JPS63209581A - Embedding culture of attachment-dependent animal normal cell - Google Patents

Abstract

PURPOSE:To make it possible to carry out high-density culture of titled cell in a large amount, by bonding attaching animal normal cells to a fine granular biological high polymer carrier and embedding the cell in gel of calcium alginate. CONSTITUTION:Attaching animal normal cell are seeded in a microcarrier consisting of a biological high polymer such as gelatin, collagen, etc., in such a cell density that 5-10 cells are bonded to one carrier and cultivated in 5% carbonic acid gas concentration atmosphere for 1-3hr. to bond the cells to the carrier. The microcarrier is blended with 1.6% solution alginic acid obtained by dissolving alginic acid in 0.85% sodium chloride injection in such a way that the final concentration of alginic acid gel is 0.3-1.0%. The mixed solution is added from a nozzle or a sterilized pipette to a solution obtained by adding 0.001-0.01% Tween80 to sterile calcium chloride to give a cell embedded microcarrier, which is cultivated in a medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an embedding culture method for culturing adherent normal animal cells in large quantities.

Jubei Saban: Conventionally, in order to mass culture cultured cells that grow in an adherent manner, that is, adhesion-dependent cells, cells are attached to the surface of a cylindrical bottle with a large surface area, and the bottle is rotated for cultivation. It is known that so-called microcarrier culture, in which cells are attached to the surface of a dish culture or a particulate carrier and cultured while stirring, is effective.

However, animal normal cells, such as normal 2 derived from human IIN tissue, which is a t-blasminogen activator producing cell.
When culturing the above-mentioned microcarriers in the case of diploid fibroblasts, the cells tend to peel off from the microcarriers due to the bridging phenomenon that occurs with cell proliferation and the shearing action caused by stirring. In addition, serum-free media for the production of useful substances have the problem that the shearing force caused by agitation causes the cells to detach from the carrier and die, making stable substance production impossible.

There is also a known method for mass culturing free or adherent cancer cells or transformed cells by embedding them in a gel body such as calcium alginate. No cell proliferation is observed even when cultured in a manner similar to normal adherent animal cells.

However, in recent years, as seen in the production of t-plasminogen activator, with the development of technology to produce useful physiologically active substances using the culture of normal animal cells,
There is a need to establish a technology for mass culture of normal animal cells.

The present invention was made in view of the above-mentioned circumstances, and an object thereof is to provide an embedding culture method for culturing adherent animal normal cells in large quantities at high density. shall be.

The present inventor has discovered that by embedding adherent normal animal cells attached to a microparticulate biopolymer carrier in a gel body of calcium alginate, the above-mentioned cell detachment phenomenon does not occur. We succeeded in culturing in large quantities at high density, leading to the present invention.

The present invention will be explained in detail below.

Kokai (2) 1-bottom A feature of the present invention is that adherent normal animal cells adhered to a particulate biopolymer carrier are mixed with an alginic acid solution that has been sterilized by dissolving alginic acid in physiological saline. The final result of alginate gel? The concentration was adjusted to 0.3 to 0%, and then the mixture was dropped into a sterilized calcium chloride solution containing a nonionic surfactant to prepare alginic acid in which normal cells were embedded. The purpose is to prepare microparticle carriers made of calcium and to culture the microparticle carriers in a medium supplemented with serum or the like.

In the present invention, adherent animal normal cells are mixed with a carrier made of biopolymers such as gelatin, collagen, etc. in the form of fine particles.
That is, it is important to embed what is attached to the microcarrier in a gel body of calcium alginate.

Microcarriers made of the above biopolymer can be formed by conventionally known methods. To attach the normal cells to the microcarriers, seed them at a cell density such that 5 to 10 cells adhere to each microcarrier (in a furan vessel with a 5% carbon dioxide atmosphere). Pre-cultivation is preferably performed for 1 to 3 hours.

The cells attached to the microcarriers as described above are mixed with an alginic acid solution prepared as described below.

Preparation of alginic acid solution 20. Dissolve alginic acid to a concentration of 1.6% in 85% physiological saline, and sterilize this solution by filtering. This sterilization was initially carried out using a 0.8 μm filter, then a 0.8 μm filter.
It is recommended to use a 45 μm filter and a 0.22 μl filter for R1&.

