Abstract:
A temperature regulating container handles multiple samples which can be optically measured in real time while regulating the temperature of the samples. The samples can be moved at high speed by, for example, shaking without requiring a complicated mechanism. A cell culture container is a combination of three kinds of members: a temperature regulating liquid-holding member, a sample-holding member, and a lid. The cell culture container ensures that multiple samples are contacted with a temperature regulating liquid so that the temperature of the samples can be maintained at a uniform temperature, or quickly increased. The lid contacts the surface of the samples, preventing the samples from drying and contamination and, preventing dew formation on an optical path for performing optical measurement. The distance the light passes through the samples can be held constant to thereby assure accurate optical measurement; and sample containers can be easily moved.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a cell culture container which can stably hold a liquid whose temperature is regulated between multiple sample containers. 
       BACKGROUND ART 
       [0002]    As for cell culture, temperature management is one of the most important technical issues. In order to quickly and stably apply a culture temperature to cells, a temperature-controlled liquid is generally brought into contact with a sample container for culturing the cells. Advantageous effects of using the liquid for temperature regulating mainly include the following two points. (1) The liquid is more likely to transfer heat, compared to gas. (2) The liquid is deformed, and comes into close contact with an outer surface of the sample container. Accordingly, a contact area thereof can be maximized. Owing to these advantageous effects, heat is easily transferred to the sample container itself and a sample inside the container. 
         [0003]    According to PTL 1, the liquid is injected to a portion between a heat block serving as a heat source and the sample container, thereby obtaining the advantageous effects (1) and (2). 
         [0004]    Here, in order to monitor a culture process of the cells, optical measurement and culture are concurrently performed in many cases. For example, time-lapse imaging is performed in order to observe a differentiation process of the cells. According to PTL 1, if the sample container is detached from the heat block during optical measurement, an optical measurement surface is brought into a state where the liquid adheres to the optical measurement surface. Therefore, in order to perform the optical measurement, it is necessary to remove or clean the liquid on the optical measurement surface. However, if the optical measurement surface is frequently cleaned, there is a possibility that the optical measurement surface may be scratched. 
         [0005]    According to PTL 2, in order to observe a state of the sample inside the sample container, the sample container is not separated from the temperature-regulated liquid, and the optical measurement is performed through the liquid. 
         [0006]    Here, in order to perform the optical measurement through the temperature-regulated liquid, the temperature-regulated liquid needs to maintain a stable and high transmittance rate. In a state where the temperature-regulated liquid is released to the atmosphere in order to dip the sample container into the temperature-regulated liquid, the temperature-regulated liquid is held inside a device for a long time. Accordingly, due to contamination such as bacteria in the air, the temperature-regulated liquid becomes turbid, and the transmittance rate is changed. According to PTL 2, in order to prevent the transmittance rate from being changed due to the contamination, there is provided a device for sterilizing the contaminating bacteria. In addition, the sample container is dipped into the temperature-regulated liquid. Accordingly, if the sample container is moved or shaken fast, the temperature-regulated liquid bubbles up. Consequently, the temperature-regulated liquid cannot be stably held inside the device. An operation of uniformly mixing the sample by shaking the sample container is one of important operations in performing the cell culture. 
       CITATION LIST 
     Patent Literature 
       [0007]    PTL 1: JP-A-2004-294130 
         [0008]    PTL 2: JP-A-2013-134141 
       SUMMARY OF INVENTION 
     Technical Problem 
       [0009]    In a method of removing a liquid on an optical measurement surface or cleaning the optical measurement surface, if the liquid on the optical measurement surface of a sample is quickly and automatically removed or cleaned so as not to affect optical measurement, a device requires a complicated configuration, and manufacturing cost of the device increases. Accordingly, it is difficult to perform the optical measurement in real time. According to a method of performing the optical measurement through a temperature-regulated liquid, in order to maintain a transmittance rate of the temperature-regulated liquid, it is necessary to provide a disinfection device of the temperature-regulated liquid, as a countermeasure against contamination such as bacteria, thereby resulting in mechanically complicated configuration. In addition, if a sample container is dipped into the temperature-regulated liquid, or if the sample container is moved or shaken fast, the temperature-regulated liquid cannot be stably held inside the device. 
