JPH07313151A - Sequential culture method for animal cells using porous carrier - Google Patents

Abstract

(57) [Summary] [Objective] In animal cell culture using a porous carrier composed of a natural polymer, cells are transferred from carrier to carrier without using trypsin or a chelating agent for the cells,
To provide a method for continuously growing cells without impairing cell activity. [Structure] In animal cell culture using a porous carrier for cell culture, which comprises a natural polymer and / or a derivative thereof, an old porous carrier having cells attached to the surface and / or the inside thereof is replaced with a new one having no cells attached. A method of sequential culture of animal cells, which comprises transferring cells from one carrier to another by mixing with a porous carrier and continuously growing the cells.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a cell culture technique using a porous carrier, which is developing in the field of bioindustry, and more specifically, by continuously growing cells without impairing cell activity. Cell culture scale-up technology.

[0002]

2. Description of the Related Art Conventionally, in cell culture using a carrier,
As a method of passage of cells from carrier to carrier, cells are recovered from the carrier by an enzyme treatment represented by trypsin, and then trypsin is inactivated in a medium containing serum,
A method is generally known in which cells are recovered from a carrier by a chelating agent treatment, a medium is added to neutralize the chelating agent, and then a new carrier is added and transferred (Ph.
rmcia LKB Biotechnology,
Microcarrier cell culture
principles & method (19
88) 80, Yutaka Matsutani, Introduction to Animal Cell Culture Method (1993)
215-223).

However, trypsin changes the viability of cells and removes surface-binding molecules of cells (Kelly RO et al., On the Natu).
reof the external surface
of culture human embryo
fibroblasts. Differentiati
It is known that there is a drawback that the cells are greatly damaged, such as on 3). In addition, in the case of large-scale culture, an auxiliary device for detaching / collecting cells is required, and the cell detachment process is complicated or requires a long time. In addition, trypsin is not preferable because trypsin may be contaminated with viruses, nucleic acids and the like from the animal from which it is derived, and the product (medicine etc.) is contaminated by these.

In addition, as a method of substituting cells from carrier to carrier, there is known a method of adding a new carrier when the culture approaches a confluent state. For example, "DEAE-Se
"Padex particles" (manufactured by Phrmcia) (Manousos.M. et al., Feasibili)
ty studios of oncornaviru
s production in microcarr
ier cultures. In Vitro 15
(1980) 507-515) and an example of a carrier comprising a hydrophilic synthetic polymer such as an acrylic acid ester or a methacrylic acid ester (Japanese Patent Laid-Open No. 5-91872). However, the former is limited to the case of using special cells (RD: human rhabdomyosarcoma) and requires an operation to disperse the cell mass released from the old carrier, and the transfer ratio is 1.4- There is a problem in that it has a low ratio of 2.0 times, and the latter has a low transfer ratio of 15 times or less,
It was insufficient.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides an animal cell culture using a porous carrier composed of a natural polymer,
Without using trypsin or chelating agent for cells,
It is an object of the present invention to provide a method for transferring cells from carrier to carrier and continuously proliferating them without impairing cell activity.

[0006]

The present inventors have completed the present invention as a result of intensive studies to achieve the above object. That is, in the present invention, in a culture method in which cells are fixed in their three-dimensional structure using a porous carrier and suspended in a culture solution, an old carrier with attached cells and a new carrier without attached cells are mixed. Thus, by transferring cells from an old carrier to a new carrier, it has succeeded in enabling the passage of cells without using an enzyme such as trypsin or a chelating agent, and has reached the present invention.

That is, the present invention relates to a natural polymer and / or
Alternatively, in animal cell culture using a porous carrier for cell culture comprising a derivative thereof, by mixing an old porous carrier having cells attached to the surface and / or the inside thereof with a new porous carrier having no cells attached, The method for sequential culture of animal cells is characterized in that cells are transferred from carrier to carrier and continuously grown. Hereinafter, the present invention will be described in detail.