The alginate solution prepared once in this way is mixed with the microcarriers to which the above-mentioned cells have been attached, and at this time,
Mixing is performed so that the final concentration of alginate gel is (), 3-1.0%. In this case, if the final 4 degree of the alginate gel after mixing is less than 0.03%, the gel strength will be low and will not be able to withstand stirring, while if it exceeds 1.0%, the growth of cells on the microcarriers in the gel will be inhibited. suppressed.

Next, the above liquid mixture is dropped into a calcium chloride solution prepared as described below to prepare a microparticle embedding carrier (macrocarrier) made of calcium alginate.

Preparation of calcium chloride solution: 50-100+1 with the addition of a non-ionic surfactant, preferably an ethylene oxide condensate of sorbitan monooleate (Tween 80: o, ooi% to 0.01%)
M? After sterilizing the calcium chloride aqueous solution in an autoclave, it is allowed to cool to room temperature. The nonionic surfactant added to the calcium chloride aqueous solution used here serves to lower the surface tension of the calcium chloride solution to facilitate the formation of an embedding gel upon dropping the sodium alginate solution.

-1- The above-mentioned mixed solution may be added dropwise to the calcium chloride solution in accordance with conventional methods for preparing macrocarriers used for embedding culture of cancer cells and the like.

That is, to create the macrocarrier, the above-mentioned mixture of sodium alginate and cell-attached microcarriers is added to a solution of sterile calcium chloride with 0.001 to 0.01% Tween 80 added through a nozzle (or sterile pipette). Created by dripping. The size of the macro piece can be controlled by freely changing the diameter of the nozzle. Microcarriers with attached cells are embedded in the generated macrocarriers. The cell concentration in the above microcarrier is such that 5 to 10 cells are embedded in each microcarrier. Because they are fixed in the gel, they do not come off during culture due to bridging or agitation, unlike cells attached to microcarriers as described above.
In addition, it is less affected by external environmental factors, so
longevity of the cell itself
is also longer.

Therefore, by culturing this macrocarrier in, for example, a serum-supplemented medium, it becomes possible to culture adhesion-dependent normal animal cells in large quantities at high density.

Note that cells can be attached to microcarriers in the following manner.

Microcarriers made of biopolymers (gelatin, collagen, etc.) were swollen with PBS and then incubated at 121'C.
Sterilize in an autoclave for 10-15 minutes.

PBS of microcarrier suspension sterilized by autoclave was mixed with cell culture medium (DM) supplemented with 10% FC3.
I? :M or MEM), then inoculate adherent normal cells in a microcarrier spinner flask so that about 5 to 10 cells adhere to each microcarrier, and inoculate them in a 5% COt atmosphere. Stir vigorously at ~4 o'clock (leave for 30 minutes, stir for 1 minute, repeat for 1 to 4 hours). After the cells have completely attached to the microcarriers, a predetermined concentration of sodium alginate i8? fl (final concentration of sodium alginate in the mixture 0.
3-1.0%, and the cell-attached microcarriers can be freely set to the desired concentration).

As described above, according to the present invention, by culturing adhesion-dependent animal normal cells as macrocarriers prepared by adhering them to microcarriers made of biopolymer and embedding them in a gel body of calcium alginate. The present invention can be said to be extremely useful in terms of technology for producing useful physiologically active substances by culturing normal cells, since it becomes possible to culture the above-mentioned normal cells in large quantities, which has conventionally been regarded as a national fortune.

EXAMPLES The present invention will be specifically described below with reference to Examples.

Example 1 Human fetal lung-derived normal diploid fibroblast mMR-90 (A
TCC, CCL-186) in a cell culture flask (T-
175 enl) until 80% confluence. The obtained double-length cells were washed with sterilized PBS to remove medium components, and then treated with trypsin to reduce the total number of cells to 5XlO.
'I got a piece. The obtained cells were added with 10% FC3.
MEM (Dulbetzko's modified Eagle's medium, old +1bec
eo'sModified Eagle Mediuw
l) Suspended at 1.5 m&).

