JPH04148676A - Perfusion culture - Google Patents

Abstract

PURPOSE:To forcibly eliminate multiplication inhibition factor and decomposition factor in subjecting cells to perfusion culture using hollow fibers by sending a medium for cell culture from a cell existing space side to a cell nonexisting space side of hollow fibers. CONSTITUTION:In subjecting cells to perfusion culture using hollow fibers, a medium for cell culture is made to flow from a cell existing space (extra capillary space)(ECS) side to a cell nonexisting space (inner capillary space)(ICS) side of hollow fibers so that waste material, dead cells, multiplication inhibition factor of live cells and/or decomposition factor of the objective product accumulated in ECS are forcibly discharged out of ECS.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a new method for perfusion culture of cells using holofibers, more specifically, a method that can supply macromolecular nutrients as a cell culture medium and improve the aging of the cultured cell environment. The present invention relates to an improved perfusion culture method that enables high-density cell culture over a long period of time while preventing deterioration of the cell quality as much as possible, and also enables production of desired products at higher yields.

Conventional technology The perfusion culture method, which uses a holofiber (hollow fiber) as a culture tank, has been known as one of the methods for culturing various types of cells such as animal cells, microbial cells, and plant cells at high density and in large quantities. There is.

This method uses the selective permeability of the holofiber wall as a semipermeable membrane to place cells on the outer surface of a large number of bundled holofibers (capillaries) packed in an appropriate column. and the inner space of the capillary (I
By perfusing cell culture media such as nutrients into the inner Capillary Space IC8),
The above nutrients, etc. are sent to the cells through the capillary wall, and conversely, they are delivered to the outer space of the capillary where the cells exist (Extra C
apilary 5pace.

The cell products produced in EC8) are transferred to the above IC through a solid wall.
8 into the flowing perfusate.

The above method is superior in that it can maintain a microenvironment suitable for in vitro culture of cells and can grow cells three-dimensionally into a so-called organism-like structure. The nutrients, etc. that should be supplied to the cells are limited to a size that can pass through the semipermeable membrane of the capillary, that is, below the molecular weight cutoff, and therefore it is impossible to continuously supply macromolecular substances larger than that, which is a fatal problem. There were certain shortcomings.

In addition, in the above method, waste products and dead cells produced by cultured cells accumulate over time in the EC8 where cells exist, and furthermore, due to the decomposition of dead cells, cell growth inhibitory factors such as proteases and the desired production Decomposition factors, etc. of substances accumulate over time in the EC8, which deteriorates the cultured cell environment relatively quickly, and has the disadvantage of not being able to achieve a high density of cultured cells, making it difficult to culture cells for a long period of time. There were also disadvantages in that it was impossible to improve the yield of the desired product.

Problems to be Solved by the Invention The purpose of the present invention is to eliminate the drawbacks of the above-mentioned conventional cell perfusion culture method using holofibers, and in particular, to solve the problems of the above-mentioned methods for perfusion culture of cells using holofibers. In addition to being able to constantly supply other cell culture reagents,
Waste products such as lactic acid and ammonium ions produced by proliferating cells are discharged from the cell space as quickly as possible, thus constantly maintaining a fresh culture environment, allowing rapid and smooth cell proliferation, and ensuring that cells reach the final stage. The objective is to improve the density, prolong the cell growth maintenance period, and ultimately improve the yield and yield of the desired product.

Means for Solving the Problems The present inventors have conducted intensive research for the above-mentioned purpose, and as a result, in the conventional holofiber perfusion culture method, culture is performed from the cell-existing space (ECS) side to the cell-free space (IC8) side. The present inventors have devised a method of flowing a medium for use, and have discovered that all of the above objects can be achieved by this method, thus completing the present invention.

That is, according to the present invention, when perfusion culturing cells using a holofiber, a cell culture medium is allowed to flow from the cell presence space (ECS) side to the cell non-existence space (IC8) side of the holofiber. A perfusion culture method characterized by the following, and a cell existence space (E
The above-mentioned perfusion culture method is provided, in which at least one of waste products, dead cells, living cell growth-inhibiting factors, and target product decomposition factors accumulated in CS) is forcibly discharged from the EC3.