The natural polymer in the present invention includes cellulose, gelatin, collagen and the like, and among these, cellulose is particularly preferable. In the present invention, it is important to use a porous carrier made of a natural polymer and / or its derivative as a carrier for cell culture. By using a porous carrier, it is possible to transfer cells at a high rate (scale up), which could not be performed with a carrier having no porous structure. This is because the porous carrier has a larger number of fixed cells per carrier particle than the mere spherical carrier, so that the number of cells that proliferate and are released from the old carrier is relatively large, and that the porous carrier has a three-dimensional structure. Therefore, it is considered that the new carrier can easily acquire the released cells.

The porous carrier has a particle size of 100 μm.
˜5 mm is preferable, and further 180 μm-210
mm is preferable. The pore diameter is preferably 10 μm or more, more preferably 20 μm to 50 μm. The positive charge density of the porous carrier is 0.1.
Although the thing of -4.0 mEq / g can be used, the range of 0.5-2.5 mEq / g is preferable from a cell proliferation viewpoint. The present invention utilizes the property that cells actively move from an old carrier to a new carrier, but the transfer efficiency of cells is limited by the release efficiency of cells from the old carrier. So, in seed culture (culture before cell transfer),
By using a carrier having a low charge density, the migration rate of cells from the old carrier to the new carrier is increased. this is,
This is because the lower the charge density of the carrier is, the smaller the charge attractive force for attaching the cells is, and thus the cells grown in the seed culture are frequently released from the carrier.

Further, the higher the density of adherent cells per carrier of the old carrier, the more frequently newly proliferated cells are released, resulting in higher cell transfer efficiency.
In the present invention, the transfer ratio of the cell culture carrier is 1.1 to 10.
Good cell transfer is observed up to 00 times, but if the transfer ratio is too small, the scale-up efficiency is poor and it is not practical, so 16-1000 times is preferable.
Further, by enabling the scale-up at a high ratio up to 1000 times, it is possible to reduce the number of scale-ups when performing large-scale culture, which leads to a reduction in cell culture equipment cost. Here, the transfer ratio is represented by the following formula.

Transfer ratio = (a + b) / a where a = dry weight of the old culture carrier b = dry weight of the new culture carrier In the present invention, the culture solution is suspended or pseudo-suspended from the old carrier and then new. It is preferable to transfer the cells to a carrier. This means that cells do not have to be subjected to an operation step in which cells are once settled and transferred from carrier to carrier, and cells are transferred while maintaining a uniform state as a culture system. This causes the cells to move uniformly from the old carrier to the new carrier.

[0012]

EXAMPLES The present invention will be specifically described below with reference to examples.
However, the present invention is not limited to the following examples.
Yes. The charge density, particle size, and pore size of the porous carrier in the examples
Was measured by the following method. (1) Charge Density The porous carrier was 0.3N HCl aqueous solution, 10^-FourNHC
1 aqueous solution, 10% Na ₂SO_FourAqueous solution (At this time, for titration
Of the filtrate), washed with water and dried,
Add 2 ml of 0.1M potassium chromate aqueous solution and add 1M
AgNO₃Titrate with aqueous solution. Titration, porosity after drying
The weight of the carrier was substituted into the following formula to calculate the charge density.

Charge density = titration amount (ml) / weight of porous carrier after drying (g) [meq / g] (2) Particle size and pore size The particle size remains undried in an optical microscope (Olympus Optical Industry). It was measured by IMT-2) manufactured by the company. At this time, 50
For fine particles having a size of μm or less, the undried particles after washing are rapidly cooled with liquid nitrogen and frozen while maintaining the structure, and then freeze-dried in a vacuum, and a scanning electron microscope (SEM) (S-manufactured by Hitachi Ltd. Observation measurement using 570) was also used.

The pore diameter was measured by observing the sample, which had been freeze-dried, by subjecting it to gold sputtering and magnifying it to an appropriate magnification with SEM. When the carrier was not a perfect sphere or a perfect circle, the smallest diameter was measured.

