JP2004024179A - Warm water circulation type micro-culturing chamber - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a micro-culturing chamber capable of readily carrying out exchange of a cultured solution of a cell. <P>SOLUTION: A warm water circulating chamber 1 is composed of opposed two sheets of optical glass 3, an optical glass fixing means 5 for fixing two sheets of optical glass 3 so that a first closed space 4 for circulating warm water is formed between two sheets of optical glass 3, a warm water flow inlet 6 for allowing warm water to flow into the first closed space 4 and a warm water flow outlet 7 for allowing the warm water to flow out the first closed space. A cell culturing chamber 2 is composed of a cover glass 10 for culturing a cell 9, a cover glass-fixing means 12 for fixing the cover glass 10 so that a second closed space for culturing the cell 9 is formed between the surface of the optical glass 3 of the warm water circulating chamber 1 and the surface of the cover glass 10 and a culture solution exchange port 13 for exchanging a cell culture solution of the second closed space 11. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The invention of this application relates to a hot water circulation type microculture chamber. More specifically, the invention of this application is applicable to a general erecting microscope system, and relates to a warm water circulation type microculture chamber capable of easily exchanging a cell culture solution.
[0002]
[Prior Art and Problems of the Invention]
Most conventional cell culture systems for microscopes require an inverted microscope, and the entire observation stage or the entire microscope is covered with a heat-retaining chamber. The cell culture system for microscopes with such a configuration is large and has no standardized configuration or shape, so there is no compatibility between different manufacturers, and once installed, it can be used with other microscope systems. There is a problem that it is difficult to change the name. In addition, these microscope cell culture systems do not have a mechanism for exchanging cell culture media, and it is necessary to attach a dedicated system separately in order to exchange cell culture media during observation. was there.
[0003]
At present, a product manufactured by Warner Corporation in the United States is sold as a culture chamber that can be installed in both an upright microscope and an inverted microscope. This is to perfuse the cell culture solution pooled outside the observation chamber into the observation chamber, and the temperature of the cell culture solution is controlled by a heater incorporated in the middle of the perfusion tube. Although the cell culture medium can be exchanged, it is necessary to pay sufficient attention to the heat denaturation of the culture medium additives and the like since the heat of the heater is directly applied to the cell culture medium. In addition, since the temperature in the chamber was not measured, observation was performed in a state where the actual environmental temperature of the cells was unknown.
[0004]
In order to observe animal cultured cells in a living state with a fluorescence microscope, it is necessary to install a culture chamber in the microscope and strictly control the temperature environment. In particular, in order to observe the reaction when a chemical stimulus is applied, it is necessary that the cell culture medium can be exchanged without changing the observation field.
[0005]
In order to satisfy these requirements and to be compatible with various microscope systems such as fluorescence microscope, confocal laser microscope, differential interference microscope, and cooled CCD camera, it has a configuration that can be installed on a general upright microscope. The development of a micro-culture chamber was also required.
[0006]
Accordingly, the invention of this application has been made in view of the circumstances described above, and provides a new microculture chamber that can be easily replaced with a cell culture solution, corresponding to a general erecting microscope. Is an issue.
[0007]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the invention of the present application firstly provides a microculture chamber having a two-layer structure of a warm water circulation chamber arranged in a lower layer and a cell culture chamber arranged in an upper layer. An optical glass in which a hot water circulation chamber fixes two optical glasses so that a first closed space for circulating hot water between the two optical glasses and the two optical glasses is formed. A cover glass for culturing cells, comprising: fixing means; a hot water inlet for flowing hot water into the first closed space; and a hot water outlet for flowing hot water into the first closed space. A cover glass fixing means for fixing the cover glass such that a second closed space for culturing cells is formed between the surface of the optical glass of the hot water circulation chamber and the surface of the cover glass; The cell culture in the second closed space And culture exchange ports for conversion, to provide a microscopic culture chamber, characterized in comprising a.
[0008]
In addition, the invention of this application, as an aspect of the second invention, states that the hot water circulation means for circulating the hot water in the hot water circulation chamber is connected to the hot water inlet and the hot water outlet. According to a fourth aspect of the present invention, the hot water circulating means for circulating hot water in the hot water circulating chamber is connected to the hot water inlet and the hot water outlet via bubble removing means for removing bubbles in the hot water. According to a fifth aspect of the present invention, the temperature measuring means is a micro thermocouple having a diameter of 0.5 mm or less. As a seventh aspect of the present invention, there is provided a hot water temperature control means for reading a signal output by the temperature measuring means in accordance with the measured temperature and controlling the temperature of the hot water by PID. Introduced culture solution Culture exchange means for derivation is that it is connected to the culture liquid exchange opening, there is provided the above microscopic culture chamber, wherein, respectively.
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
The invention of this application has the features as described above, and embodiments thereof will be described below.
