WO2013011962A1 - 細胞培養装置、細胞培養長期観察装置、細胞長期培養方法、および細胞培養長期観察方法 - Google Patents
細胞培養装置、細胞培養長期観察装置、細胞長期培養方法、および細胞培養長期観察方法 Download PDFInfo
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
Definitions
- the present invention relates to a cell culture device, a cell culture long-term observation device, a cell long-term culture method, and a cell culture long-term observation method.
- Non-Patent Document 1 Non-Patent Document 1
- this method has a small number of cells that can be moved at a time, and it is necessary for the experimenter to select and remove each individual cell. Culture was the limit.
- Non-patent Document 2 a method for culturing and observing cells for a long period of time.
- a wide groove (100 ⁇ m width) and a narrow groove (1 ⁇ m width, 25 ⁇ m length) are formed in a whisker shape on the substrate. Then, the cells are put into the narrow groove, the culture solution is poured from the wide groove, and the cells pushed out from the narrow groove to the wide groove with cell proliferation are washed away with the culture solution. It is said that unnecessary cells can be removed and the cells in the narrow groove can be cultured for over 200 generations.
- the culture environment around the cells is performed by exchanging the culture medium in the narrow groove caused by diffusion from the wide groove.
- the length of the narrow groove is long, there is a problem that the environmental conditions greatly change in the narrow groove and in the region close to and far from the wide groove. Therefore, for example, when observing the response of a cell to a drug or the like, it is possible that the result of the response of the cultured cell fluctuates due to the difference in environmental conditions, making accurate verification difficult.
- the present invention has been made in view of the circumstances as described above, and continuously cultures and observes cells for a long period of time under uniform environmental conditions without accompanying changes in the physiological state of the cultured cells with aging. It is an object of the present invention to provide a cell culture device, a cell culture long-term observation device, a cell long-term culture method, and a cell culture long-term observation method capable of tracing the history (lineage) of a specific cell.
- a cell culture device of the present invention is a cell culture device having a cell culture substrate, a semipermeable membrane, and a culture solution supply means.
- a thin culture groove for holding and culturing the cells, and a thick discharge groove for discharging the cells cultured and held in the culture groove, and both ends of the culture groove are connected to the discharge grooves.
- the discharge groove is thicker and deeper than the culture groove, and the semipermeable membrane is used to cover the culture groove and the discharge groove of the cell culture substrate.
- the culture medium can be continuously supplied to the cell culture substrate covered with the semipermeable membrane.
- the semipermeable membrane can cover the cell culture substrate via a biotin-avidin bond.
- the culture medium supply means has a liquid feeding pad.
- the cell culture long-term observation apparatus of the present invention is characterized by comprising the above-described cell culture apparatus and a microscopic observation means capable of observing cells on the cell culture substrate.
- the microscopic observation means is preferably an inverted microscope.
- the cell long-term culture method of the present invention is a method for culturing cells for a long time using the cell culture apparatus. Furthermore, a step of holding desired cells in the culture groove of the cell culture substrate, a step of covering the culture groove and the discharge groove of the cell culture substrate with a semipermeable membrane, and continuously supplying the culture solution by the supply means The culture solution is fed to the cell culture substrate and supplied to the cells held in the culture groove of the cell culture substrate through the semipermeable membrane, and the culture solution flows through the discharge groove connected to both ends of the culture groove. And a step of discharging a part of the cells in the culture groove to the discharge groove.
- the cell culture long-term observation method of the present invention is a method for culturing and observing cells for a long period of time using the cell culture long-term observation apparatus. Furthermore, a step of holding desired cells in the culture groove of the cell culture substrate, a step of covering the culture groove and the discharge groove of the cell culture substrate with a semipermeable membrane, and continuously supplying the culture solution by the supply means The culture solution is fed to the cell culture substrate and supplied to the cells held in the culture groove of the cell culture substrate through the semipermeable membrane, and the culture solution flows through the discharge groove connected to both ends of the culture groove. And a step of discharging a part of the cells in the culture groove to the discharge groove, and a step of observing the cells on the cell culture substrate by the microscopic observation means.
- the present invention long-term continuous culture is possible, and cells are continuously cultured under uniform environmental conditions or controlled environmental change conditions without accompanying changes in the physiological state of cultured cells with aging.