[0015]

[Example 1] As a porous carrier for animal cell culture, a porous carrier made of cellulose "Asahi Kasei Microcarrier" (trade name; manufactured by Asahi Kasei Corporation, diameter 200μ
m, pore size 30 μm, charge density 0.98 mEq / g), and 10% calf serum in a 100 ml spinner flask (Belco) so that the carrier has a dry weight of 2.0 g / l. E-RD including (Hyclone)
Prepared by suspending in 100 ml of F medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) and genetically modified CHO cells (mouse IL-4 producing strain)
To a density of 2.0 × 10 ⁵ cells / ml and 37
The stirring culture was carried out at 0 ° C for 5 days. The cell density on day 5 was 6.
It was 4 × 10 ⁶ cells / ml. This culture will be called a seed culture. The medium used here will be referred to as a growth medium. In another 100 ml spinner flask, add "Asahi Kasei Microcarriers" to 0.1
Prepare by suspending it in the growth medium so that it becomes 9 g / 95 ml,
5 ml of the above-mentioned culture medium on the 5th day of seed culture (including the porous carrier to which cells were attached) was added, and the culture was continued. The transfer ratio in this example is 20 times.

During the culture after the transfer, the cell density and the adhesion rate represented by the following formula were measured every day. The increase in attachment rate represents the efficiency of cell migration from the old carrier to the new carrier. Adhesion rate = x × 100 / (x + y) where x = number of porous carriers with attached cells y = number of porous carriers with no attached cells As a result, after transfer, as shown in FIG. The cells smoothly repeated logarithmic growth. Further, from the proliferation of the adhesion rate (Fig. 2), it was confirmed that the cells were transferred from the old porous carrier to the new porous carrier.

[0017]

Example 2 As an animal cell culture porous carrier, four types of "Asahi Kasei Microcarriers" having different charge densities as shown below were prepared. 0.58 mEq / g 0.98 mEq / g 1.59 mEq / g 2.02 mEq / g The above-mentioned and two types of charge density porous carriers are added to a dry weight of 2.0 g / l to separate 100 ml each.
Growth medium 100 as in Example 1 in a spinner flask.
The cells were suspended and prepared, and the genetically modified CHO cells as in Example 1 were inoculated at a density of 2 × 10 ⁵ cells / ml and agitated and cultured at 37 ° C. for 4 days. This culture will be called a seed culture. The cell density on the 4th day of seed culture was 4.0 × 1 in the culture using the above porous carrier.
0 ⁶ cells / ml, 6.0 × 10 ⁶ cells / ml in culture using the above porous carrier.

In addition, 0.19 g of each of the above three kinds of porous carriers and 95 m of the growth medium are added.
Two 100 ml spinner flasks each prepared by suspending in 1 l were prepared, and 5 ml of the above-mentioned culture medium on the 4th day of seed culture (including the porous carrier to which the cells were attached) was transferred in the following combination. The culture was continued. The transfer ratio in this case is 20 times. (1) 0.58 mEq / g before transfer → 0.98 mEq / g after transfer (2) 0.58 mEq / g before transfer → 1.59 mEq / g after transfer (3) 0.58 mEq / before transfer g → 2.02 mEq / g after transfer (4) 0.98 mEq / g before transfer → 0.98 mEq / g after transfer (5) 0.98 mEq / g before transfer → 1.59 mEq / g after transfer (6) Before transfer 0.98 mEq / g → after transfer 2.02 mEq / g During the culture after transfer, the cell density and the adherence rate (the definition is the same as in Example 1) were measured every day. The increase in attachment rate represents the efficiency of cell migration from the old carrier to the new carrier.

As a result, as shown in FIG. 3 (Table 1), the cell growth after cell transfer was logarithmic growth without being affected by the charge density of the microcarriers. However, as shown in FIG. 4 (Table 2), the variation in the adhesion rate, which represents the cell migration efficiency, was 0.98 mEq when the porous carrier having a charge density of 0.58 mEq / g was used in the seed culture. /
The increase was faster than the case of using g. This indicates that the lower the charge density of the microcarriers used in seed culture (when 0.58 mEq / g), the better the cell migration efficiency after cell transfer.

