Abstract:
A microchamber including a glass substrate which is transparent to a specific wavelength, an absorbent region which absorbs the specific wavelength, and a melting substance region which does not absorb the specific wavelength, is solid at room temperature and melts when heated, which regions are layered on the glass substrate. The absorbent region, is irradiated with a focused light beam of the specific wavelength and locally heated in the vicinity of the converging rays, so that the melting substance region is locally melted at a portion adjacent to the absorbent region, thereby forming a cavity as the focused light beam moves. Accordingly, the shape of the microchamber can be arbitrarily changed in accordance with the process of cell culture.

Description:
TECHNICAL FIELD 
     The invention of the present application relates to a novel microchamber for cell culture capable of culturing cells one by one while observing the state of the cells under a microscope. 
     BACKGROUND ART 
     A change in the state of cells or response of cells to a chemical or the like has conventionally been observed as if the average of a cell group represented the property of one cell. In an actual cell group, cells which are synchronized in a cell cycle are not so many and cells express a protein at cycles different from each other. A method of synchronized culture has been developed with a view to overcoming these problems. Since the cells cultured are not cells derived from exactly the same cells, however, there is a possibility of causing a difference in the protein expression due to a difference in genes among the derived cells before cultivation. Upon actual analysis of a response to stimulation, it is very difficult to find whether fluctuations appearing in the results are attributable to the fluctuations in response which are common in the reaction mechanism of the cell itself or to a difference among cells (that is, a difference in genetic information). Since a cell line is usually not obtained by cultivating one cell, it is also very difficult to find whether or not the fluctuations in the reproduction of a response to stimulation are attributable to a genetic difference among cells. Stimulation (signal) to cells can be classified into two groups, that given by the amounts of a signal substance, nutrition and dissolved gas contained in a solution surrounding the cells therewith; and that given by a physical contact with another cell, which also makes the judgment on the fluctuations difficult. 
     When cell observation is carried out in the biotechnological research field, it is common practice to temporarily take out a portion of a cell group cultured using a large-sized incubator and observe the cells set on a microscope, or to control the temperature of the microscope while surrounding the entire microscope with a plastic container and carry out microscopic observation at a carbon dioxide concentration and humidity controlled using another small container inserted in the plastic container. Upon this observation, the conditions of a culture solution are kept constant by replacing a wasted culture solution with a fresh one, while culturing cells. According to the method as disclosed in Japanese Patent Laid-Open No. 10-191961, for example, the nutrition condition is kept constant by a mechanism in which a circulation pump moves the level of a medium relative to the surface of a base material up and down between a level higher than the upper end height of the base material and a level lower than its bottom end height, whereby a new medium is fed when the level is below the above-described low level and the medium is discharged when the level exceeds the high level. Disclosed in Japanese Patent Laid-Open No. 08-172956 is a culture apparatus, in which one end of each of an inlet pipe for introducing a new culture medium into a culture vessel, an outlet pipe for discharging the culture medium in the culture vessel to the outside, and a gas pipe permitting a gaseous portion in the culture vessel and a pump to communicate with each other is inserted in the culture vessel and filters for preventing the invasion of bacteria into the culture vessel are installed to the inlet pipe, outlet pipe and gas pipe, respectively. Thus, the nutrition conditions in a culture tank can be kept constant. In either one of these inventions, however, an example of culturing cells while controlling their solution environment and physical contact between cells is not known. 
     With a view to overcoming these problems, the inventors of this application invented a technology of selecting only one specific cell and cultivating it as a cell line, a technology of controlling the conditions of a solution environment of cells and keeping the cell density in the vessel constant upon cell observation, and a technology of cultivating and observing cells, which interact with each other, while specifying them and applying for patent on them as Japanese Patent Application No. 2000-356827. 
     The microchamber newly proposed by the present inventors has novel characteristics in its constitution. The microchamber is formed utilizing a microfabrication technology of glass or the like. Prior to the initiation of cultivation, the microchamber is formed on the glass surface and cell culture can be carried out by making use of its shape. It is therefore difficult to change, depending on the state after cultivation, the pattern of a flow path between the microchambers which path determines interaction between the microchambers. It is also difficult to change the shape of the microchamber itself in accordance with the progress of the cultivation. 