- the growth state can be observed for a long time, and the growth of specific cells can be traced and observed over a long period of time, so the cell size frequency distribution, the growth rate frequency distribution, the generation time frequency distribution, the protein expression level
- the autocorrelation function and cell lineage can be measured.
- FIG. 1B is a cross-sectional view taken along line A-A ′ in FIG. 1A
- FIG. 3C is a cross-sectional view taken along line B-B ′ in FIG. It is the elements on larger scale which illustrated another embodiment of the cell culture substrate which comprises the cell culture apparatus of this invention.
- (A) is the whole figure which illustrated one Embodiment of the cell culture apparatus of this invention, and has shown one part in the cross section.
- (B) is the schematic which illustrated the usage method of the semipermeable membrane.
- FIG. 1 It is the schematic diagram which illustrated the state of the cell at the time of supplying a culture solution to the cell culture substrate covered with the semipermeable membrane.
- A) is a plan view and
- B) is a cross-sectional view. It is the whole figure which illustrated another embodiment of the cell culture device of the present invention, and a part is shown in section. BRIEF DESCRIPTION OF THE DRAWINGS It is the whole figure which illustrated one Embodiment of the cell culture long-term observation apparatus of this invention, and has shown one part in the cross section. It is a part of continuous image which recorded the mode of the cell which exists in the groove
- the cell culture apparatus of the present invention has a cell culture substrate, a semipermeable membrane, and a culture solution supply means.
- FIG. 1 (A) is a partially enlarged view illustrating an embodiment of a cell culture substrate constituting the cell culture device of the present invention.
- FIG. 1B is a cross-sectional view taken along the line A-A ′ of FIG. 1A
- FIG. 1C is a cross-sectional view taken along the line B-B ′ of FIG.
- a lattice-like groove pattern in which both ends of a thin culture groove L are connected to a thick discharge groove M is formed.
- the culture groove L and the discharge groove M intersect at substantially right angles.
- the culture groove L functions as a region that allows continuous culture by holding cells in the groove, and the discharge groove M appropriately drains the cells cultured in the culture groove L. By doing so, it functions as a region for adjusting the environment in the culture groove L.
- continuous culture refers to cell culture for a plurality of generations, and in the present invention, long-term cell culture for 200 generations or more can be realized by controlling the environment around the cells.
- the cell to be the subject of the present invention may be a desired cell, and the type of cell is not limited. Specifically, for example, stem cells, skin cells, mucosal cells, hepatocytes, pancreatic islet cells, nerve cells, chondrocytes, endothelial cells, epithelial cells, bone cells, muscle cells, etc. isolated from human or non-human animal tissues.
- stem cells for example, stem cells, skin cells, mucosal cells, hepatocytes, pancreatic islet cells, nerve cells, chondrocytes, endothelial cells, epithelial cells, bone cells, muscle cells, etc. isolated from human or non-human animal tissues
- cells such as plant cells, insect cells, and bacterial cells such as E. coli are included, and one or more of these cells can be cultured.
- the groove depth L1 and the groove width L2 are larger than the size of the cells to be cultured.
- the groove depth L1 of the culture groove L can be designed to be 1 to 2 times, preferably 1 to 1.5 times larger than the cell size.
- the groove width L2 of the culture groove L can be designed to be 1 to 3 times, preferably 1 to 2 times larger than the cell size.
- cell size is based on the diameter (maximum length) of a cell if it is a substantially spherical cell.
- it can be determined based on the thickness (width) of the cells in a state where the cells are placed on the substrate.
- the culture groove L The groove depth L1 can be designed to be 1 to 2 times larger than the cell thickness (width), and the groove width L2 can be designed to be 1 to 3 times larger than the cell thickness (width).
- the cell size can be determined by referring to a known size described in literature or the like, or based on the result of actually measuring the cell size with a microscope or the like.
- the cells can be stably held in the culture groove L, and the cells can be continuously cultured and observed.
- the groove depth L1 of the culture groove L is larger than twice the cell size, the cells may flow out to the discharge groove M without staying in the culture groove L, and the culture groove It is difficult to keep cells in L and continuously culture them.
- the groove width L2 of the culture groove L is more than three times larger than the cell size, the semipermeable membrane adheres to the culture groove and crushes the cells, or the cells flow out into the discharge grooves. It is difficult to continue culturing.