On the other hand, microcarriers formed from gelatin (Jelibis, manufactured by KC Biological) were swollen with PBS, sterilized in an autoclave,
The microcarrier concentration was adjusted to 24 μg/ml by replacing it with I) MEM supplemented with C3. 50++ 15m7 of the above cell suspension in a 1 volume MA・FRIROCARRIER 11"rJ culture spinner bottle! (Number of cells: 5
Add 5 mJ of microcarrier suspension (120 mg of microcarriers), replace the space with 5% CO□, let it stand for 30 minutes, and repeat the operation for 1 minute twice. Stirring culture was carried out repeatedly for ~3 hours. After the cells have completely attached to the microcarriers,
Cell-adhered microcarriers were collected by sinking the microcarriers by low-speed centrifugation (500-600 rpm, 5 minutes) or by standing still, and removing the supernatant medium.

This cell (1 microcarrier containing 30% FC3 was suspended in 15 ml!
It was mixed with a 0.48% sodium alginate solution that had been sterilized by filtration.

The resulting mixed liquid was 40mjl! into a sterile syringe fitted with an 18-gauge needle, and while applying pressure, inject i/i!
Alginate gel macrocarriers were prepared by embedding cell-attached microcarriers by dropping them into a 50 d calcium chloride solution containing 0.01% Tween 80. The final sodium alginate concentration was 0.3%.

Next, 40 m Q of a mixture of the cell-attached microcarrier suspension and sodium alginate solution was added to each group, 5
A total of 8 groups of macrocarriers were formed by equally dividing each macrocarrier into al. After each macrocarrier was prepared, the calcium chloride solution was replaced with physiological saline for washing within 30 to 60 minutes, then 10% FC3 was added, and the stomach was washed with DMEM.

The macrocarriers washed with the medium as described above are
250s I sterilized with T-ray! polystyrene bottle (
Corning) and added 10% FC3 I) M
50 tae of rEM was added to each group, 5% COz was blown into the headspace, and the mixture was placed in a shaking incubator for bacterial culture and incubated at 37°C while shaking with a 40RP steel.
Culture was performed for 10 days.

After culturing, 4 of the above 8 groups were used to measure the number of cells, and the other 4 groups were
The group was used for the production of L-blasminogen activator. To produce t-blasminogen activator, only the medium is removed from the macrocarrier culture solution (cultivated at 37°C for 10 days) using a bibet, and the macrocarrier is washed with physiological saline or PΔS to remove the medium components. After removing 1% proteose bebutone and 0.01% T
i) Add MEM10P to which ween80 was added, 3
The cells were cultured at 7° C. for 7 days with shaking at 40 rpm. The reason why 0.01% Tween 80 is added to the medium is to prevent the t-plasminogen activator from being adsorbed in the macrocarrier and to smoothly release it from the macrocarrier into the medium.

In addition, to measure the number of cells after the above culture, after dissolving 100mM of macrocarriers made of alginate gel with EDTA (ethylenediaminetetraacetic acid), centrifuging the cell-attached microcarriers (600~QOOrp Akebono,
The microcarriers were harvested for 10 minutes, treated with trypsin, and the cells were detached from the microcarriers and counted.

As a result of the above measurement, the average number of cells in each bottle was 5 x 10'/bottle (average 8 cells per microcarrier) at the start of culture, and 3 cells after 10 days of culture at 37°C.
．． It was 5 x 10"/bottle.

On the other hand, the production status of (-blasminogen activator) according to the number of culture days is as shown in Table 1.

Table 1 Example 2 Human fetal lung-derived normal 2 (i'W body fibroblast mMR-9
0 (ATCC, CCL-186) as in Example 1.
- culture in flasks to obtain 4.5 x 10' seed cells;
It was attached to 120 mg of microcarriers (jelly beads, manufactured by KC Biological). This cell-attached microcarrier (microcarrier amount 120mg)
suspension of 15mj! , 25mj of filter sterilized 0.8% sodium alginate solution! Mix with 40% of the mixed liquid
I got m i.