     In addition, a technology of heating the microchamber to change its shape by making use of a focused beam or the like cannot be applied to the heating in a three-dimensional local region smaller than the wavelength of infrared light. 
     An object of the present invention is therefore to provide a novel microchamber whose shape can be changed depending on the cultivation stage, based on the detailed investigation on the above-described microchamber developed by the present inventors. Another object of the present invention is to provide a novel microchamber for cell culture which permits spot heating of a nanoscale fine region. 
     SUMMARY OF THE INVENTION 
     The invention of the present application provides, in order to overcome the above-described problems, a microchamber for cell culture which comprises a substrate transparent to light of a specific wavelength, and a region which absorbs light of the specific wavelength and another region made of a solid which does not absorb light of the specific wavelength and has a melting point lower than the boiling point of water, for example, a substance which is a solid at normal temperature but is melted by heating, both regions being laid over the substrate. Further, the invention provides a cell culture apparatus with a unit of irradiating light having a specific wavelength, along with the microchamber. 
     More specifically, the cell culture apparatus according to the present invention has a unit of irradiating a focused beam having the specific wavelength to a specific region of the above-described microchamber for cell culture. The region of a solid substance which does not absorb light of the specific wavelength locally emits heat in the absorption region when exposed to a focused beam having the specific wavelength and by the resulting heat, the region of the solid substance existing adjacent to the absorption region is partially molten and dispersed. With the movement of a focused beam, it forms a space. 
     Also provided is a spot heating unit of a nanoscale fine region. It can carry out microscopic level drawing to form a nanoscale fine pattern of the absorption region, and expose the pattern to a focused beam, thereby spot-heating a region limited to a size as fine as the line width of a thin film layer and finer than the wavelength of the exposed light. 
     The microchamber for cell culture further comprises a semipermeable membrane which covers therewith the upper surface of the microchamber so as to block the cells from coming out of the chamber, has a pore size small enough to disturb the passage of cells through the film, and is optically transparent to the focused beam, and a unit permitting the replacement of a solution in the solution replacement section, in which a culture solution is circulated, on the upper surface of the semipermeable membrane. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic view illustrating one example of the fundamental constitution of the invention of the present application; 
         FIG. 2  is a schematic view illustrating the constitution of the microchamber for cell culture as illustrated in  FIG. 1 ; 
         FIG. 3  is a schematic view illustrating one example of the constitution of an apparatus for observing the microchamber for cell culture as illustrated in  FIG. 1  and spot heating using a focused beam; 
         FIG. 4  is a schematic view for explaining the processing of the microchamber for cell culture by spot heating using a focused beam; 
         FIG. 5  is a schematic view illustrating another example of the constitution of the microchamber for cell culture; 
         FIG. 6  is a schematic view illustrating a further example of the constitution of the microchamber for cell culture; 
         FIG. 7  is a schematic view illustrating a still further example of the constitution of the microchamber for cell culture; 
         FIG. 8  is a microphotograph for explaining one example of the processing of the microchamber for cell culture by spot heating using a focused beam; 
         FIG. 9  is a schematic view illustrating a still further example of the constitution of the microchamber for cell culture; 
         FIG. 10  is a schematic view illustrating a still further example of the constitution of the microchamber for cell culture; and 
         FIG. 11  is a schematic view illustrating a still further example of the constitution of the microchamber for cell culture. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention of the present application has characteristics as described above. The embodiment of the invention will next be described. 