[0020]

[Table 1]

[0021]

[Table 2]

[0022]

Example 3 Asahi Kasei Microcarriers (charge density 0.98 mEq / g) as a porous carrier for animal cell culture
Prepared. This was suspended and prepared in 100 ml of a growth medium similar to Example 1 in a 100 ml spinner flask so as to have a dry weight of 0.5 g / l, and 1.25 cells of genetically modified CHO cells similar to Example 1 were prepared. × 10 ⁵ ce
The seeds were inoculated to a density of 11s / ml and cultured at 37 ° C. for 5 days with stirring. The cell density on the 5th day was 3.1 × 10 ⁶ cells.
It was s / ml. This culture will be referred to as a first generation seed culture.

In a separate 100 ml spinner flask, 0.0495 g of the same "Asahi Kasei Microcarriers" was suspended in 99 ml of a growth medium to prepare a second generation culture. (Including the porous carrier to which was attached) was added and the culture was continued. The transfer ratio in this example is 100 times. Further, when the cell density increased to about 100 times after the transfer, the cells were transferred again at a 100-fold ratio by the same method. This was repeated 3 times and the culture was continuously continued until the 4th generation.

The cell density was measured every day during the culture.
As a result, the cell growth after the cell transfer was repeated logarithmic growth even if the passage number was increased, as shown in FIG.

[0025]

Example 4 Asahi Kasei Microcarriers (charge density 0.98 mEq / g) as a porous carrier for animal cell culture
Prepared. This was suspended in 100 ml of a growth medium similar to Example 1 in a 100 ml spinner flask so as to have a density of 0.5 g / l dry weight, and 3 × 10 3 recombinant CHO cells similar to Example 1 were prepared. ⁵ cells
Inoculation to a density of 1 ml / ml was carried out at 37 ° C. for 3 days with stirring. The cell density on the 3rd day was 3.8 × 10 ⁶ cells / m 2.
It was l. This culture is called seed culture.

In a separate 100 ml spinner flask, the same "Asahi Kasei Microcarriers" was prepared by suspending it in a growth medium at a density of 1.0 g / l, and a seed culture medium (a porous carrier having cells attached thereto was prepared). 0.2 ml) was added and the culture was continued. The transfer ratio in this case is 10
It is 00 times. As a result, the cell density immediately after transfer was 7.6 × 10 ³ cells / ml, whereas
The cell density on the day reached 1.3 × 10 ⁶ cells / ml. In addition, the adhesion rate was changed from 0.1% immediately after transfer to 9%.
It increased to 95% on the day, and cells were transferred from the old porous carrier to the new porous carrier and proliferated even after the transfer at a high ratio of 1000 times.

[0027]

Example 5 As a porous carrier for animal cell culture, "Asahi Kasei Microcarriers" (1.0
mEq / g and 1.8 mEq / g) were prepared. An e-RDF containing 10% fetal bovine serum (manufactured by Flow laboratory) was placed in each 100 ml spinner flask so as to have a dry weight of 0.5 g / l.
Suspended in 100 ml of medium. HeLa cells to 1.1
It was inoculated to a density of × 10 ⁵ cells / ml and stirred at 37 ° C for 3 days. This culture will be called a seed culture.

In another 100 ml spinner flask, the same two kinds of "Asahi Kasei Microcarriers" with the same charge density as described above were prepared by suspending them in a growth medium at 0.05 g / 100 ml, respectively. 1m of culture solution (including porous carrier with attached cells) on day 3 of seed culture
1 was added and the culture was continued. However, the same charge density was used for the porous carrier before and after transfer.

After transfer, changes in cell density and adhesion rate were measured. As a result, as shown in FIG. 6, all the cells grew logarithmically after the transfer, and as shown in FIG. 7, the adhesion rate also increased. At this time, the increase in the adhesion rate is
It started when the HeLa cell density per porous carrier used in the seed culture exceeded a certain value (3 days after transfer).

[0030]

[Example 6] As a porous carrier for animal cell culture, "Asahi Kasei Microcarriers" (1.0
mEq / g and 1.8 mEq / g) were prepared. E-R containing 10% fetal bovine serum (manufactured by Flow laboratory) in each 100 ml spinner flask so as to have a dry weight of 0.5 g / l
It was suspended in 100 ml of DF medium. BHK-21 cells,
Inoculate to a density of 3.5 × 10 ⁵ cells / ml and
The stirring culture was carried out at 7 ° C. for 3 days. This culture will be called a seed culture.