FIG. 1 is a schematic diagram showing the configuration of a microculture chamber according to the invention of the present application. As shown in FIG. 1, the microculture chamber according to the invention of this application has a two-layer structure of a warm water circulation chamber (1) arranged in a lower layer and a cell culture chamber (2) arranged in an upper layer.
[0010]
In the lower warm water circulation chamber (1), two opposing optical glasses (3) are arranged so as to form a first closed space (4) for circulating warm water therebetween. The two optical glasses (3) are fixed by optical glass fixing means (5). The optical glass fixing means (5) has a hot water inlet (6) for flowing hot water into the first closed space (4), and a hot water outlet for flowing hot water into the first closed space (4). (7) is provided. It is preferable to arrange the anti-friction table (8) so that the optical glass (3) at the bottom of the microculture chamber does not contact the installation surface of the microculture chamber.
[0011]
In the upper cell culture chamber (2), a cover glass (10) for culturing the cells (9) is placed between the upper surface of the optical glass (2) in the hot water circulation chamber (1) and a second closed space ( 11) is fixed. The cover glass (10) is fixed by cover glass fixing means (12). The cover glass fixing means (12) is provided with a culture medium exchange port (13) for exchanging the cell culture medium in the second closed space (11).
[0012]
FIG. 2 is a schematic diagram showing the configuration of the entire system when the microculture chamber according to the invention of the present application is operated as a cell culture system.
[0013]
A hot water circulation means (24) for circulating hot water is connected to the hot water inlet (22) and the hot water outlet (23) in the hot water circulation chamber (21). Circulate. The hot water circulating means (24) may be connected to the hot water inlet (22) and the hot water outlet (23) via a bubble removing means (25) for removing bubbles mixed in the hot water. preferable. Various pumps such as a tube pump are appropriately selected and used as the hot water circulation means (24).
[0014]
As the bubble removing means (25), for example, as shown in FIG. 3, a container (37) in which the hot water inlet (35) is located above the hot water outlet (36) is used. Bubbles in the warm water flowing into the vessel (37) from the warm water inlet (35) are separated into the upper part of the vessel (37), and the warm water flows out of the warm water outlet (36) with the bubbles removed. . A gas vent (38) is opened in the upper part of the container (37), and a tube (39) is connected to the gas vent (38). As the tube (39), a Teflon tube or the like is used. A pinch cock (40) is attached to the distal end of the tube (39). When air bubbles are accumulated in the container (37), the pinch cock (40) is released to release the container (37). Releases gas to the outside of the
[0015]
Further, the microculture chamber according to the invention of the present application is provided with a temperature measuring means (26) for measuring the temperature of the cell culture chamber, as shown in FIG. Specifically, a fine thermocouple having a diameter of 0.5 mm or less is used for the temperature measuring means (26). The signal output by the temperature measuring means (26) according to the measured temperature is input to the hot water temperature control means (27) connected to the hot water circulation means, and is input to the hot water temperature control means (27). PID control for adjusting the temperature of the hot water to an appropriate temperature is performed according to the signal.
[0016]
Specifically, the hot water temperature control means (27) includes a PID temperature control device (28) corresponding to the temperature measurement means (26) and capable of outputting a signal for a solid state relay, a solid state relay (29), And a heater (30). A signal from the temperature measuring means (26) is input to the PID temperature controller (28), the solid state relay (29) is controlled based on the PID control theory, and power is supplied to the heater (30). The heater (30) is wound around a hot water bottle connected to a tube for hot water circulation. As the heater (30), for example, a silicon rubber heater is used. At this time, it is preferable that the hot water bottle is installed on a magnetic stirrer (31) so that the temperature inside the bottle is agitated so that the temperature inside the bottle becomes uniform.
[0017]
A culture medium exchange means (34) for introducing or extracting a cell culture medium is connected to a culture medium exchange port (33) in the cell culture room (32). As shown in FIG. 4, the culture medium exchange means (34) has two culture medium storage tubes (41) storing the cell culture medium and a piston portion (42) attached thereto so as to be interlocked. And a configured injection cylinder (43). The amount of the cell culture solution to be injected into the cell culture chamber (44) by the syringe barrel (43) is equal to the amount of the cell culture solution to be aspirated.
[0018]
The above is an example of the embodiment of the microculture chamber according to the invention of the present application, and the invention of the present application is not limited to the above-described embodiment, and it is considered that the details thereof can take various forms. Needless to say, it should be done.
[0019]
In the microculture chamber according to the invention of this application, the temperature of the cell culture solution in the cell culture room can be precisely controlled by measuring the temperature of the cell culture room in real time and performing PID control of the temperature of the circulating hot water based on the measured value. Can be controlled. Even after replacement of the cell culture solution, the solution temperature can be returned to the set temperature in a short time of several seconds, and stable temperature control without any hunting can be realized. Because of the indirect heating with warm water, chemical denaturation of the additive in the cell culture is also unlikely to occur.