In the method of the present invention, any of the various types of holofibers conventionally used for perfusion culture of cells of this type can be used.

Examples include melt spinning, dry spinning, wet spinning, etc. using cellulose, cellulose esters, polysulfone, polyacrylonitrile, polymethyl methacrylate, polyamide, polybenzimidazole 1, silicone polycarbonate, glass, etc. An example is a hollow fiber manufactured by. These are usually capillaries with a diameter of less than 1 annular, and they have in common the property that the hollow fiber wall functions as a semipermeable membrane. Among the above, cellulose acetate membrane made by Dow Chemical Company (inner diameter: 180-200 μm 1 outer diameter = 230-250 μm 1 molecular weight cut-off: approximately 30,000)
Polysulfone membrane manufactured by Amicon (inner diameter: approx. 200 mm)
μm, outer diameter: approximately 350 μm.

Molecular weight cutoff: approx. 10,000. Approximately 30,000, approximately 5,000
0) is preferred. Further, the above-mentioned holofiber may be coated with a substrate suitable for cell retention such as collagen or fibronectin (FN), and this is particularly suitable for culturing adhesion-dependent cells.

The method of the present invention is basically carried out as follows.

That is, a plurality of the above-mentioned holofibers are bundled and packed into a suitable column to form a cell culture tank, and first, cells for culture are placed on the outer surface of the holofibers in the above-mentioned column, and this cell existence space (E CS) is introduced with cell culture medium. The medium is continuously supplied to the cells in the cell-existing space (ECS), passes through the wall of the holofiber, reaches the internal space (cell-free space, IC8), and passes through C8 to the column. Continuously discharged outside. Cell products such as lactic acid, ammonium ions, and protease are discharged from the column through the hollow fiber wall and the cell-free space (IC8) as the medium is discharged. The method of the present invention adopts a configuration opposite to the above, that is, a configuration in which cells are allowed to exist in the internal space of the holofiber, a cell culture medium is continuously supplied thereto, and the medium is discharged to the external space through the holofiber wall. You can also do that.

The cell culture medium used in the above method of the present invention can be determined as appropriate depending on the type of cells to be cultured.
Any of these may contain the various nutrients required for cell culture, and specific examples include ME.
M medium, DMEM medium, RPMI-1640 medium, F-
12 medium, NCTC-109 medium, Waymouth's MB medium, Williams' E medium, and the like.

In particular, the method of the present invention is based on the above configuration, that is, the configuration in which the cell culture medium is perfused from the cell-existing space (ECS) through the holofiber wall into the cell-free space (IC8). Polymeric substances can also be used as a cell culture medium, which has the advantage of being constantly supplied to the cells. The polymeric substances include substances with a molecular weight larger than the molecular weight cutoff of the hollow fiber used, such as transferrin (molecular weight cutoff: 280,000), serum albumin (molecular weight cutoff: 69,000), α-cyclodextrin (α-CD, serum Cohn's fraction of globulin (complex of transferrin, ceruloplasmin, haptoglobulin, etc.), hepatocyte growth factor (HGF, 10300
0), platelet-derived growth factor (PDGF, 30,000)
, nerve growth factor (NGF, 26,000), fibronectin (FN, 220,000 to 250,000), interleukin 2 (IL-2, 13,000 to 1,400)
0), granulocyte colony stimulating factor (G-C3F, 300
00), macrophage colony stimulating factor (M-C8F
, 60,000 to 85,000), and granulocyte/macrophage colony stimulating factor (GM-C8F, 20,000).