     First, one example of the fundamental constitution of the microchamber for cell culture according to the invention of the present application will be described based on the example illustrated in  FIG. 1 . For example, as illustrated in  FIG. 1 , the microchamber  100  for cell culture according to the invention of the present application has an optically transparent substrate  101  such as slide glass and a thin film layer  102  laid thereover as an absorption layer exhibiting optical absorption such as a chromium deposited layer. Upon observation through a transmitted light, the thin film layer  102  is preferably thick enough not to absorb light completely and at the same time is thin without irregularities in thickness. When the absorption layer is made of chromium, it has a thickness of 50 Å and transmits about 70% of light in the visible range. Over the light absorption thin film layer  102 , a region  103  of a substance such as agarose is laid which is optically transparent, has a low melting point and has no toxicity to cells such as. The substance of this region  103  is a solid substance, as defined by the invention of the present application, which does not absorb light of a specific wavelength and at the same time, having a melting point lower than the boiling point of water. Agarose is a typical example of it. The solid substance having a melting point not greater than 45° C. is usually preferred. In particular, agarose is harmless to cells, has less influence on the culture test data and is therefore the most appropriate substance, because it does not exhibit adhesion to cells and is not a signal substance for cells. In the region  103 , a plurality of cavities  105  for introducing a sample such as cell  104  are formed in a mold upon formation of the region  103 . In each cavity  105 , a specific cell  104  is cultivated. The surface of the light absorption thin film layer  102  such as a chromium deposited layer may be subjected to silane formation treatment, followed by application and fixing thereto a cell absorptive factor such as collagen to permit stable adhesion of the cell  104  to the bottom surface of the cavity  105 . As in this example, by covering the upper surface of the region  103  with an optically transparent semipermeable membrane  109  such as cellulose, contamination from outside world such as that by microorganisms can be prevented and at the same time, escape of the cell from the cavity  105  can also be prevented. When the region  103  is made of agarose and the semipermeable membrane  109  is cellulose, a portion of each of their saccharide chains is ring-opened, the —CHO residues are modified with avidin and biotin having an amino terminal, respectively, and the semipermeable membrane  109  is connected with the region  103  via the resulting avidin-biotin linkage. When circulation of a culture solution is necessary upon cultivation of the cell  104 , an optically transparent container  106  large enough to cover the entire region  103  is laid over. The culture solution may be introduced from a tube  107 , while the waste is collected from the tube  108 . 
       FIG. 2  illustrates one example of the arrangement of the cavities  105  formed in the region of a substance, such as agarose, which is optically transparent, has a low melting point and has no toxicity to cells and the like. As is apparent from this diagram, a plurality of cavities  105  are arranged in the region  103  and cells can be cultivated in these cavities after introduced therein. 
       FIG. 3  illustrates one example of the constitution of an apparatus for introducing a focused beam to change the shape of the region  103  of the microchamber  100  for cell culture. This apparatus is equipped with a microscopic observation system for observing a change in the state of a sample such as cell while cultivating it in the microchamber  100  for cell culture, a culture solution circulating system, and a focused beam irradiation system for changing the shape of the microchamber  100  for cell culture during cultivation. As is apparent from  FIG. 3 , the microchamber  100  for cell culture is placed on a light path of the microscopic observation system and culture solution feeding and discharging sections are connected to this microchamber  100  for cell culture. First, the constitution of the microscopic observation system will be described. Light irradiated from a light source  301  is adjusted to a specific wavelength by a filter  302 , collected by a condenser lens  303 , and irradiated to the microchamber  100  for cell culture. The light thus irradiated is used in the observation through an objective lens  305  as a transmitted light. The transmitted optical image inside of the microchamber  100  for cell culture passes through the filter  312  by a mirror  311 , induced by a camera  313 , and then focused onto the acceptance surface of the camera. The chamber  100  for cell culture is desirably made of an optically transparent material to light of a wavelength selected by the filter  302 . Specific examples include glasses such as borosilicate glass and quartz glass, resins or plastics such as polystyrene, solid substrates such as silicon substrate and high molecular substances such as agarose. When a silicon substrate is employed, use of light having a wavelength of 900 nm or greater is taken into consideration. As described above in relation to the light absorption layer  102 , selective use of a film thick enough not to permit 100% light absorption or selective use of a wavelength where no absorption is detected is desired. 