This mixture '111 (40 ml) was added to 8
While dividing into equal parts, add 0.01% from the tip of a 5-liter pipette.
They were each added dropwise to a 50++M CaCl solution containing Tween 80 to prepare alginate bead macrocarriers. The final concentration of sodium alginate in the carrier was 0.5%, and the microcarriers attached to the cells were embedded and fixed in the macrocarriers.

In addition, 5m as stated! ! When the tip of the pipette 1- was used as a nozzle for dropping, the diameter of the macrocarriers formed was about 4 to 5 mta.

Next, the macrocarriers of each group thus obtained (groups divided into 8 equal parts) were washed in the same manner as in Example 1, suspended in 50 mIt of DMEM supplemented with 10% FC3, and then
The mixture was transferred to a 0 ml polystyrene bottle (manufactured by Corning) and cultured at 37°C for 10 days while shaking at 100 rpm in a shaking incubator for bacterial culture.

After culturing, 4 of the 8 groups were used for cell count measurement;
The group was used for the production of t-plasminogen activator.

Toblasminogen activator was produced in the same manner as described in Example 1, except that the rotational speed was 1100 rpm.

The results of cell number measurement and t-plasminogen activator production are shown in Tables 2 and 3.

Example 3 Human fetal kidney-derived normal diploid fibroblast cell Flow 400
0 was cultured in a T-flask in the same manner as in Example 1 to obtain seed cells. , I X 10'' and microcarrier (
Collagen beads (manufactured by Funai Yakuhin Co., Ltd.) (168 mg) were attached.

18.5 m/ml of a suspension of this cell-attached microcarrier (Mike [J-Toyaria amount: 168 mg) and 31.5 mf/filter-sterilized 1.6% sodium alginate solution.
Mix f, total 50117! A mixed solution of

Divide this mixed solution (5 (bwi) into 10 equal parts of 5 mf each and pipette with 0.01%
Alginate bead macrocarriers were prepared by dropping each into a 50mM CaCIz solution containing Tween 80. The final concentration of sodium alginate in this macrocarrier was 1.0%, and the cell-attached microgearia (collagen beads) were embedded and fixed in the macrocarrier. The diameter of macro beads is approximately 4 m.
It was 111.

After washing the macrocarriers of each group (10 groups) obtained as described above, DMEM50s containing 10% FC3 was added.
I! After suspending the suspension in 250a+ 1 volume polystyrene bottle (manufactured by Corning), it was incubated at 37°C for 1 hour while shaking at 100 rpm in a shaking incubator for bacterial culture.
Cultured for 0 days.

After culturing, 5 of the 10 groups were used to measure the number of cells, and the other 5 groups were used to produce L-plasminogen activator.

Toblasminogen activator production is performed at a rotational speed of 1
The same procedure as described in Example 1 was followed except that 00rps+ binding was performed.

The results of cell number measurement and t-blasminogen activator production are shown in Tables 4 and 5.

Claims (4)

[Claims] (1) Adhesive normal animal cells adhered to a particulate biopolymer carrier are mixed with an alginic acid solution that has been sterilized by dissolving alginic acid in physiological saline, and the final concentration of the alginate gel is adjusted to 0. 3 to 1.0%, and then the mixture was dropped into a sterilized calcium chloride solution containing a nonionic surfactant to embed the normal cells attached to the carrier. 1. A method for embedding adherent animal normal cells, which comprises preparing microparticle carriers (macrocarriers) made of calcium alginate and culturing the microparticle carriers in a medium. (2) 1 x 10 of the above animal normal cells on a biopolymer carrier
The embedding culture method according to claim (1), wherein the seeds are seeded in 5%/ml carrier or more, precultured for 1 to 3 hours in a 5% carbon dioxide atmosphere, and then attached to the carrier. (3) The embedding according to claim (1), wherein the alginic acid solution is obtained by dissolving alginic acid at a concentration of 1.6% in 0.85% physiological saline and sterilizing it by filter filtration. Culture method. (4) The calcium chloride solution is a solution containing 0.001 to 0.01% of an ethylene oxide condensate of sorbitan monooleate as a nonionic surfactant and having a calcium chloride concentration of 50 mM. (1)
Embedded culture method described in section.