JPS5894384A - Cultivation device - Google Patents

Abstract

PURPOSE:To prevent the reduction in quality of a culture solution for a long time, by making a constitution wherein a bath for culture solution for preserving it at low temperature is provided and a heater is set in a flow path between the bath for culture solution and an incubator to heat the culture solution to a given temperature. CONSTITUTION:The main body 20 of a cultivation device is provided with the incubator 21 having a cell to be cultivated, the baths 22, 23, and 24 for culture solution are charged with a nutrient medium, an enzyme solution, and a buffer solution, put in the cooling bath 26 and preserved at low temperature. The culture solution is fed from the baths 22, 23, and 24 for culture solution to the incubator 21 as occasion demands, the heater 25 is set in a flow path for culture solution, and the low-temperature culture solution is heated to 37 deg.C. Consequently, the nutrient medium, the enzyme solution and the buffer solution required for cultivation operation are preserved at low temperature until just before use, so that vitamin, amino acid, etc. in the culture solution are prevented from being decomposed, the deactivation of enzyme is prevented, and cultivation is carried out continuously for a long period.

Description

[Detailed description of the invention] This invention relates to an automatic culturing device.

A conventional culture apparatus of this type will be described in detail with reference to FIGS. 1 and 2.

Figure 1 shows a schematic diagram of 14 culture devices. FIG. 2 is a diagram showing the process of culturing operation in the culturing apparatus of FIG. 1. First, with reference to Figure 1, the w4I of this device is
2I2, will be explained in detail. In Figure 1, there is a culture vessel l containing cells to be subcultured, an observation device & λ detection unit for observing the proliferation state of cells in this culture vessel l, and a liquid flowing into the culture vessel l. The nozzle tip of the distributor 3 for injecting the liquid and discarding the liquid from inside the culture vessel l is housed in the culture vessel main body μ maintained at constant gas atmosphere, temperature, and humidity conditions. In order to maintain the gas atmosphere inside the incubator main body 1 in a predetermined state, a CO2O2 gas pump device is used to control opening and closing, thereby maintaining the gas atmosphere in the main body μ in a predetermined state.
A humidity controller 10 is connected to the main body q, and the temperature and humidity inside the main body μ are maintained at a predetermined state by controlling the temperature controller 9 and the humidity controller 10f by a control device wLr.

Culture solution @ // , /2, /3 in tubes //
a, /2a, pulp by IJ&//b, t
! It is connected to the distributor 3 in the main body μ via bll3b.

The culture solution tank // contains a nutrient medium for growth, and the culture solution tank
The fermentation solution tank 2 contains an enzyme solution, and the culture solution tank l3 contains a buffer solution.The opening and closing of the pulps t/b, t2b, /Jb is controlled by the control device r, and a predetermined solution is stored in accordance with the culture operation. The pulp is opened so that the liquid in the culture liquid tank can be supplied to the culture vessel l via the distributor 3.

Next, the culturing operation in the culturing apparatus shown in FIG. 1 will be explained according to the process diagram in FIG. 2. First, before carrying out the culture operation, the subculture 11f: the culture vessel containing the cells to be carried out is housed in the incubator main body μ, and the observation device I! Observe the state of cell proliferation using this method. At this time, the temperature, humidity, pH of the culture solution, gas atmosphere information, and information on the f&sensing results in the main body q are input manually to the control device r, and based on these tlI information, commands from the control device l are sent. Pulp j+), l
, 7b and the operation of the regulator 9 and humidity regulator 10 are controlled to maintain a constant gas atmosphere, temperature, and humidity. For example, 5% CO2, humidity 10
0%, temperature 37C1, sterile conditions [The trowel culture operation is unstable.

Cells are electrostatically cultured in a constant atmosphere in a heather t4-ψ, and after a predetermined period of time the cells are observed using an observation device. When the cells have proliferated to the fullest in the culture container, the control device The distributor 3 is activated by the command from the rxr to suck the culture solution from inside the culture container l and discard it, after which pulp//
'D, with 12b closed, open the pulp/Jb and introduce the buffer stored in the culture solution tank 11 through the tube/jeL and inject it into the culture container/with the distributor 3 to wash the cells in the container. Do the following. After this washing, the buffer solution in the culture container/ is sucked and discarded by the distributor 3, and then the pulp//b
, /)bfM Sivalp/2bfi-An enzyme solution (e.g., trypsin) contained in an open culture solution tank 11 is introduced through the tube t2a and injected into the culture container 1 by the distributor 3, and the culture ζ; The cultured cells implanted on the bottom surface of the container are peeled off 1111.