- the length of the culture groove L can be appropriately designed according to the size of the cells, the number of cells to be retained and cultured in the culture groove L, and the like. Specifically, for example, a range of about 10 ⁇ m to 100 ⁇ m, preferably a range of about 30 ⁇ m to 100 ⁇ m can be exemplified.
- the discharge groove M is thicker and deeper than the culture groove L. That is, the groove depth M1 and the groove width M2 of the discharge groove M are formed larger than the groove depth L1 and the groove width L2 of the culture groove L.
- the groove depth M1 of the discharge groove M refers to the distance from the height position of the groove bottom of the culture groove L to the groove bottom of the discharge groove M as shown in FIG.
- the groove depth M1 and the groove width M2 of the discharge groove M depend on the size and number of cells to be retained and cultured in the culture groove L and the speed of the cell culture solution flowing on the cell culture substrate. It can design suitably in the range which can wash out a cell.
- the groove depth M1 of the discharge groove M is preferably 3 to 50 times the cell size.
- a depth that is 1.5 to 50 times the groove depth L1 of the culture groove L can be exemplified.
- the groove depth M1 of the discharge groove M is preferably exemplified by a range of about 5 ⁇ m to 50 ⁇ m.
- the groove width M2 of the discharge groove M has the same size as or larger than the groove depth M1.
- a length 10 to 100 times the groove width L2 of the culture groove L can be exemplified, and specifically, a range of 10 ⁇ m or more, preferably 30 ⁇ m or more is exemplified. be able to.
- the groove depth (L1, M1) and groove width (L2, M2) of the culture groove L and the discharge groove M can be appropriately designed according to the size of a desired cell to be cultured.
- E. coli is known to have a size of about 0.5 ⁇ m to 1.0 ⁇ m (width) ⁇ 1.5 ⁇ m to 7.0 ⁇ m (length).
- the culture groove L of the cell culture substrate has a groove depth L1: 1.0 ⁇ m to 1.5 ⁇ m and a groove width L2: 1.0 ⁇ m to 3
- the discharge groove M has a groove depth M1: from 5 ⁇ m to 20 ⁇ m and a groove width M2: 20 ⁇ m to 100 ⁇ m.
- the material for the cell culture substrate examples include glass such as borosilicate glass and quartz glass, resin and plastic such as polystyrene, and silicon substrate.
- a glass substrate is preferable because it is excellent in workability and handleability.
- the shape of the culture groove L and the discharge groove M is patterned into a photoresist on one surface of the glass plate by, for example, a photolithography method.
- the cell culture substrate can be formed by forming the culture groove L and the discharge groove M by etching in step (1). Moreover, laser processing etc. can also be given to a glass plate suitably.
- As patterning in addition to the photolithography method, an electron beam direct writing method or the like can also be used.
- the surface of the cell culture substrate may be treated with a surface treatment agent or may be subjected to physical surface processing.
- the cell culture substrate surface or a part of the surface can be treated to impart functions such as hydrophilicity, lipophilicity and water repellency.
- a silicon coat, a functional group coat such as an amino group, an isocyanate group, an epoxy group, a carboxyl group, a hydroxyl group, an SH group, or a silanol group can be provided on the surface.
- a functional group coat such as an amino group, an isocyanate group, an epoxy group, a carboxyl group, a hydroxyl group, an SH group, or a silanol group
- those whose surface is modified with any of biotin, avidin, streptavidin and the like are preferable.
- the surface of the cell culture substrate can be coated with a cell adhesion substrate such as collagen, fibronectin or gelatin.
- the groove pattern of the culture groove L and the discharge groove M is not limited to the shape illustrated in FIG. In the cell culture substrate, the culture groove L and the discharge groove M only have to be connected at a substantially right angle. For example, 1 between two discharge grooves M as illustrated in FIG. The form which has the groove
- emission is formed in the square shape, it is not limited to this shape, For example, even if the bottom part of a groove
- the semipermeable membrane of the cell culture device of the present invention is used so as to cover the culture groove and the discharge groove of the cell culture substrate.
- the semipermeable membrane for example, a known membrane such as a cellulose membrane can be employed.
- the semipermeable membrane is preferably modified with any one of biotin, avidin, and streptavidin.
- the surface of the cell culture substrate is either biotin, avidin, or streptavidin. It is preferably modified.
- a semipermeable membrane modified with avidin or streptavidin is used.
- the cell culture substrate is modified with avidin or streptavidin, it is modified with biotin.