In another 100 ml-volume spinner flask, two "Asahi Kasei Microcarriers" of the same charge density as described above were prepared by suspending them in a growth medium at 0.05 g / 100 ml, respectively. 1m of culture solution (including porous carrier with attached cells) on day 3 of seed culture
1 was added and the culture was continued. However, the same charge density of the microcarriers before and after the transfer was used.

After transfer, changes in cell density and adhesion rate were measured. As a result, as shown in FIG. 8, all the cells were logarithmically proliferated after the transfer, and as shown in FIG. 9, the adhesion rate was also rapidly increased.

[0033]

Example 7 Asahi Kasei Microcarriers (1.0 mEq / g) was prepared as a porous carrier for animal cell culture. 1 to give a dry weight of 0.5 g / l
E-R containing 10% fetal calf serum (manufactured by Flow laboratory) in a 00 ml spinner flask
It was suspended in 100 ml of DF medium. Vero cells were added to 2.
Inoculate to a density of 6 × 10 ⁵ cells / ml and incubate at 37 ℃
The stirring culture was carried out for 3 days. This culture will be called a seed culture.

0.05 g / 100 of the same "Asahi Kasei Microcarrier" was added to another 100 ml spinner flask.
The suspension was prepared by suspending it in a growth medium to a volume of 1 ml, and 1 ml of the above-mentioned culture medium on the 3rd day of seed culture (including the porous carrier to which cells were attached) was added to continue the culture. After transfer, changes in cell density and attachment rate were measured.

As a result, as shown in FIG. 10, the cells were logarithmically proliferated after the transfer, and as shown in FIG. 11, the adhesion rate was gradually increased.

[0036]

Example 8 Asahi Kasei Microcarriers (1.0 mEq / g) was prepared as a porous carrier for animal cell culture. 1 to give a dry weight of 0.5 g / l
E-R containing 10% fetal calf serum (manufactured by Flow laboratory) in a 00 ml spinner flask
It was suspended in 100 ml of DF medium. Human x human hybridoma cells (HF10B4) were treated with 1.0 x 10 ⁵ c
Inoculate to a density of ells / ml and stir culture at 37 ℃ for 3
Went for a day. This culture will be called a seed culture.

In another 100 ml spinner flask, 0.05 g / 100 of the same "Asahi Kasei Microcarrier" was added.
The suspension was prepared by suspending it in a growth medium to a volume of 1 ml, and 1 ml of the above-mentioned culture medium on the 3rd day of seed culture (including the porous carrier to which cells were attached) was added to continue the culture. After transfer, changes in cell density and attachment rate were measured.

As a result, cells showed logarithmic growth after transfer as shown in FIG. 12, and the adhesion rate gradually increased as shown in FIG.

[0039]

[Example 9] As a porous carrier for animal cell culture, "Asahi Kasei Microcarriers" (1.0
mEq / g and 1.8 mEq / g) were prepared. E-R containing 10% fetal bovine serum (manufactured by Flow laboratory) in each 100 ml spinner flask so as to have a dry weight of 0.5 g / l
It was suspended in 100 ml of DF medium. Mouse x mouse hybridoma cells (HyGPD, YK-1-1) were added to 3.0
It was inoculated to a density of × 10 ⁵ cells / ml and stirred at 37 ° C for 3 days. This culture will be called a seed culture.

In another 100 ml-volume spinner flask, two "Asahi Kasei Microcarriers" of the same charge density as described above were prepared by suspending them in a growth medium at 0.05 g / 100 ml, respectively. 1m of culture solution (including porous carrier with attached cells) on day 3 of seed culture
1 was added and the culture was continued. However, the same charge density was used for the porous carrier before and after transfer.

After transfer, changes in cell density and adhesion rate were measured. As a result, as shown in FIG. 14, all the cells proliferated logarithmically after the transfer, and as shown in FIG. 15, the adhesion rate also increased sharply.

