JPH02119772A - Cell culturing device - Google Patents

Abstract

PURPOSE:To optimize culturing condition from small amount of culture solution and cell and culture in a large amount by measuring cell density with density sensor and automatically varying amount of culture solution, amount of medium changing and concentration of dissolved oxygen from cell density and growing rate. CONSTITUTION:Density of culture solution is automatically measured by density sensor 70 and growing rate is calculated in growing rate-calculating part 72 as variation of the density sensor 70 in control part. Increment of culture solution is decided in deciding part 74 of increment of culture solution from output of the density sensor 70 and the growing rate and driving of actuator 76 is controlled, then amount of medium changing is decided in deciding part 78 of amount of medium changing, thus driving of medium changing pump 80 is controlled. For output of the density sensor 70 larger than a fixed value, concentration of dissolved oxygen is increased by deciding part 82 of concentration of dissolved oxygen through gas control system 84.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an apparatus for culturing cells, and in particular to temperature, p +
- [Relates to a cell culture device that maintains an appropriate dissolved oxygen concentration (Do) and automatically exchanges a culture solution for supplying nutrients and removing waste products.

Such a cell culture device is used, for example, as a high-density cell culture device for culturing cells at high density.

(Prior Art) In a high-density cell culture device, a gas is supplied and brought into contact with the upper surface of a culture solution containing cells and housed in a culture tank. A pH sensor, a dissolved oxygen concentration sensor, and a temperature sensor are immersed in the culture solution, and a level sensor is provided to detect the liquid level.The amount of CO2 gas supplied to the top of the culture solution is determined by the detection signal of the pH sensor. The amount of 02 gas supplied to the top of the culture medium is controlled by the detection signal of the dissolved oxygen concentration sensor, and the energization to the heater that heats the culture medium is controlled by the detection signal of the temperature sensor, and a portion of the culture medium is taken out. When the fresh medium pump is activated by the detection signal of the level sensor, fresh medium is supplied to the culture tank, and the amount of culture solution in the culture tank is kept constant.

As the culture progresses, the cell density increases.

To set the environmental conditions as the cell density increases, humans remove a portion of the culture solution from the system, measure the cell density, and change the environmental conditions.

(Problems to be Solved by the Invention) There is a close relationship between growth rate and environmental conditions when culturing cells, and incorrect setting of environmental conditions will have a negative impact on subsequent cell growth.

Factors of environmental conditions include temperature, pH1 dissolved oxygen concentration, amount of medium exchanged, stirrer rotation speed, amount of culture solution, etc. It is a tedious task for humans to optimize these conditions while measuring cell density.

The present invention aims to automatically change such environmental conditions.

(Means for solving problems) The present invention will be explained with reference to FIGS. 1 and 3.

In the cell culture apparatus of the present invention, gas is supplied to and contacts the upper surface of the culture solution 4 housed in the culture tank 2, and the dissolved oxygen concentration sensor 12 is immersed in the culture solution 4.
of the culture solution 4 via the control unit 42 according to the detection signal of 2.
- A gas control system 84 that controls gas supply to the surface, and a culture medium exchange pump 80 (5
0° 54) operates to supply fresh culture medium to the culture tank 2, an optical density sensor 70 that irradiates the culture medium with light and measures cell density from its absorption, and instructions from the control unit 42. [4] having an actuator 76 that changes the set value of the liquid level according to the level sensor 76. Control unit 4
In addition to the above-mentioned functions, 2 includes a growth rate calculation unit 72 that calculates the growth rate as the amount of change in the output of the density sensor 70, and an actuator 76 that determines the amount of increase in the culture medium from the output of the density sensor 70 and the growth rate. and a culture medium exchange amount determining unit 74-, which controls the drive of the culture medium exchange pump 80 (50° 54), which determines the culture medium exchange amount from the output of the density sensor 70 and the growth rate, and controls the drive of the culture medium exchange pump 80 (50° 54). part 78 and density sensor 7
The dissolved oxygen concentration determination unit 82 increases the dissolved oxygen concentration via the gas control system 84 when the output of zero becomes equal to or greater than a predetermined value.

(Effect) Figure 2 shows the results of a high-density continuous culture experiment using a serum-free medium. The vertical axis is viable cell density, and the horizontal axis is culture time.