- a thin film of a material that is silane-coupled with a silanol group such as silicon, silicon oxide, chromium, aluminum, iron, or titanium, is deposited or sputtered on the upper surface of the cell culture substrate.
- a silanol group such as silicon, silicon oxide, chromium, aluminum, iron, or titanium
- an amino group, a carboxyl group, or an SH group is introduced into the surface of the thin film by a bivalent reagent having a silanol group at one end and an amino group, a carboxyl group, or an SH group at the other end, Make a covalent bond.
- the upper surface of the cell culture substrate can be sealed by modifying a semipermeable membrane such as a cellulose membrane with avidin or streptavidin, bringing the semipermeable membrane into contact with the thin film and bonding with biotin-avidin.
- a semipermeable membrane such as a cellulose membrane with avidin or streptavidin
- Avidin or streptavidin can be bound to the surface of the thin film of the cell culture substrate, and biotin can be bound to the semipermeable membrane.
- the semipermeable membrane is not limited to a form using a biotin-avidin bond, as long as it can cover the discharge groove and the culture groove of the cell culture substrate from above with good adhesion.
- FIG. 3A is an overall view illustrating an embodiment of the cell culture device of the present invention, and a part thereof is shown in cross section.
- FIG. 3B is a schematic view illustrating the method of using the semipermeable membrane.
- 4A and 4B are schematic views illustrating the state of cells when a culture solution is supplied to a cell culture substrate covered with a semipermeable membrane.
- FIG. 4A is a plan view and FIG. 4B is a cross-sectional view.
- the cell culture apparatus 1 includes a cell culture substrate 2, a semipermeable membrane 3, and a culture solution supply means 4.
- the cell culture substrate 2 is formed with the culture groove L and the discharge groove M, and the upper surface thereof is covered with the semipermeable membrane 3.
- the semipermeable membrane 3 only needs to be covered at least in the upper region of the culture groove L.
- the both ends of the discharge groove M are opened without being covered with the semipermeable membrane 3, so that the culture solution can be supplied from one end of the discharge groove M and the culture solution can be discharged from the other end. it can. Cells are held in the culture groove L covered with the semipermeable membrane 3.
- a frame seal S is disposed on the surface of the cell culture substrate 2 around the culture groove L and the discharge groove M.
- the frame seal can be preferably exemplified by those having an appropriate thickness and having adhesiveness on the front and back surfaces, but are not particularly limited.
- the culture medium supply means 4 includes a liquid feed pump 41 (syringe), a liquid feed pad 42, and a waste liquid tank 43.
- the liquid feed pump 41 is connected to a through hole at one end of the liquid feed pad 42 by a liquid feed pipe, and can send out a culture solution for cells and supply the culture solution to the cell culture substrate 2.
- the liquid feeding pad 42 is disposed on the cell culture substrate 2 and covers the culture groove L, the discharge groove M, and the semipermeable membrane 3, and the culture groove L, the discharge groove M, and the semipermeable membrane. 3 is arranged so as to be in close contact with and sealed through a frame seal S around a range including 3.
- the liquid feeding pad 42 penetrates the pad surface at least one through hole through which the culture solution flows into one end and a through hole through which the waste culture solution is discharged to the other end. Is provided.
- Each of these through holes is arranged so that the culture solution flows from the culture solution inflow through hole to the discharge through hole in a space formed between the liquid feeding pad 42 and the cell culture substrate 2 and filled with the culture solution. It should be done.
- the liquid feeding pad 42 has a function of always holding a fresh culture solution on the cell culture substrate 2.
- the liquid feeding pad 42 can be obtained by using a hard material such as glass or acrylic or a flexible material such as rubber or elastomer without any particular limitation.
- a transparent material is suitable when transmitted light from the upper surface of the cell culture device 1 is required for microscopic observation, but when transmitted light is not required for fluorescence observation or the like. Need not be transparent.
- the liquid feeding pad 42 is bonded to the upper side of the cell culture substrate 2 via the frame seal S.
- a space is formed between the cell culture substrate 2 and the liquid feeding pad 42, and this space is filled with the culture solution supplied from the liquid feeding pump during the culture.
- the culture solution that has passed over the cell culture substrate 2 is sent out to the waste solution tank 43 connected to the solution supply pad 42 through the through hole of the solution supply pad 42.