         [0010]    A problem to be solved is to provide a temperature regulating container in which each state of multiple samples can be optically measured in real time while each temperature of the multiple samples is regulated, and in which the sample can be shaken or moved fast without requiring a complicated mechanism. 
       Solution to Problem 
       [0011]    A temperature regulating container according to the present invention includes a temperature regulating liquid container that accommodates a temperature regulating liquid, a sample-holding member that can be accommodated inside the temperature regulating liquid container, and that has multiple recess portions for holding a sample, and a lid member that has multiple projection portions for putting a lid on each recess portion. 
       Advantageous Effects of Invention 
       [0012]    Each temperature of multiple samples can be uniformly regulated at any desired timing. Each state of the samples can be optically measured while the temperature is regulated. A sample container can be moved or shaken fast. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0013]      FIG. 1  is a perspective view illustrating characteristics of a cell culture container according to Embodiment 1. 
           [0014]      FIG. 2  is a perspective view illustrating a sample setting method of the cell culture container according to Embodiment 1. 
           [0015]      FIG. 3  is a perspective view illustrating a sample observation method of the cell culture container according to Embodiment 1. 
           [0016]      FIG. 4  is a perspective view illustrating characteristics of a cell culture container according to Embodiment 2. 
           [0017]      FIG. 5  is a perspective view illustrating a sample setting method of the cell culture container according to Embodiment 2. 
           [0018]      FIG. 6  is a perspective view illustrating a sample observation method of the cell culture container according to Embodiment 2. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0019]    The present invention provides a container which can hold a temperature regulating liquid around a sample container by interposing the temperature regulating liquid between a container for accommodating multiple samples and the other member. In addition, the present invention provides an introduction method of the sample and the temperature regulating liquid. 
         [0020]    In addition, the present invention provides a container which has a portion for accommodating multiple samples and a portion for accommodating the temperature regulating liquid, in which one or more holes for a temperature regulating liquid inlet and an air hole are disposed in the portion for accommodating the temperature regulating liquid, and which can inject the temperature regulating liquid into the portion for accommodating the temperature regulating liquid through the inlet. In addition, the present invention provides an introduction method of the sample and the temperature regulating liquid. 
         [0021]    Furthermore, the present invention provides a lid which removes an air layer on an optical path for optical measurement so as not to cause dew formed due to a temperature difference between the external air and the inside of the sample container, which can maintain a constant distance in which light passes through the inside of the sample, and which has a structure partially dipped into the sample. 
         [0022]    A sample to be measured may be a chemical reagent in addition to a cell suspension. 
         [0023]    Hereinafter, with regard to preferred embodiments for embodying the present invention, two embodiments will be described with reference to the drawings. In each embodiment, the following items will be described. 
       1. Outline of Each Member 
     2. Sample Setting Method 
     3. Incubation and Observation Method 
       [0024]    The embodiments described herein are merely examples of representative embodiments of the present invention. The embodiments do not allow the scope of the present invention to be narrowly interpreted. 
       Embodiment 1 
     1. Outline of Each Member 
       [0025]      FIG. 1  is a perspective view illustrating a configuration and characteristics of a cell culture container according to the present invention. 
         [0026]    The cell culture container is configured to include three members of a temperature regulating liquid-holding member  110 , a sample-holding member  120 , and a lid  130 . 
         [0027]    The temperature regulating liquid-holding member  110  illustrated in  FIG. 1( a )  has a structure surrounded by an edge having a temperature regulating liquid-holding portion  111  which is one partitioned region for holding a temperature regulating liquid. A size and a shape of the overall temperature regulating liquid-holding portion  111  are aligned with an outer edge of the sample-holding member  120  illustrated in  FIG. 1( b ) . In this manner, a structure is employed in which the temperature regulating liquid is less likely to be scattered. 