The use of such polymeric substances in the method of the present invention has the following advantages, for example. That is, transferrin is essential for the survival, proliferation, and differentiation of most cultured animal cells, and it binds iron as a nutritional substance to two atoms per molecule (K8.=10
-IOM)L, and also has the function of efficiently providing iron to cells through binding with receptors (T f R). Serum albumin serves as a carrier for long-chain unsaturated fatty acids (oleic acid, linoleic acid, etc.) that are essential fatty acids for cultured cells, and controls the supply of fatty acids to the cells, thereby promoting cell proliferation. rL-2 can be used in various cells that have receptors for IL-2 on the cell membrane surface, such as NK cells and T cells.
Induces proliferation of cellular leukemia cells, T cells, etc. H.G.
F promotes proliferation of mature hepatocytes in the presence of insulin and EGF (epidermal growth factor).

The cells to be cultured by the method of the present invention are not particularly limited, but typical examples include human fibroblasts, smooth muscle cells, glial cells, 3T3 cells, NK cells, and T cells. .

The method of the present invention will be explained below with reference to the accompanying drawings.

1 to 3 are schematic diagrams of an apparatus suitable for carrying out the method of the present invention, and FIG. 1 shows a circulating culture system (C1osed Cu
Figure 2 shows the IC8 circulation culture + fresh medium supply system (IC5Overflow Culture).
Figure 3 shows IC8 and EC8 circulating culture + fresh medium supply + culture product recovery system (ICS & EC5 OverflowC
culture) are shown respectively.

In each figure, (1) shows the circulation pump, (2) shows the gas exchanger, (3) shows the Do sensor (introduction side), and (4) shows the Do sensor.
Sensor (discharge side), (5) pH sensor (introduction side), (6) pH sensor (discharge side), (7) reserve tank, (8) fresh culture tank, (9) ) is the spent culture medium tank, (10) is the product recovery tank, (11
) indicates the holofiber culture tank (column), respectively.

The perfusion culture conditions, culture method, control of culture environment, etc. in the method of the present invention using the apparatus shown in the above figure can be determined as appropriate depending on the cells to be used, etc., and are not particularly limited, but for example, as described below. The following is a detailed description of the culture of antibody-producing cells shown in Examples.

That is, the culture temperature can generally be 37°C, which can be kept constant during the culture period.

The culture circulation flow rate is approximately 2011/min [from the initial stage of culture].

(introduction side) - until DO (discharge side) > 50111 mHg], and DO (introduction side) -D.

(Discharge side) Every time >50mmHg, increase the circulation flow rate by 10x.
l! It is preferable to increase the time by /minute. The medium was replaced every hour using the circulating culture system (C1osed C) shown in Figure 1 above.
IC3 circulation culture + fresh medium supply system (IC5Overflow Culture) shown in Figure 2
It is desirable to alternately perform these steps, and gradually increase the amount of medium exchanged in the IC8 circulation culture + fresh medium supply system. p
For H control, the set value of the PID controller is set to pH=7.
2, preferably controlled by the controller. still,
During the middle stage of culture, the amount of CO2 supplied to the gas exchanger (PID
After the value calculated by the controller) reaches 0, there is a dangerous zone for cell growth and material metabolism.
)L = 6.8 or less, but if the cumulative amount of NaHCO3 in the medium becomes 3 times the concentration of the reference medium, stop maintaining the pH by adding NaHCOs and periodically pump circulation. It is desirable to increase the flow rate and exchange the entire culture medium. Further, Do control is preferably performed by setting the set value of the PID controller to DO=150 mmHg, and maintaining Do (discharge side) > 100 mmHg by changing the culture medium circulation flow rate.

The above-mentioned temperature control can maintain a constant temperature of the culture solution in the circuit by placing the entire perfusion circuit in a clean circulation constant temperature bath using a HEPA filter.

The above pH control uses N2 02 vented to a hollow fiber type gas exchanger as a detector for the pH sensor (introduction side).
The CO2 composition ratio in the three types of CO2 mixed gas is measured using a dedicated P.
This can be carried out by sequentially determining and supplying air using an ID controller. Furthermore, the fresh culture tank (8) in the circuit,
A plurality of them are connected in parallel, and at least one of them is supplied with a pH buffer, for example, NaHCO3-PBS which is 500 times more concentrated than the medium.
By adding the solution whenever the pH value changes, it is possible to prevent the pH from decreasing as the culture progresses.