- a transparent one formed of PDMS polydimethylsiloxane
- the liquid delivery pad 42 by PDMS is formed by, for example, applying a photoresist on a silicon wafer, producing a convex mold in which the groove is inverted by a lithography method, pouring PDMS resin into the mold and heating it. Can be made into a box-lid shape.
- the liquid feeding pad 42 may be provided with a bubble trap groove or the like for removing bubbles in the culture solution on the bottom surface of the inner surface of the box lid.
- the culture solution supply means 4 is such that the supply direction of the culture solution supplied from the liquid feed pump 41 to the cell culture substrate 2 is the length of the discharge groove M of the cell culture substrate 2. It is arranged so as to coincide with the vertical direction.
- the cell culture device 1 of the present invention can constitute a cell culture long-term observation device together with the microscopic observation means 5 capable of observing cells on the cell culture substrate 2.
- the microscopic observation means 5 include an apparatus including an optical system such as a lens for enlarging an image of a cell to be observed. Specifically, an inverted microscope, an optical microscope, a fluorescence microscope, a video recording, and the like. A device, a camera, etc. can be illustrated. Further, these microscopic observation means can be connected to a personal computer or the like to perform image processing. Moreover, you may use with the light irradiation apparatus for making observation of a cell easy.
- the liquid feeding pump 41 of the supply means 4 is driven to send the culture liquid onto the cell culture substrate 2. Since the liquid feeding direction from the liquid feeding pump 41 to the cell culture substrate 2 matches the direction of the discharge groove M of the cell culture substrate 2, the culture liquid supplied from the liquid feeding pump 41 is discharged. It flows smoothly from one end of the groove M and passes over the cell culture substrate 2 while filling the internal space between the liquid feeding pad 42 and the cell culture substrate 2. For example, the culture solution can be fed at a rate of about 0.5 ml / hr to 200 ml / hr.
- the cell culture substrate 2 has thick discharge grooves M connected to both sides of the thin culture groove L.
- the flow rate of the culture solution flowing through the two discharge grooves M facing each other is slightly different. is there.
- the semipermeable membrane 3 is not used, the culture solution may flow into the culture groove L at a high flow rate, and the cells may flow out from the culture groove L to the discharge groove M.
- the culture solution flowing above the semipermeable membrane 3 is supplied by permeating and diffusing through the semipermeable membrane 3. For this reason, for example, even when the culture groove L is formed longer than 30 ⁇ m, stable and uniform environmental conditions are maintained in the culture groove L. In addition, since the culture environment is uniform, the response of cells to drugs and the like can be verified more accurately.
- FIG. 4 is a schematic view illustrating the state of cells when a culture solution is supplied to a cell culture substrate covered with a semipermeable membrane.
- the boundary line of the connecting portion between the culture groove L and the discharge groove M is not shown.
- 4A is a plan view and FIG. 4B is a cross-sectional view.
- the cells in the culture groove L are stably cultured and proliferate geometrically.
- both ends of the culture groove L discharge grooves M
- a part of the cells located near the connection portion of the culture groove L) is washed out into the discharge groove M, discharged together with the culture medium, and collected in the waste liquid tank 43.
- the number of cells in the culturing groove L can be maintained at a predetermined number, and the conventional problems such as cell nutrient consumption, waste accumulation, and changes in the environment surrounding the cells have occurred over time.
- the cell culture long-term observation apparatus using the microscopic observation means 5 can continuously cultivate and observe cells for 200 generations or more. This makes it possible to measure information such as the growth rate of one cell line, the fluctuation level of gene expression level, and autocorrelation functions based on unprecedented long-term time series data. Since the time over 100 generations corresponds to a time scale in which an evolutionary state accompanied by a change in genotype occurs, the cell culture device and the cell culture long-term observation device of the present invention have been experimentally verified so far. It can be applied to evolutionary research that was difficult to verify.
- channel M is connecting the both ends of the culture
- channel L has fluidity
- the cell culture device and the cell culture long-term observation device of the present invention can also be used for detecting gene mutations.
- a gene mutation such as a frameshift mutation or point mutation occurs at a specific site
- a cell line designed so that the fluorescent protein placed at that site is expressed in the correct sequence.
- the mutation occurs in the cells present in the culture groove, it can be detected that the mutation has occurred without killing the cells in the form that the cells start to emit fluorescence due to the expression of the fluorescent protein.
