 
% of CaCl.sub.2 and 0.1 wt % of polyethylene imine, coating the formed 
gel-core beads with 0.05 wt % polylysine solution and followed by washing 
with water and sodium citrate solution. 
The encapsulated Murein hybridoma CT04 cells were then placed in a T-flask 
and cultivated in a state of 20 microcapsules per ml of culture medium for 
10 days. The rate of glucose uptake, lactate prodution and IgG production 
of Murein hybridoma CT04 cells after the cultivation are shown in Table 8 
below. The amount of glucose uptake, lactate production and IgG production 
from the encapsulated cells is shown in FIG. 5. 
TABLE 8 
______________________________________ 
glucose 
lactate IgG 
uptake production production 
______________________________________ 
Free Suspension 
0.25* 0.28* 0.12** 
Alginate gel-bead 
0.26 0.22 0.15 
Polylysine-coated 
0.40 0.35 0.12 
liquid-core capsules 
Type A microcapsules 
0.25 0.24 0.091 
Type B microcapsules 
0.16 0.20 0.090 
______________________________________ 
*.mu.moles/10.sup.6 cellshr 
**.mu.g/10.sup.6 cellshr 
Example 10 
Type A microcapsules prepared in Example 8 were placed in a reverse 
funnel-shaped air-lift fermenter and incubated at a air flowing rate of 50 
ml/min for 21 days. The cell concentration was measured to be 
2.times.10.sup.8 cells/ml and no ruptured cells were observed. The amount 
of the glucose uptake, lactate production and IgG production from the 
encapsulated Murein hybridoma CT4 cells is shown in FIG. 6.