The above Do control uses 3 of the N2-O2CO2 vented to the holofiber type gas exchanger as a Do (introduction side) detector.
This can be carried out by sequentially determining the 02 composition ratio in the seed mixed gas using a dedicated PID controller and supplying air.

Further, the control of the medium circulation flow rate can be changed by controlling the rotation of the drive motor of the medium circulation pump (which may be a besteric pump, a syringe pump, a bellows pump, etc.).

To explain in more detail the method of the present invention using the apparatus shown in each of the above figures, the apparatus shown in each figure uses the outer space of the holofiber as a space for cells to exist, and the culture medium is continuously fed into this space during culture. The main constituent circuits are a supply circuit and a circuit for continuously discharging the culture medium supplied from the space into the inner space of the holofiber.

Further, as shown in FIG. 2, an apparatus suitable for carrying out the method of the present invention is such that a tank (8) containing fresh culture medium is
A sub-component circuit (hereinafter referred to as " Further, as shown in Figure 3, in addition to the main flow of the culture medium from the cell-existing space to the cell-free space in the main circuit, Waste products and dead cells that may accumulate in the cell space as the cell progresses, as well as proteolytic enzymes (proteases, etc.) leaked from decomposed dead cells, growth inhibitory factors for living cells, and decomposition factors for target products, etc. A sub-configuration circuit (hereinafter referred to as "circuit configuration (3)") for periodically forcibly discharging to the outside of the column may be provided.

In the method of the present invention using the apparatus shown in each of the figures above, for example, first, the components of the culture circuit are sterilized in advance by an appropriate sterilization operation such as high pressure steam sterilization (using an autoclave), ethylene oxide gas sterilization, radiation sterilization, etc. The entire circuit is assembled in a sterile environment such as a clean bench, the culture perfusion circuit is initialized as described below, and then cells are seeded and cultured.

The above initialization can be carried out, for example, by installing the circuit in a constant temperature bath maintained at 37°C (the constant temperature bath is preferably a clean constant temperature bath with a ventilation circulation system using a HEPA filter), and %FBS (fetal bovine serum)
This can be carried out by circulating a suitable medium such as RPMI-1640 medium containing . In the above, three types of gases, oxygen gas, carbon dioxide gas, and nitrogen gas, can be supplied to the gas exchanger. The amount of oxygen gas supplied is determined by a Do controller, and the amount of carbon dioxide is determined by a pH controller.
Depending on the control output, the mass flow controller for oxygen gas and mass flow controller for carbon dioxide gas can be adjusted, and the nitrogen gas supply amount can be adjusted by computer processing.
The total gas supply amount is determined to be a predetermined constant value, and can be adjusted by a mass flow controller dedicated to nitrogen gas. In this way, the gas supply is controlled to maintain the pH at 7.2 and the Do (introduction side) at 150 mmHg, and the culture solution is perfused for 48 hours to complete the initialization.

Following the above initialization (cell seeding is carried out, for example, at a predetermined cell density, preferably 4X1 in the case of the RPMI-8226 strain)
Cell suspension 5011 adjusted to approximately 06 cells/7A'
This can be carried out by taking a needleless syringe and injecting it into the empty outer space (or inner space) of the hollow fiber through the port.
It is appropriate to seed the cells with 0B of RPMI-8226.

Furthermore, to culture the cells seeded above, for example, after changing the orientation of the holofiber several times approximately every hour to adjust the cells so that they are not localized within the column, the circulation rate of the culture medium is adjusted to 111/1/2 for several hours. The culture solution is circulated through the main component circuit for 1 minute, and D
o (introduction side).

If the difference (on the discharge side) does not seem to decrease compared to the difference at the time of seeding, increase the circulation speed of the culture medium by 20zl! /min. The capacity of the reserve tank (7) was set to about 21, and from 48 hours after the start of culture, the circulating culture system (C1
osed culture) and the IC3 circulation culture + fresh medium supply system (IC5Overflow Cu) shown in Figure 2D.
The culture is continued by continuously exchanging the spent culture medium with fresh culture medium at an exchange rate of 500 zl/day for up to one week of culture by alternating between 1 ture and 500 zl/day and adjusting the time ratio.