- By observing these cells continuously for a long time in a cell culture device it is possible to acquire information such as the occurrence frequency of the gene mutation, the generation timing in the division cycle, and the like.
- FIG. 5 is an overall view illustrating another embodiment of the cell culture device of the present invention, and a part thereof is shown in cross section. The description of the parts common to those shown in FIGS. 3 and 4 is omitted.
- a plurality of liquid feeding pumps 41 are connected to a liquid feeding pad 42, and the culture solution is supplied from each liquid feeding pump to the cell culture substrate 2 covered with the semipermeable membrane 3.
- the culture grooves L of the cell culture substrate 2 are switched to the culture grooves L.
- Various changes in environmental conditions can be imparted to the retained cells, and the influence of the environmental conditions on the cells can be verified over a long period of time.
- FIG. 6 is an overall view illustrating an embodiment of the cell culture long-term observation apparatus of the present invention, and a part thereof is shown in cross section. A description of portions common to the embodiment shown in FIGS. 3 and 5 is omitted.
- the cell culture long-term observation apparatus 1 a illustrated in FIG. 6 includes the cell culture apparatus 1 and the microscopic observation means 5.
- an inverted electric microscope having the function of a fluorescence microscope is used as the microscopic observation means 5 suitable for the time-lapse observation of the cell growth rate and the expression of the fluorescent protein as described above.
- the inverted electric microscope includes a bright field observation light source 51a, a fluorescence observation light source 51b, an automatic shutter 52a, an automatic shutter 52b, a condenser lens 53, a dichroic mirror 54, an objective lens 55, and an XY stage 56. ing.
- the XY stage 56 has an opening A, and the cell culture substrate 2 of the cell culture device 1 is placed on the XY stage 56 so that the culture groove L in which the cells are held is located in the opening A.
- the cells held in the culture groove L can be automatically moved to a position to be observed by an XY stage 56 driven by an electric motor.
- An objective lens 55 is disposed adjacently below the opening A of the XY stage 56.
- the relative positions of the XY stage 56 and the objective lens 55 are moved by individually moving in the X and Y axis directions crossing the optical axis of the XY stage 56 and moving the objective lens 55 in the vertical direction in the Z axis direction. Adjustment is possible. Further, the XY stage 56 can be moved independently in the Z-axis direction in addition to the X- and Y-axis directions, and thereby the relative distance from the objective lens 55 can be adjusted.
- the inverted electric microscope includes a bright-field transmission illumination system, an epi-illumination system, and an imaging system inside a main body case (not shown).
- the bright field transmission illumination system is disposed above the height position of the XY stage 56, and the epi-illumination system and the imaging system are disposed below the height position of the XY stage 56.
- the bright-field transmission illumination system is used for observation with transmitted light.
- the bright field transmission illumination system includes a bright field observation light source 51a, an automatic shutter 52a, and a condenser lens 53.
- a halogen lamp or the like can be used as the bright field observation light source 51a.
- the light emitted from the bright field observation light source 51a passes through the condenser lens 53 with the automatic shutter 52a opened, and is cultured on the cell culture substrate 2 placed on the XY stage 56 downward in the vertical direction.
- the cells held in the groove L are irradiated.
- the epi-illumination system is used for observation by fluorescence.
- the epi-illumination system includes a fluorescence observation light source 51b, an automatic shutter 52b, and a dichroic mirror 54.
- a mercury lamp or the like can be used as the fluorescence observation light source 51b.
- the epi-illumination system can further include an optical system such as a heat absorption filter, a collector lens, and an excitation filter for converting light from the fluorescence observation light source 51b into excitation light of a specific short wavelength band.
- the light emitted from the fluorescence observation light source 51b passes through the dichroic mirror 54 and the objective lens 55 in a state in which the automatic shutter 52b is opened, and the cells placed on the XY stage 56 are directed upward in the vertical direction.
- the cells held in the culture groove L of the culture substrate 2 are irradiated from the opening A.
- the imaging system includes a camera 57 mounted facing the dichroic mirror 54.
- a camera 57 mounted facing the dichroic mirror 54.
- the camera 57 for example, a CCD camera can be used.
- the inverted electric microscope is equipped with a power supply unit, a motor drive circuit board, and the like.
- the power supply unit includes a power source for the bright field observation light source 51a, a power source for controlling a system such as a motor incorporated in the inverted electric microscope, and a power source for the fluorescence observation light source 51b.