[0042]

Example 10 As a porous carrier for animal cell culture, “Asahi Kasei Microcarrier” (1.
0 mEq / g and 1.8 mEq / g) were prepared. E-R containing the additive RD-1 (manufactured by Kyokuto Pharmaceutical Co., Ltd.) for serum-free cell culture in each 100 ml spinner flask such that the dry weight of the product was 0.5 g / l.
It was suspended in 100 ml of DF medium. Mouse x mouse hybridoma cells (HyGPD, YK-1-1) were added to 2.5
Inoculate to a density of × 10 ⁵ cells / ml and incubate at 37 ℃
The stirring culture was carried out for 3 days. This culture will be called a seed culture.

In another 100 ml-volume spinner flask, "Asahi Kasei Microcarriers" of the same two types of charge density as described above were suspended and prepared in a growth medium at 0.05 g / 100 ml, respectively. 1m of culture solution (including porous carrier with attached cells) on day 3 of seed culture
1 was added and the culture was continued. However, the same charge density was used for the porous carrier before and after transfer.

After transfer, changes in cell density and adhesion rate were measured. As a result, as shown in FIG. 16, all cells proliferated logarithmically after the transfer, and as shown in FIG. 17, the adhesion rate also rapidly increased.

[0045]

INDUSTRIAL APPLICABILITY According to the present invention, a new carrier is mixed with a carrier to which cells are attached without using an enzyme such as trypsin or a chelating agent in animal cell culture using a porous carrier composed of a natural polymer. As a result, it became possible to transfer cells from carrier to carrier and continuously grow the cells. As a result, the following effects are obtained, and the present invention enables industrial high-density mass culture of animal cells.

The trypsin treatment step is unnecessary. Cell activity can be maintained. The risk of contamination by microorganisms etc. is reduced.

[Brief description of drawings]

FIG. 1 is a diagram showing a cell growth curve after transfer in Example 1 of the present invention.

FIG. 2 is a diagram showing a change in an adhesion rate after transfer in Example 1 of the present invention.

FIG. 3 is a diagram showing a cell growth curve after transfer in Example 2 of the present invention.

FIG. 4 is a diagram showing a change in an adhesion rate after transfer in Example 2 of the present invention.

FIG. 5 is a diagram showing a cell growth curve after transfer in Example 3 of the present invention.

FIG. 6 is a diagram showing a cell growth curve after transfer in Example 5 of the present invention.

FIG. 7 is a diagram showing a change in adhesion rate after transfer in Example 5 of the present invention.

FIG. 8 is a diagram showing a cell growth curve after transfer in Example 6 of the present invention.

FIG. 9 is a diagram showing changes in the adhesion rate after transfer in Example 6 of the present invention.

FIG. 10 is a diagram showing a cell growth curve after transfer in Example 7 of the present invention.

FIG. 11 is a diagram showing a change in an adhesion rate after transfer in Example 7 of the present invention.

FIG. 12 is a diagram showing a cell growth curve after transfer in Example 8 of the present invention.

FIG. 13 is a diagram showing a change in an adhesion rate after transfer in Example 8 of the present invention.

FIG. 14 is a diagram showing a cell growth curve after transfer in Example 9 of the present invention.

FIG. 15 is a diagram showing a change in an adhesion rate after transfer in Example 9 of the present invention.

FIG. 16 is a diagram showing a cell growth curve after transfer in Example 10 of the present invention.

FIG. 17 is a diagram showing a change in the adhesion rate after transfer in Example 10 of the present invention.

Claims (2)

[Claims] 1. In an animal cell culture using a porous carrier for cell culture, which comprises a natural polymer and / or a derivative thereof, an old porous carrier having cells adhered on the surface and / or inside is adhered with cells. Transfer cells from carrier to carrier by mixing with fresh, non-porous carrier,
A method for sequentially culturing animal cells, which comprises continuously growing the cells. 2. The method for sequentially culturing animal cells according to claim 1, wherein the transfer ratio of the carrier for cell culture calculated by the following formula is 16 to 1000 times. Transfer ratio = (a + b) / a where a = dry weight of old culture carrier b = dry weight of new culture carrier