         [0028]    The sample-holding member  120  illustrated in  FIG. 1( b )  has a structure provided with multiple recessed sample-holding portions  121  which can dispense and hold the sample from above in a lid-shaped top plate. A cross-sectional shape of the sample-holding portion  121  may be circular or square. It is desirable that a bottom surface of the sample-holding portion  121  is flat and smooth. A size and a shape of the overall sample-holding member  120  are aligned with the temperature regulating liquid-holding portion  111  illustrated in  FIG. 1( a ) . In this manner, the sample-holding member  120  is shaped so that the temperature regulating liquid is less likely to be scattered. In addition, the top plate of the sample-holding member  120  has one or more air holes  122  serving as through-holes. The air hole  122  is provided with an air exchange function when the temperature regulating liquid-holding member  110  and the sample-holding member  120  are combined with each other, and a function to maintain high humidity so as to prevent the sample from being dried. The air hole  122  may be covered with a waterproof moisture-permeable material so that the temperature regulating liquid is not scattered. 
         [0029]    The lid  130  illustrated in  FIG. 1( c )  has a structure having multiple recessed sample contact portions  131  in the top plate having a normal lid shape. A size and a shape of the overall lid  130  are aligned with the sample-holding member  120  illustrated in  FIG. 1( b ) . The number of the sample contact portions  131  is the same as the number of the sample-holding portions  121  illustrated in  FIG. 1( b ) , and the sample contact portion  131  and the sample-holding portion  121  are disposed at the same position. A cross-sectional shape of the sample contact portion  131  is smaller than a cross-sectional shape of the sample-holding portion  121 . The sample contact portion  131  is accommodated inside the sample-holding portion  121 . A depth of the sample contact portion  131  is set to a depth which allows contact with the surface of the sample when the temperature regulating liquid-holding member  110 , the sample-holding member  120 , and the lid  130  are combined with each other. 
         [0030]    The temperature regulating liquid-holding member  110 , the sample-holding member  120 , and the lid  130  employ a transparent member for a portion through which light passes during microscopic observation or optical density measurement or for all members. 2. Sample Setting Method 
         [0031]      FIG. 2  schematically illustrates a sample setting method of the cell culture container. 
         [0032]    As illustrated in a three-dimensional view (a) in  FIG. 2  and a sectional view (a′) thereof, a sample  140  is put in the sample-holding portion  121  of the sample-holding member  120 . Thereafter, a temperature-regulated temperature regulating liquid  141  which raises the temperature of the sample  140  up to a target temperature is dispensed to the temperature regulating liquid-holding portion  111 . It is desirable to use a fluid with large specific heat such as water for the temperature regulating liquid  141 . 
         [0033]    As illustrated in a three-dimensional view (b) in  FIG. 2  and a sectional view (b′) thereof, the sample-holding member  120  and the temperature regulating liquid-holding member  110  are caused to overlap each other, and the temperature regulating liquid  141  is brought into contact with the sample-holding portion  121 . Here, the air is discharged from the air hole  122 . In this manner, the temperature regulating liquid  141  is introduced into and held in a portion between the sample-holding portions  121 . 
         [0034]    As illustrated in a three-dimensional view (c) in  FIG. 2  and a sectional view (c′) thereof, the lid  130  is put thereon from above the sample-holding member  120 . Here, in a case where the air is exchanged under a culture condition of the sample  140 , the sample-holding member  120  and the lid  130  are set to have a shape which is not sealed. In a case where the air is not exchanged, the sample-holding member  120  and the lid  130  are set to have a shape which is sealed. 