- the motor drive circuit board drives, for example, a motor for driving the XY stage 56 in the X and Y axis directions, a motor for driving the electric zoom mechanism of the imaging system, and a diaphragm of a fluorescence observation light source 51b such as a mercury lamp. To control the motor and so on.
- the inverted electric microscope allows a user to perform various operations and settings using a keyboard and a mouse by a computer 58 such as a personal computer.
- the transmitted illumination system and the epi-illumination system are selectively used.
- the transmission illumination system is selected, the image of the cell held in the culture groove L obtained by the transmission illumination light from the bright field observation light source 51 a passes through the objective lens 55 from the opening A, and passes through the dichroic mirror 54. It is reflected and captured by the camera 57 arranged in the horizontal direction.
- the image of the fluorescence of the cells held in the culture groove L obtained by the irradiation of the excitation light from the fluorescence observation light source 51b passes through the objective lens 55 from the opening A. Then, the light is reflected by the dichroic mirror 54 and taken into the camera 57 arranged in the horizontal direction.
- the inverted electric microscope includes an eyepiece for observing cells with eyes, and an optical system that optically couples the eyepiece and the objective lens 55.
- This optical system includes a mirror, a relay lens, and an optical path switching prism.
- an optical path switching prism is inserted on the observation optical axis, and the primary image of the objective lens is reflected by the optical path switching prism so that it can be observed with the camera 57.
- the optical path switching prism is removed from the observation optical axis, and the primary image of the objective lens is reflected toward the eyepiece by the mirror. Further, the primary image of the objective lens is relayed by a relay lens and can be observed with the eye by an eyepiece.
- the following cell observation can be performed using the cell culture apparatus 1 having the above configuration.
- the liquid feeding pad 42 is provided with the above-described foam trap groove 44 that is a groove for removing the foam in the culture solution.
- the observation position of the microscopic observation means 5 is located below the cell culture device 1 by using an inverted electric microscope as the microscopic observation means 5.
- bubbles generated in the cell culture apparatus 1 are prevented from interfering with observation by the microscopic observation means 5, and long-term continuous cell observation, for example, 200-generation long-term continuous cell observation is also possible.
- the cells in the culture groove L are stably cultured and proliferate geometrically. Then, when the cells fill the culture groove L, the cells located near both ends of the culture groove L by the culture solution flowing through the discharge grooves M connected at substantially right angles on both sides of the culture groove L. Is washed out into the discharge groove M and discharged together with the culture solution.
- the cells cultured in the culture groove L in this way are irradiated with either the transmission illumination system or the epi-illumination system, and are observed with the camera 57 or the eyepiece as an enlarged image using the objective lens 55. Can do.
- the cell size as described above in particular, the cell size change or growth rate in one cell line can be observed.
- the fluorescence image of the stained cell and the change thereof can be observed.
- a time-lapse measurement can be performed by acquiring a fluorescent image of a fluorescent protein with the camera 57 over time and recording it in the computer 58.
- a time-lapse image By analyzing this time-lapse image with the computer 58 using dedicated image analysis software, it is possible to acquire time-series information about the size of cells included in the acquired image, the average of the internal fluorescence luminance, and the like. This makes it possible to measure information such as the growth rate of one cell line, the fluctuation level of gene expression level, and autocorrelation functions based on unprecedented long-term time series data.
- Example 1 Preparation of Cell Culture Substrate
- a glass substrate 60 mm (length) ⁇ 24 mm (width) ⁇ 0.17 mm (thickness)
- Groove L Groove depth L1: 1.0 ⁇ m
- groove width L2 3.0 ⁇ m
- discharge groove M (M1: groove depth 17 ⁇ m
- M2 groove width 60 ⁇ m, length 5000 ⁇ m, 20) were provided in a grid pattern (see FIG. 1).
- Example 2 Coating with semipermeable membrane
- the surface of the cell culture substrate prepared in Example 1 was modified with biotin.
- a cellulose semipermeable membrane having a width of 1 mm and a length of 1 mm was used as the semipermeable membrane, and the surface was modified with streptavidin.
- 1 ⁇ l of a culture solution containing Escherichia coli is dropped from above the culture groove L and the discharge groove M on the surface of the cell culture substrate, and then the central portion of the cell culture substrate above the culture groove L and the discharge groove M is removed. Some areas were sealed with a semipermeable membrane.