     The light irradiated from a light source  308  is introduced to an objective lens  305  by a dichroic mirror  310  after selection of the wavelength by a filter  309 , and is used as an excitation light for fluorescent observation inside of the microchamber  100  for cell culture. The fluorescence emitted from the microchamber  100  for cell culture is collected by the objective lens  305  again. Only the fluorescence and transmitted light remaining after removal of the excitation light by the filter  312  can be observed by a camera  313 . Only the transmitted light, only the fluorescence or both of the transmitted light image and fluorescence image can be observed by the camera  313  by changing the combination of the filters  302 ,  309  and  312 . 
     The optical path has, inside thereof, a mechanism for introducing a laser light generated by a laser light source  307  into the objective lens  305  by a movable dichroic mirror  306 . This laser light becomes a focused beam by the objective lens  305  and is able to spot-heat the microchamber  100  for cell culture. With regards to the transfer of a beam focus point, the laser beam focusing position within the microchamber  100  for cell culture can be transferred by moving the movable dichroic mirror. The laser preferably has a wavelength which is not absorbed by water and has no photochemical action. For example, at 1064 nm of an Nd:YAG laser, remarkable light absorption by water, glass or agarose does not occur, but laser beam absorption occurs selectively in the thin chromium film layer. Heat is generated only in the vicinity of the beam focus point of the thin chromium film layer in which light absorption has occurred. By this heating, as described later in full detail referring to  FIG. 4 , the shape of the microchamber  100  for cell culture can be changed during the cultivation. 
     The image data in the camera is analyzed by an image analyzer  314 . In order to control, based on the various analysis results, the position of the movable dichroic mirror  306  or a movable XY stage  304  equipped with a temperature controlling function on which the microchamber  100  for cell culture has been placed, a stage moving motor  315  capable of moving the stage freely in the X-Y direction can be driven. This makes it possible to recognize the shape of the cell, keep the cell in the center of the image by pursuing the cell after recognition, or adjust the distance from the objective lens to focus to a specific cell. It is also possible to control, at a constant cycle, the movable dichroic mirror  306  or the stage  304  equipped with a temperature controlling function on which the microchamber  100  for cell culture has been placed, or to drive the stage moving motor  315  at regular intervals. 
     The culture solution feeding or discharging section will next be described. Plural kinds of culture solutions or culture solutions different in concentrations are fed to the microchamber  100  for cell culture from a culture solution tank  316  by using a feeder  317  which has a feeding function. The culture solution is fed to the microchamber  100  for cell culture while its temperature is adjusted by a temperature controlling mechanism in the feeder, its components of dissolved air are adjusted by a dissolved air exchange mechanism, and its flow rate is also adjusted. The culture solution, on the other hand, can be suctioned from the container  100  through a pump  318  and then sent to a waste reservoir  319 . 
     The shape change procedure of the region  103  by a focused laser beam will next be explained based on  FIG. 4 . A focused laser beam  402  irradiated to the microchamber  100  for cell culture by the objective lens  305  is selectively absorbed by the light absorption layer  102  and it locally generates heat in the vicinity of the irradiating position. Heat emission due to direct absorption does not occur in the other regions  101  and  103 , because these regions do not absorb a focused laser beam. Owing to the heat emission of the heat absorption layer  102  at the beam focus point, the region  103  in the vicinity thereof is locally molten and molten components are diffused in an aqueous solution of the culture solution. When the position of the focused beam is transferred in the direction of an arrow  401 , the region  103  in the vicinity of the light absorption layer  102  is selectively molten, leading to the formation of a tunnel  403 . The diameter of the tunnel can be changed, depending on the diameter or strength of a laser to be irradiated, or a transfer rate. 