         [0035]    As illustrated in a three-dimensional view (d) in  FIG. 2  and a sectional view (d′) thereof, a bottom surface of the sample contact portion  131  is brought into contact with a surface of the sample  140 . 
         [0036]    In this way, sample setting is completed. 
       3. Incubation and Observation Method 
       [0037]    With regard to an incubation method, in the sample container in which three members of the sample-holding member  120  having the sample  140  introduced thereto in  FIG. 2 , the temperature regulating liquid-holding member  110  having the temperature regulating liquid  141  introduced thereto, and the lid  130  are combined with each other, the temperature of the sample container is allowed to reach the target temperature by the temperature regulating liquid  141 . In order to hold the temperature, the sample container is stored in an air thermostat which holds the temperature to be the same as the target temperature. In this way, the sample container is prevented from losing heat. In addition, in a case where the sample is to be mixed, the sample container is shaken. 
         [0038]    With regard to an observation method, as illustrated in  FIG. 3 , light emitted from a light source  151  in the thermostat  153  holding the temperature is condensed by a lens. The light is transmitted through the sample inside the sample container  150  used for incubation. The light is received by a photodetector  152 . An observation value of optical density or turbidity of the sample is acquired from a signal obtained from the photodetector  152 . In order to observe a culture state, the measurement is performed at any desired time interval such as one hour interval, thereby observing how cells proliferate. In addition, instead of the photodetector  152 , an imaging element and an objective lens are disposed, and microscopic observation is performed on the sample. It is also possible to observe a proliferative process of the cells by comparing the observation images. 
         [0039]    According to the present embodiment, even if a temperature environment is changed when the sample container is detached, dew is not formed on an optical path. In addition, it is possible to maintain a constant distance in which light passes through the inside of the sample, and it is possible to maintain a state where the temperature of the sample is less changed. 
       Embodiment 2 
     1. Outline of Each Member 
       [0040]      FIG. 3  is a perspective view illustrating a configuration and characteristics of a cell culture container according to the present invention. 
         [0041]    The cell culture container is configured to include two members of a sample-holding member  210  and a lid  220 . 
         [0042]    The sample-holding member  210  illustrated in  FIG. 3( a )  has a structure provided with multiple sample-holding portions  211  having a recessed shape which the sample enters on an upper surface of a hollow box. A cross-sectional shape of the sample-holding portion  211  may be circular or square. It is desirable that the bottom surface of the sample-holding portion  211  is flat and smooth. A depth of the bottom surface of the sample-holding portion  211  may be equal to that of the bottom surface of the sample-holding member  210 . Alternatively, a space may be disposed between the bottom surface of the sample-holding portion  211  and the bottom surface of the sample-holding member  210 . An outer space of the sample-holding portion  211  inside the sample-holding member  210  serves as temperature regulating liquid-holding portions  212 . In order to uniformly and quickly raise the temperature of the sample inside the multiple sample-holding portions  211 , it is desirable to install multiple temperature regulating liquid inlets  214  on the upper surface or the side surface of the sample-holding member so that the temperature regulating liquid can be introduced fast. The temperature regulating liquid inlet  214  may be provided with a structure having a check valve so that the liquid does not leak when the liquid is injected. An air hole  213  is installed on the upper surface of the sample-holding member, and is provided with an air exchange function when the temperature regulating liquid is introduced, and a function to maintain high humidity so as to prevent the sample from being dried. The air hole  213  may be covered with a film made of a waterproof moisture-permeable material so that the temperature regulating liquid is not scattered. 
         [0043]    The lid  220  illustrated in  FIG. 3( b )  has a structure having multiple recessed sample contact portions  221  in the top plate having a normal lid shape. A size and a shape of the overall lid  220  are aligned with the sample-holding member  210  illustrated in  FIG. 3( a ) . The number of the sample contact portions  221  is the same as the number of the sample-holding portions  211  illustrated in  FIG. 3( a ) , and the sample contact portion  221  and the sample-holding portion  211  are disposed at the same position. A cross-sectional shape of the sample contact portion  221  is smaller than a cross-sectional shape of the sample-holding portion  211 . The sample contact portion  221  is accommodated inside the sample-holding portion  211 . A depth of the sample contact portion  221  is set to a depth which allows contact with the surface of the sample when the sample-holding member  210  and the lid  220  are combined with each other. 