The cells used were NAT-30 (human lymphoid cells), and the basic medium was (RPMI-1640) + DME + (F-
12) Growth factors include insulin, transferrin, ethanolamine, selenium, lipoproteins, and serum albumin. The setting conditions are pH 7.2 and temperature 3.
The temperature was 7°C, the stirring was 60 rpm, and the dissolved oxygen concentration was t in the figure.
The concentration is 5.0 ppm in periods a and tb, and higher than that in period tc. Assuming that the maximum capacity of the culture solution in the L1i tank is 1.0Q, the culture solution exchange rate is 0 in period ta and 0 in period t.
2 for b and tc. OQ was 7 days.

The results shown by the solid line in FIG. 2 were obtained through manual control based on the judgment of the experimenter, and were obtained when the environmental conditions were properly set.

If cells are seeded in the medium and the density is low (period t
a)j If the medium is replaced at this point, the cells will die and decrease as shown by the broken line A.

Furthermore, if the culture medium is not replaced during the logarithmic growth phase (until the viable cell density is saturated during periods tb and tc), the cells will still die as shown by the broken line B.

Furthermore, if the dissolved oxygen concentration is not increased during the period tc when the living cell density becomes high, the living cell density will not be sufficiently high and will reach a saturated state, as shown by the broken line C.

Therefore, in the present invention, the cell density is automatically measured by the density sensor 70, and control is automatically performed based on the cell density and growth rate so that the cell density increases as shown by the solid line in FIG. .

Therefore, the control unit 42 is given data based on the experimental results as shown in FIG.
18 Nutrient solution air, medium exchange amount, and dissolved oxygen degree are automatically set based on the cell density and growth rate.

The increase in the amount of culture solution is determined by the 1g nutrient solution increase amount determining section 74 or J':
) actuator 7 of the level sensor 14
This is realized by driving G. When the level sensor 14 is at the current level of 1f (below the liquid level of the nutrient solution 4), the level sensor 14 is detected by the htf ground exchange pump 80.
Nutrient solution 4 is increased.

When changing the amount of medium exchange, the medium exchange pump 80 is driven by a signal from the medium exchange date determination section 78.

When increasing the dissolved oxygen concentration, the gas control system 84 is controlled by a signal from the dissolved oxygen concentration determination section 82, and the oxygen pressure in the atmosphere in contact with the 4th side of the culture solution 4 is increased.

(Example) FIG. 3 represents one embodiment.

2 is a culture tank in which a culture solution 4 is stored.

If (Nutrient solution 4 contains cells. Culture tank 2) -
The section is sealed with a lid 6, and the culture solution 4 and the gas come into contact in the -L section space of the lt'γf nutrient solution 4.

A temperature sensor 8, a pH sensor 10, and a dissolved oxygen concentration sensor 12 are immersed in the culture solution 4 through the lid 6.

The lid 6 is also provided with a supply pipe 16 for supplying gas to the upper space of the culture solution 4 and an outlet pipe 18 for discharging waste.

A level sensor 14 is provided outside the culture tank 2 to detect the level of the culture solution 4 .

Gas supply pipe] 6 is supplied with 02, C○~ and N= (or air) through reculators 22-1~22-3, solenoid valves 24-1~24~3 and check valves 261~26-3, respectively. Supplied. 28 is a filter, and 40 is a safety valve.

An exhaust nozzle 30, a reverse well valve 32, an orifice 34, and a solenoid valve 36 are connected to the exhaust pipe 18. 38 is a pressure gauge provided in the exhaust pipe.

The opening and closing of the electromagnetic valve 24-1 for supplying 02 is controlled by the control unit 42 in response to a detection signal from the dissolved oxygen concentration sensor 2 so that the dissolved oxygen concentration reaches a set value. C.O.
The opening and closing of the electromagnetic valve 242 that supplies := is controlled by the control unit 42 based on the detection signal of the pH sensor 10 so that the pH becomes the set value. Electromagnetic valve 1 for supplying N3
24-3 is 02 and CO? in culture tank 2. In order to lower the partial pressure, the 02. It is used to replace COC gas, and is controlled to open and close by the control unit 42 according to detection signals from the p+-(sensor 10 and the dissolved oxygen concentration sensor 12).