After this, the enzyme solution in the culture container l is distributed by the distributor 3. Then, close the pulp /2b, /Jt), open the pulp //b, introduce the nutrient medium for growth contained in the culture liquid tank 11 through the tube //&, and transfer it to the culture vessel by the distributor 3. /, dilute the proliferating cells, distribute I:rl overlappingly to at least λ other culture vessels, and roughly proliferate the cells.

By the way, in such automatic culture equipment, maintaining the sterility of the proliferating cells as well as the gas atmosphere, temperature, and humidity are essential conditions for culturing cells, and during the culture operation, it is necessary to keep the cultured cells such as bacteria and rickettsia. also increase + f
Aseptic operation must be performed at all times to prevent contamination by highly potent microorganisms. For this reason, only sterilized culture solutions are used, and the culture solution once stored in the culture solution tank is not replaced even after the culture operation is completed, which may cause contamination.
Is it a method to keep setting it in the culture device until it is used up? taking it. In this way, culture fluids are kept for several days, sometimes even for several tens of days. Placed at a temperature of 7'C.

But something like this? When the nutrient medium for growth is placed at a temperature of 7-C, its components, vitamins and amino acids, begin to deactivate after one week, and the enzyme activity of trypsin, an enzyme solution, also deactivates within a few days. Put it away. For this reason (1), the conventional automatic culture apparatus as described above has the disadvantage that continuous, long-term culture cannot be carried out because it does not have a culture solution.

The purpose of the present invention is to solve the drawbacks of such conventional @culture devices and to deactivate the enzyme activity of the nutrient medium and enzyme solution as the culture solution even during continuous and long-term culture operations. The objective is to obtain a culture device that can be used without any problems.

In order to achieve this object, the culture device of the present invention is a cell culture device that automatically performs a series of culture operations, and includes a culture solution tank in which the culture solution is stored at a low temperature, and a culture device from which the culture solution is stored at a low temperature. n1 provided in the flow path leading to the culture container in
The apparatus includes a heater for heating the culture solution to a predetermined greenhouse temperature.

The culture apparatus of the present invention will be described in detail below with reference to the drawings. FIG. 3 shows an embodiment of the culture solution supply mechanism of the culture apparatus to which the present invention is applied. The culture machine main body J is controlled by a predetermined gas regulator, temperature controller, and moisture controller (not shown) to maintain the internal atmosphere at a constant gas atmosphere, temperature, and humidity conditions suitable for cell culture. , a culture vessel J containing predetermined cells to be cultured in this main body r
jrr-accommodate. Air filters 22a and #a are provided in culture solution tanks 22, 27, and 244, respectively.

Ventilation pipes 22b, nb, a with 24IeL attached
b, culture solution supply tube 22C+ 2JC', 2'lO
Connect with. Culture solution supply nozzles ne and 276 are made by inserting roller pumps nd113d and 21a into the culture solution supply tubes no, Pc, and 2G C, respectively, and communicating them through the warmer B and into the culture vessel main body.

Connect to 211f3. The culture solution tank contains a nutrient medium for growth, the culture solution tank n contains an enzyme solution, and the culture solution tank 2 contains a buffer solution. These culture solution tanks n,
11 and 211 are housed in the cold MJ register, and this cooling tank U
Store at a predetermined temperature (a low temperature at which the culture solution will not be deactivated or frozen, e.g. 10°C to 10°C) in a cooling tank made of a material with good thermal conductivity such as aluminum. A cooling pipe l made of metal and carrying refrigerant is wound around the peripheral wall.Both ends of the cooling pipe I are connected to a compressor I, and refrigerant cooled to a predetermined temperature is supplied from the compressor J into this cooling pipe r. !2.Also, in order to cool the refrigerant, the heat radiation pipe 30 wrapped around the heat sink 3 is connected to the compressor d.A temperature sensor 31 is installed in the cooling tank 1.
, a cooling m variable control circuit 3 that controls the cooling temperature in the cooling tank m
The detection output of the temperature sensor 31 is supplied to λ, and the cooling operation by the compressor 1 is controlled by the control circuit 32 according to the detection value of the temperature sensor 31. roller pump 22d
, ffd, and 2IId are controlled by a control device 33 that controls the gas atmosphere, temperature, and humidity inside the main body and controls the operation of the entire culture apparatus.