[0020]
In the hot water circulation, air bubbles are generated due to the pressure difference inside the pump which is the hot water circulation means, but by interposing the air bubble removing means immediately before flowing into the microculture chamber, the mixing of the air bubbles into the hot water circulation chamber is completely completed. Can be prevented.
[0021]
Since only the optical glass and warm water of the warm water circulation chamber are present in the lower part of the cell culture room, clear Nomarski differential interference images can be taken without disturbing bright-field illumination. The microculture chamber according to the invention of the present application has characteristics sufficient for performing normal fluorescence time-lapse analysis and bright-field time-lapse analysis such as differential interference and phase difference.
[0022]
Conventionally, all the cell culture systems for microscopes provided by scientific instrument manufacturers are expensive and have a large configuration, so there is a problem that one microscope system is occupied for cultured cells. Was. The microculturing chamber of the invention of the present application has a simple configuration, so that it is possible to reduce the cost sufficiently.Also, the entire configuration is compact, and the installation can be easily performed. It has various advantages such as easy use. Also, unlike conventional cell culture systems for microscopes, it can be applied to erect microscopes.In addition to ordinary bright-field microscope systems, epi-fluorescence devices, laser confocal systems, differential interference optical systems , And a phase difference optical system.
[0023]
【The invention's effect】
According to the invention of this application, as described in detail above, a new microculture chamber is provided which is compatible with a general erecting microscope and can easily exchange cell culture media. The microculture chamber according to the invention of the present application is easy to set up, can easily perform cell culture, and can be applied to many microscopes. Is expected to be put to practical use as a cultured cell observation technology with a [Simplified explanation of drawings]
FIG. 1 is a schematic diagram showing a configuration of a microculture chamber according to the invention of the present application.
FIG. 2 is a schematic diagram showing a configuration of a microculture chamber according to the invention of this application.
FIG. 3 is a schematic diagram showing a configuration of a bubble removing unit in the microculture chamber according to the invention of the present application.
FIG. 4 is a schematic diagram showing a configuration of a culture solution exchange means in the microculture chamber according to the invention of the present application.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Hot water circulation room 2 Cell culture room 3 Optical glass 4 First closed space 5 Optical glass fixing means 6 Hot water inflow port 7 Hot water outflow port 8 Anti-friction table 9 Cell 10 Cover glass 11 Second closed space 12 Cover glass fixing means 13 Culture Fluid Exchange Port 21 Hot Water Circulation Room 22 Hot Water Inlet 23 Hot Water Outlet 24 Hot Water Circulation Mean 25 Bubbles Removal Means 26 Temperature Measurement Means 27 Hot Water Temperature Control Means 28 PID Temperature Control Device 29 Solid State Relay 30 Heater 31 Magnetic Stirrer 32 Cells Culture room 33 Culture solution exchange port 34 Culture solution exchange means 35 Hot water inlet 36 Hot water outlet 37 Container 38 Gas vent 39 Tube 40 Pinch cock 41 Culture solution storage tube 42 Piston part 43 Injection cylinder 44 Cell culture room

Claims (7)

A microculture chamber having a two-layer structure of a warm water circulation chamber arranged in a lower layer and a cell culture chamber arranged in an upper layer,
Optical glass fixing for fixing two optical glasses such that a warm water circulation chamber forms two opposing optical glasses and a first closed space for circulating hot water between the two optical glasses. Means, a hot water inlet for flowing hot water into the first closed space, and a hot water outlet for flowing hot water into the first closed space,
A cell culture chamber covers the cells so as to culture the cells, and a cover such that a second closed space for culturing the cells is formed between the surface of the optical glass and the surface of the cover glass in the warm water circulation chamber. A microculturing chamber comprising: a cover glass fixing means for fixing glass; and a culture medium exchange port for exchanging a cell culture medium in the second closed space. 2. The microculture chamber according to claim 1, wherein a hot water circulating means for circulating hot water in the hot water circulating chamber is connected to the hot water inlet and the hot water outlet. 3. The hot water circulating means for circulating hot water in the hot water circulating chamber is connected to the hot water inlet and the hot water outlet through bubble removing means for removing bubbles in the hot water. Microculture chamber. The microculture chamber according to claim 2 or 3, further comprising a temperature measuring means for measuring a temperature in the cell culture chamber. The microculture chamber according to claim 4, wherein the temperature measuring means is a micro thermocouple having a diameter of 0.5 mm or less. The microculture chamber according to claim 4 or 5, further comprising a hot water temperature control means for reading a signal output by the temperature measuring means according to the measured temperature and performing PID control of the temperature of the hot water. 7. The microculture chamber according to claim 1, wherein a culture medium exchange means for introducing or taking out the cell culture medium into or from the cell culture chamber is connected to the culture medium exchange port.

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