The dissolved oxygen concentration sensor 12 and the solenoid valve 24-1.24-3 constitute a gas control system, the pI-1 sensor 10 and the solenoid valve 24-2.24-3 constitute a pI-1 control system, I'm here.

A heater 44 is provided at the bottom of the culture tank 2. Based on the detection signal from the temperature sensor 8, the control unit 42 controls the energization of the heater 44 so that the temperature is constant.

The culture solution 4 is circulated between the culture tank 2 and the Talosflow filter 48 by a perfusion pump 46.

The Talos flow filter 48 is a cylindrical filter made of ceramic, and the culture solution containing cells passes through the inside of the filter 48. A portion of the culture solution is filtered out of the filter 48 and discharged. The filtered culture solution is taken out of the culture apparatus by the span 1 medium pump 50. 5
2 is the spent medium taken out. Culture tank 4
A fresh M medium 56 from outside the system is supplied by a fresh medium pump 54. The perfusion pump 46 , the spent medium pump 50 and the fresh medium pump 54 are controlled by the controller 42 .

The level sensor 14 detects the level L of the culture solution 4, and when the culture solution is discharged through the cross-flow filter 48, the level of the culture solution 4 in the culture tank decreases. A fresh culture medium pump 54 is operated in response to a detection signal from the sensor 14, and a fresh culture medium 56 is supplied to the culture tank 2 to control the liquid level of the culture solution 4 to be at the position of the level sensor 14.

The level sensor 14, the control unit 42, the perfusion pump 46, the spent medium pump 50, and the fresh medium pump 54 constitute a level control system.

A starter 58 is provided in the culture solution 4, and a magnet 62 is provided at the bottom of the culture tank 2.

The starter 58 is activated by rotating the magnet 62 via the starter motor 60.

Starter motor 60 is also controlled by control section 42 .

64 is a display section of the control section 42, which displays temperature, pH1 dissolved oxygen concentration, etc. Reference numeral 66 denotes a setting section for setting set values such as temperature in the control section 42.

The flow path including perfusion pump 46 and crossflow filter 48 is provided with an optical density sensor 70 shown in FIG.

In the density sensor shown in FIG. 4, a cell 89 made of transparent glass is provided between silicone rubber tubes 86 and 87 constituting a flow path, and the culture solution passes through the cell 89. Light is emitted from one side of the cell 89 by the LED 90, and the cell 8
The light transmitted through the photodiode 91 is received by the photodiode 91. A sensor that uses light to determine the density of a suspension from light absorption by a suspension sample is generally used as a turbidity sensor. The density sensor in this embodiment uses the same principle as such a turbidity sensor.

FIG. 5 shows the relationship between the cell density and the output of the density sensor 70, and there is a one-to-one relationship.

The level sensor 14 is capable of automatically changing the set value of the liquid level, and an example thereof is shown in FIG.

92 is an LED, and 93 is a photodiode, which are supported by a support member 94 so as to face each other with the culture tank 2 in between. The support member 94 is supported movably in the vertical direction by a guide 95, and a screw 96 is provided on the support member 94, and a dedicated screw 97 rotated by a pulse motor 98 is screwed into the screw 96.

The support member 94, screws 96, 97, and pulse motor 98 are actuators for the level sensor 14. When the support member 94 moves vertically due to the operation of the pulse motor 98, the moved position becomes the new liquid level setting. value, and the liquid level of culture medium 4 is 50.54 at the medium exchange pump.
moved by

This liquid level sensor 14 utilizes the fact that the intensity of light emitted from the LED 92 and incident on the photodiode 93 changes depending on the liquid level position of the culture liquid 4. Liquid level is LE
If it is below the optical path of the light emitted from D92 and incident on the photodiode 93, the received light intensity will be strong, and when the liquid level rises and the optical path passes through the liquid, the optical path will be bent due to refraction and the light intensity will decrease. become weak.

FIG. 7 shows the connection relationship between the control section 42 and various sensors and drive parts in this embodiment.

The control unit 42 includes a CPU 42a and a knowledge database 42b that uses experimental results as shown in FIG. 2 as setting values for setting environmental conditions. Further data can be added to the knowledge database 42b to the culture conditions that have already been set.

Next, the operation of one embodiment will be explained with reference to FIG.