         [0044]    The sample-holding member  210  and the lid  220  employ a transparent member for a portion through which light passes during microscopic observation or optical density measurement or for all members. 
       2. Sample Setting Method 
       [0045]      FIG. 4  schematically illustrates a sample setting method of the cell culture container. 
         [0046]    As illustrated in a three-dimensional view (a) in  FIG. 4  and a sectional view (a′) thereof, a sample  230  is put in the sample-holding portion  211  of the sample-holding member  210 . 
         [0047]    As illustrated in a three-dimensional view (b) in  FIG. 4  and a sectional view (b′) thereof, a temperature-regulated temperature regulating liquid  231  which raises the temperature of the sample  230  up to a target temperature is introduced to the temperature regulating liquid-holding portion  212  through the temperature regulating liquid inlet  214 . It is desirable to use a fluid with large specific heat such as water for the temperature regulating liquid  231 . 
         [0048]    As illustrated in a three-dimensional view (c) in  FIG. 4  and a sectional view (c′) thereof, the lid  220  is put thereon from above the sample-holding member  210 . Here, in a case where the air is exchanged under a culture condition of the sample  230 , the sample-holding member  210  and the lid  220  are set to have a shape which is not sealed. In a case where the air is not exchanged, the sample-holding member  210  and the lid  220  are set to have a shape which is sealed. 
         [0049]    As illustrated in a three-dimensional view (d) in  FIG. 2  and a sectional view (d′) thereof, the bottom surface of the sample contact portion  221  is brought into contact with the surface of the sample  230 . 
         [0050]    In this way, sample setting is completed. 
       3. Incubation and Observation Method 
       [0051]    With regard to an incubation method, in the sample container in which the sample-holding member  120  having the sample  230  introduced thereto in  FIG. 5  and the lid  220  are combined with each other, the temperature of the sample container is allowed to reach the target temperature by the temperature regulating liquid  231 . In order to hold the temperature, the sample container is stored in an air thermostat which holds the temperature to be the same as the target temperature. In this way, the sample container is prevented from losing heat. In addition, in a case where the sample is to be mixed, the sample container is shaken. 
         [0052]    With regard to an observation method, as illustrated in  FIG. 6 , light emitted from a light source  241  in a thermostat  243  holding the temperature is condensed by a lens. The light is transmitted through the sample inside the sample container  240  used for incubation. The light is received by a photodetector  242 . An observation value of optical density or turbidity of the sample is acquired from a signal obtained from the photodetector. In order to observe a culture state, the measurement is performed at any desired time interval such as one hour interval, thereby observing how cells proliferate. In addition, instead of the photodetector  242 , an imaging element and an objective lens are disposed, and microscopic observation is performed on the sample. It is also possible to observe a proliferative process of the cells by comparing the observation images. 
       REFERENCE SIGNS LIST 
       [0000]    
       
         
           
               110  temperature regulating liquid-holding member 
               111  temperature regulating liquid-holding portion 
               120  sample-holding member 
               121  sample-holding portion 
               122  air hole 
               130  lid 
               131  sample contact portion 
               140  sample 
               141  temperature regulating liquid 
               150  sample container 
               151  light source 
               152  photodetector 
               153  thermostat 
               210  sample-holding member 
               211  sample-holding portion 
               212  temperature regulating liquid-holding portion 
               213  temperature regulating liquid inlet 
               214  air hole 
               220  lid 
               221  sample contact portion 
               230  sample 
               231  temperature regulating liquid 
               240  sample container 
               241  light source 
               242  photodetector 
               243  thermostat