Warmer B is suitable for culture operation while supplying the culture solution stored at low temperature to the main body. ? This is for heating up to 7'C.The configuration of this warmer B is shown in Figure q.
As shown in (b), there is a heater body 31 made of a metal with good thermal conductivity, and a heater 35 embedded in the body 3.
And culture solution supply pipe DC, 270, 24! A heating groove 36 into which O can be fitted, and a temperature sensor embedded in the main body 3 to detect the heating temperature of the main body) (l).
7, and a culture solution supply pipe nc fitted into the heating groove 3B
, 2J0, a temperature sensor so that the liquid passing through the lac is heated to η°C after passing through the warmer,
? 7, the temperature of the main body 34I is detected, and a separate control circuit or control device 33 controls the heating of the heater 3j.
Next, a cell culturing operation using the culturing apparatus of the present invention provided with such a culture solution supply pipe structure will be explained. First, the culture vessel 2 containing cells to be subcultured is placed inside the culture vessel main body, which is controlled to maintain a predetermined gas atmosphere, temperature, and humidity condition suitable for cell culture. Culture is carried out, and the growth state of the cells is observed using a predetermined observation device having the same configuration as the conventional example described above. After a predetermined period of time has elapsed, when the results observed by the detection device show that the cells have proliferated to the fullest in the culture container 2/, a discharge pump (not shown) is activated by a command from the control device 33 to drain the cells in the culture container. @Suck up the nutrient solution and discard it. After that, the buffer solution stored in the culture solution tank 2g is passed through the culture solution supply pipe 21C, and the roller pump 2+d
After heating to a predetermined temperature (?7°C) through the warmer Bf, the culture vessel is heated through the culture solution supply nozzle 2e.
and wash the cells inside the container. After this washing, the buffer solution in the culture container 21 is sucked and discarded using a discharge pump (not shown), and then the enzyme solution contained in the culture solution tank n is transferred to the culture solution supply/supply pipe 2? Pass O through roller pump 2Jd
After heating the solution to a predetermined temperature (~7°C) through the warmer B, the culture solution is injected into the culture vessel 1sJJ from the culture solution supply nozzle Be, and the cultured cells implanted on the bottom surface of the culture vessel 1 are detached. let After that, the enzyme solution in the culture container J is sucked and discarded using a discharge pump (not shown), and then the culture solution tank N
The nutrient medium for growth contained in the culture medium supply pipe na f:
The culture solution is heated to a predetermined temperature (n″C) through a heater using a through-roller pump d, and then the culture solution is supplied to the nozzle 226.
□ Dilute the proliferated cells and distribute them into at least two or more other culture vessels in fixed positions,
Grow cells roughly.

As detailed above, the culture device of the present invention stores liquids necessary for culture work such as a nutrient medium for growth, an enzyme solution, and a buffer solution contained in a culture liquid tank in a cold MJ suitable for storage. Immediately before using the solution necessary for culture work, only the equipment used is heated to the temperature left for cell culture using a heater, so that the nutrient medium and enzyme activity are not deactivated, and the culture is maintained after each culture operation. There is no need to replace the liquid in the liquid register, the liquid used for culture is less likely to be contaminated, each liquid is kept cool until just before it is used for culture work, so fresh culture liquid can always be supplied, and it is the same for a series of experiments. This culture device has various advantages, such as high reliability of data because it can use culture fluid, and the ability to use up expensive culture fluid without wasting it.

[Brief explanation of drawings]

Fig. 1 is a diagram showing the configuration of a conventional culture device, Fig. 2 is a process diagram showing the culture work in subculture of cells in a conventional culture device, and Fig. 3 is a culture diagram of a culture device to which the present invention is applied. Diagram illustrating the configuration of an embodiment of the liquid supply mechanism, Fig.
) is a partially sectional plan view showing the configuration of the warmer B of the culture solution supply mechanism in FIG. 3, and FIG. 3(b) is a sectional view taken along the line bb of FIG. l...@culture container, 2... observation neck, 3... distributor, l... culture vessel body, 5... CO2 gas cylinder, 6
...02 To Kassan, 7-N2 gas cylinder, ja,
6a, 7a...tube, 51), 4b, 7b
···pulp,! ...control equipment! , 9... Temperature@device, /θ... Humidity controller, // , /2, /J・
・・Culture solution tank, //a, /, 2a, lj&・
...Tube, //b, t2b, /Jb--Pulp, 〃...Cultivator body, 1...Culture container, n, n. 24I... Culture 1! 'Monk, 22a, 2Ja, 2da...
・Air filter, 22t), 2Jb, 21b
... Ventilation pipe, 22c, 2JC, 2pc - Culture solution supply pipe, #d, 23d, 241d... Roller pump, ne, Be, 3e... Culture solution supply nozzle, B... Warming Vessel, soup... cold al, n...
Cooling pipe, I...Compressor, n...Radiator, 3θ
... Heat sink, 3/... Temperature sensor, 32... Control circuit, 33... Control device, 3... Warmer body, 3
j...Heater, 3t...Heating groove, q...Temperature sensor.

Claims (1)

[Claims] L Cell culture 1 that automatically performs a series of culture operations! Attire!
iI! In this method, a culture solution tank is used to store the culture solution at a low temperature, and a culture solution is heated to a predetermined temperature by pouring it into a flow path from the culture solution tank to the culture container in the culture device. A culture device characterized by a container and a mouth.