Plant cells (step S1), dissolve oxygen concentration, pH
1. Set the temperature and stirring number to predetermined values. The density is measured (step S2), and the growth rate R is calculated from the rate of change in the density (step S3).

The density is compared with a first density D+ (for example, I.times.10G cells/m.sub.Q) set by the knowledge database (step S4). While the density is below D1, the culture medium is not replaced (step S5), and the culture solution is not increased (step S6).

As the culture continues, the density eventually reaches D1) (Step S4). Second density D2 where density is set
(For example, IX 107 cells/mQ) or less, the dissolved oxygen concentration remains constant, and step S8
), if the growth rate R exceeds 50%, set the medium exchange amount to 500 mQ per day (step S10.5ll),
The increase in the amount of culture solution is set to 115, which is the total amount of culture solution per eye (step 512). However, the maximum capacity of the culture solution that can be stored in the culture tank is 500 mQ.

After further culturing, the density 1] is increased to the second density setting value of 1 and 2.
, the dissolved oxygen concentration increases from that point onwards (
Step S7. S9). To increase the dissolved oxygen concentration, increase the pressure at the top of the culture medium in the culture tank by 0.5 kg/cm. While the growth rate R exceeds 50%, increase the exchange rate by 500 kg/cm.
rn Q /day, increase the amount by 11 of the total culture volume per day
5 (step SIO, Sl 1,512), and continue culturing.

Eventually, as the cell density approaches saturation, the growth rate becomes less than 50%. Thereafter, the amount of culture medium is replaced at 1000 m Q /day without increasing the amount of culture solution (step sto, SI3.S6). From then on, culture is continued under these conditions.

(Effect of the invention) In the present invention, the density of the culture solution is automatically measured using a density sensor, and the amount of culture medium exchanged, the amount of increase in the culture solution, and the dissolved oxygen concentration are determined from the measured density and the growth rate, which is the amount of change in density. Since it is controlled to change automatically, it is possible to automate the culture process in which a small amount of culture solution and cell number is used for starting from star 1 to mass culture.

[Brief explanation of drawings]

Figure 1 is a block diagram showing parts related to the control unit of the present invention, Figure 2 is a diagram showing the results of culture experiments, Figure 3 is a configuration diagram showing one embodiment, and Figure 4 is a density sensor. Schematic front view, Figure 5 is a diagram showing the relationship between cell density and density sensor output,
FIG. 6 is a schematic perspective view showing an example of a level sensor, FIG. 7 is a block diagram showing the connection relationship between the control section, each sensor, and the drive section of one embodiment, and FIG. 8 is a flowchart showing the operation of one embodiment. Char 1 is a diagram. 2 - Culture tank, 4 - Culture solution, 12... Dissolved oxygen concentration sensor, 14 Level sensor, 42 - Control unit, 70 - Density sensor, 72 - Growth rate calculation unit, 7
4 Culture fluid increase amount determining section, 7G... Level sensor actuator, 78 Medium exchange amount = 15 Determining section, 80... Medium exchange pump, 84 Exchange system. Gas patent applicant Shimadzu Corporation Representative Patent attorney No [” ShigeruΔ1:

Claims (1)

[Claims] (1) Gas is supplied to the top surface of the culture solution containing cells and housed in a culture tank, and a dissolved oxygen concentration sensor is immersed in the culture solution. A gas control system controls gas supply to the top of the culture solution, and when a portion of the culture solution is taken out, a detection signal from the level sensor activates the culture medium exchange pump via the control unit to supply fresh culture medium to the culture tank. An optical density sensor that irradiates the culture solution with light and measures the cell density from its absorption, and a level sensor that has an actuator that changes the set value of the liquid level according to instructions from the control unit. In addition to each of the functional units, the control unit includes a growth rate calculation unit that calculates the growth rate as the amount of change in the output of the density sensor, and a growth rate calculation unit that determines the amount of increase in the culture solution from the output of the density sensor and the growth rate, a culture solution increase amount determining section that controls driving of the actuator; a culture medium exchange amount determining section that determines a medium exchange amount from the output of the density sensor and the growth rate and controls driving of the medium exchange pump; and the density sensor. A cell culture device comprising: a dissolved oxygen concentration determination unit that increases the dissolved oxygen concentration via a gas control system when the output exceeds a predetermined value.