From the 7th day of seeding, the exchange rate of the culture medium is increased to 1000 x 1 day, and the function of the sub-component circuit 2 is used to collect 10 Il of the culture medium in the cell-free space once every 2 days. When the pH value becomes 6.8 or less, p)(
Buffer solution (phosphate buffer solution containing 5% NaHCO3 (PBS)
) = A 50 times concentrated solution of the culture solution was added once during the culture perfusion circuit using a part other than the component circuit (1) shown in Figure 2 (hereinafter referred to as "the component circuit (2)"). Add 6xl per serving. However, this addition operation is automatically set so that it cannot be performed more than once within 6 hours.

Since it takes time for the added pH buffer to be sufficiently and uniformly diffused in the perfusion circuit, the pH value of the perfused culture solution should be adjusted after 2 hours and within 6 hours after the addition of the pH buffer. If it seems to be lower than 6.8, replace the spent culture medium with fresh culture medium again at a high speed of 1000//2 hours. At this time, the configuration circuit (2) is used.

Then once every 1 to 2 days, using component circuit (2),
xl perfusion culture was collected, the glucose concentration was measured,
If it is less than 50 μ/l, replace the culture medium in the same way as above.

From day 1 on day 14 of culture, the exchange ratio between fresh culture solution and spent culture solution was increased to 2000z/day, and 2511 culture solutions on the EC8 side were collected every 2 days.

Thus, according to the method of the present invention, it is possible to constantly supply cultured cells with a polymeric substance having a molecular weight larger than the molecular weight cut-off of the holofiber, and to forcefully discharge waste products produced by proliferating cells at all times during the culture period. The culture environment can be maintained at all times, therefore, the cell culture maintenance period can be extended, the maximum cell density can be improved, and the target product can be produced at high yield over a long period of time.

EXAMPLES Examples will be given below to explain the present invention in more detail.

Example 1 This example was carried out as follows under the following basic conditions using a culture apparatus using the following holofiber culture tank (see attached drawings).

(1) Target cells: RPMr-8226 cells (monoclonal antibody producing strain) (2) Target production substance: monoclonal antibody (IgG
(L-λ chain) (3) Culture solution used: RPMI-1640 medium + MEM medium (1:1) + 10% FBS (4) Detection method: IgG-λ production amount was detected by EIA method.

(5) Holofiber culture tank: Vitafiber made by Amicon (molecular weight cutoff = 30000) Holofiber (made of polysulfone) used (6) Number of seeded cells: 1 x 10B cells (seeding density = 4 x 106 cells/I) ( 7) Number of culture days = 34 days (8) Volume of space on cell side: 25 cm3 (xi) (9
) Final number of cells reached: 5 x 109 cells (10) Product amount: 430 ■ (rgG L-λ) In other words, the components of the culture circuit were sterilized with high pressure steam (using an autoclave),
After sterilization using an appropriate sterilization operation such as ethylene oxide gas sterilization or radiation sterilization, assemble the entire circuit in a sterile environment such as a clean bench, initialize the culture perfusion circuit as described below, and then seed and culture the cells. carried out.

The above initialization can be performed, for example, by installing the circuit in a constant temperature bath maintained at 37°C (the constant temperature bath is preferably a clean constant temperature bath with a ventilation circulation system using a HEPA filter), and RPMI-1640+MEM (1:1) medium containing FBS (fetal bovine serum)
This was performed by circulating in a culture perfusion circuit. In the above, three types of gases, oxygen gas, carbon dioxide gas, and nitrogen gas, are supplied to the gas exchanger.
Depending on the control output, the mass flow controller for oxygen gas and mass flow controller for carbon dioxide gas can be adjusted, and the nitrogen gas supply amount can be adjusted by computer processing.
The total gas supply amount was determined to be a predetermined constant value, and adjusted using a mass flow controller exclusively for nitrogen gas. Thus,
Gas supply was controlled to maintain pH at 7.2 and Do (introduction side) at 150 mmHg, and culture solution was perfused for 48 hours to complete initialization.