       FIG. 5  illustrates another example of the fundamental constitution of the microchamber for cell culture. In this example, the number of the region  103  which is one in the example of  FIG. 1  is increased to two, that is, regions  501  and  502  which are different from each other in melting point. When the melting point of the region  501  is lower than that of the region  502 , only the region  501  can be removed selectively by properly adjusting the strength of the focused beam necessary for heating. Both the regions  501  and  502  can be melted by heightening the strength of the focused beam. In this example, two regions different in melting point are stacked one after another to form two layers. Three or more layers may be formed by stacking materials different in melting point one after another. Regions different in melting point may be arranged after properly dividing them three-dimensionally. It is possible to select melting regions stepwise by adjusting the strength of the focused beam for heating. More specifically, such a constitution can be realized by stacking low-melting-point agaroses different in melting point or using materials different from each other such as agarose and plastic. 
       FIG. 6  illustrates a further example of the fundamental constitution of the microchamber for cell culture. In this example, heat absorption layers  601 ,  602  and  603  are placed as a fault having a specific height in the region  103 . This example is characterized in that when the light absorption layers  601 ,  602  and  603  emit heat by heating using a focused beam, the region  103  supporting these layers are molten and the light absorption layers are therefore removed simultaneously with melting. The height of the tunnel formed by light absorption and heat generation varies depending on the position of the light absorption layer in the region  103 . 
     The example shown in  FIG. 7  is similar to that in  FIG. 6  in which the light absorption layers are formed in the region  103 . In this example, however, not a light absorption layer but light-absorptive fine particles  701  are used. This makes it possible to melt the whole of the region  103  exposed to a focused laser beam by arranging the fine particles  701  in the layer form and melt the region  103  in the layer form at a specific height, or dispersing the fine particle  701  uniformly in the whole of the region  103 . 
       FIG. 8  illustrates one example of the actual results of melting of the region  103  using a focused beam. Over a substrate obtained by depositing chromium of 50 nm thick on a slide glass and then applying collagen to the resulting slide glass, agarose of 50 μm thick is laid. A microchamber  81  for cell culture having cavities  801  formed therein by applying a mold of 50 μm×50 μm to agarose prior to its coagulation is exposed to the focused beam of an Nd:YAG laser  802 . By moving the beam while irradiating it to the microchamber, a cavity is formed as shown in the illustration of the substrate  82 . After exposure, a tunnel  803  having a diameter of 5 μm is formed as shown in the illustration of the substrate  83 . By carrying out similar treatments, a tunnel  804 , a tunnel  805  and a tunnel  806  can be made successively as shown in the illustrations of the substrate  84 , the substrate  85 , and the substrate  86  successively. Moreover, a tunnel  807  connecting the tunnels thus formed can be made as shown in the illustration of the substrate  87 . 
       FIGS. 9 and 10  illustrate examples showing the possibility of not only forming a tunnel in the microchamber  100  for cell culture but also changing the shape of the cavities of the microchamber for cell culture.  FIG. 9  shows the possibility of changing a circular cavity  911  into a square cavity  912 .  FIG. 10  shows the possibility of changing a circular cavity  1011  to a star-shaped cavity  1012 . 
       FIG. 11  illustrates an example which permits spot heat generation and melting in a region smaller than the wavelength of light by using the microfabrication technology to form a light absorption region smaller than the wavelength of a focused beam. When the line width of the pattern  1102  of the light absorption layer formed by the microfabrication technology is on the submicrometer scale, exposure to a focused laser beam having a wavelength of 1064 nm selectively causes light absorption only in the pattern of the light absorption region, whereby only a region smaller than the wavelength of light can be heated locally and a tunnel  1104  is formed. This method is effective for the formation of a tunnel on the submicron scale, because a heat source can be focused only to the wavelength of light when heating is conducted using an ordinary focused beam. A similar effect can be attained by the use of light absorption submicron particles. 
     It is needless to say that the invention of the present application is not limited by the above-described illustration and description and a variety of modes can be employed for each of its details. 
     INDUSTRIAL APPLICABILITY 
     As described in full detail, the invention of the present application makes it possible to carry out cultivation of biological cells while changing the shape of the container depending on the cultivation stage, which has hitherto been impossible. In addition, it makes it possible to form the intended structure by melting a substance locally, that is, in a region not greater than the wavelength of light.