Following the above, cell seeding was carried out using a cell suspension of RPMI-8226 strain adjusted to about 4 x 106 cells/11 cells.
was taken into a needleless syringe and injected into the empty outer space of the hollow fiber through the port, thus seeding about 1×10 B cells of RPMI-8226 per column.

The above-mentioned seeded cells were cultured by changing the direction of the holofiber several times approximately every hour so that the cells were not concentrated in the column, and then the culture medium was mainly circulated at a circulation rate of 1 zl/min for several hours. circulate through the constituent circuits,D.

If the difference between Do (introduction side) and Do (output side) does not decrease compared to the difference at the time of seeding, increase the circulation speed of the culture medium to 20%.
The temperature was increased to 1 minute. Reserve tank (7)
48 hours after the start of culture, the circulating culture system (C1osedCulture) shown in Figure 1 and the second
The IC8 circulating culture fresh medium supply system (IC3Ov) shown in the figure
By alternating the erflow culture and adjusting the time ratio, the culture can be carried out for up to one week.
Cultures were continued with continuous exchange of spent culture medium with fresh culture medium at an exchange rate of 500 z/day.

From the 7th day of seeding, the exchange rate of the culture medium was increased to 1000 x 1 day, and 10 Il of the culture medium in the cell-free space was collected once every 2 days using the function of sub-component circuit 2. When the pH value is below 6.8, the pH buffer (PBS containing 5% NaHCO3 = 50 times the concentration of the culture medium) solution is automatically added to the culture perfusion circuit using configuration circuit 2.
Added 61/ml per serving. However, this addition operation was automatically set so as not to be performed more than once within 6 hours.

Since it takes time for the added pH buffer to be sufficiently and uniformly diffused in the perfusion circuit, the pH value of the perfused culture solution should be adjusted after 2 hours and within 6 hours after the addition of the pH buffer. If the value decreased below 6.8, fresh culture solution was replaced with the spent culture solution at a high speed of 1000z//2 hours. At this time, configuration circuit (2) was used.

Then once every 1 to 2 days, using component circuit (2),
1 A' of perfusion culture solution was collected and the glucose concentration was measured, and if it was less than 50 µ/l, the culture solution was replaced in the same manner as above.

From the 14th day of culture, the exchange ratio between fresh culture solution and spent culture solution was increased to 20003F/day, and 25 yJ of the culture solution on the EC8 side was collected every 2 days.

In this way, the target product was obtained.

The results of the cell culture according to the above are shown in FIG.

In the figure, the horizontal axis represents the culture time (Hr), and the vertical axis represents the purpose 1.
gGL L-λ production (■) [shown as curve (1)] and oxygen consumption (0, C, R,) (μmol/min) [shown as curve (2)].

Further, in FIG. 4, the calculated cell number (estimated, cells) based on the above oxygen consumption amount (0·C,R,) is displayed next to the oxygen consumption amount.

From the figure, it is clear that according to the method of the present invention, the cell culture maintenance period can be extended, the maximum cell density can be improved, and the target product can be produced with high yield over a long period of time. .

[Brief explanation of drawings]

1 to 3 are schematic diagrams showing an example of a holofiber perfusion culture device suitable for carrying out the method of the present invention, and FIG.
The figure is a graph showing the results of cell culture according to the method of the present invention. (that's all)

Claims (1)

[Claims] (1) A perfusion culture method characterized by flowing a cell culture medium from the cell presence space side to the cell non-existence space side of the holofiber in perfusion culture of cells using a holofiber. (2) At any time during the perfusion culture period, at least one of waste products, dead cells, growth-inhibiting factors for living cells, and decomposition factors for target products, which are accumulated in the cell space, is forced out of the cell space. 2. The method according to claim 1, wherein the waste is discharged in a vacuum.