- Example 3 Cell Culture / Observation Cells were continuously cultured and observed using the cell culture apparatus of the present invention.
- a square frame-shaped frame seal (Bio-Rad: SLF-0201), which is a double-sided tape, is sealed on the cell culture substrate so as to surround the culture groove and the discharge groove, and further the upper surface of the frame seal
- a PDMS liquid-feeding pad was affixed to.
- One of the two silicon tubes attached to the PDMS-made liquid feeding pad was connected to a syringe (liquid feeding pump), and the other was introduced into the waste liquid tank (see FIG. 3).
- the M9 minimal medium for culturing Escherichia coli continuously containing 0.2% by weight glucose as a nutrient source was continuously flowed by a syringe at a flow rate of 2 ml / h.
- a cell culture long-term observation device as shown in FIG. 6 is constructed, and the cells in the culture groove are used with a 100 ⁇ magnification phase difference objective lens. Time-lapse observation was performed at intervals of 2 minutes.
- the Escherichia coli used for observation is specifically the W3110 strain, which has a plasmid that expresses a fluorescent protein (GFP) in a form controlled by the promoter of the rpsL gene.
- GFP fluorescent protein
- FIG. 7 is a part of a continuous image in which the state of cells existing in the culture groove on the cell culture substrate is recorded.
- the culture solution is supplied to the cell culture substrate and continuously cultured, among the cells present in the culture groove, some of the cells present at both ends of the culture groove are appropriately discharged. It is washed away into the groove and disappears from the screen. It is also confirmed that the cells are stably held and cultured in the culture groove L by the groove shape and the semipermeable membrane of the cell culture substrate. Further, the culture groove L is maintained in a stable and uniform environment by supplying the culture solution to the cells via the semipermeable membrane.
- the number of cells in the culture groove L can be maintained at a predetermined number. It was confirmed that problems such as nutrient consumption, accumulation of waste products, and changes in the environment surrounding the cells can be solved, and cells can be continuously cultured and observed. It was also confirmed that the mother cells were prevented from remaining in the culturing groove L, and the cells in the culturing groove L could be cultured for a long time without a change in physiological state accompanying aging.
- FIG. 8 is a graph plotting changes in cell size for 55 generations in one cell line and changes in GFP expression level (intracellular concentration) inside the cells, obtained by image analysis.
- the cell culture apparatus of the present invention enables continuous culture of cells over a long period of time. This makes it possible to measure information such as the growth rate (change in cell size) of one cell line and the fluctuation level of gene expression based on unprecedented long-term time-series data. It was confirmed.
- Example 4 Frequency distribution of protein expression level A cell culture apparatus having a cell culture substrate, a PDMS liquid supply pad, a syringe (liquid supply pump), and a waste liquid tank similar to those in Example 3 is placed on an inverted electric microscope stage. Escherichia coli expressing the fluorescent protein GFP in the culture groove was time-lapse observed.
- the M9 minimal medium for culturing Escherichia coli continuously containing 0.2% by weight glucose as a nutrient source was continuously flowed by a syringe at a flow rate of 2 ml / h.
- the culture temperature at the time of observation was maintained at 37 ° C., and the cells in the culture groove were time-lapse observed over 5000 minutes at 1 minute intervals.
- Intracellular GFP average fluorescence brightness of all cells at all observed time points was measured, and the frequency distribution of the expression level of GFP estimated based on it was measured. The result is shown in FIG. Example 5 Cell Size Frequency Distribution In the time lapse observation of Example 4, the cell size frequency distribution was measured. The result is shown in FIG. Example 6 Growth Rate Frequency Distribution In the time lapse observation of Example 4, the growth rate frequency distribution was measured. The growth rate of one cell was obtained by fitting the change in cell size from division to division with an exponential function C ⁇ 2 kt , and the exponent k was defined as the growth rate of each cell. Where C is a constant and t is time. The result is shown in FIG.
- Example 7 Generation time distribution
- Generation time is the time required for each cell to divide from cell to cell.
- FIG. ⁇ Example 8> Autocorrelation function of protein expression level
- the autocorrelation function of protein expression level was measured. Assuming that the protein expression level at a certain time t is x (t), the autocorrelation A (t) with the time t is determined by calculating the following equation.
- E [] and V [] represent the expected value and variance, respectively.
- the result is shown in FIG. This figure plots A ( ⁇ ) against ⁇ .
- Example 9 Distribution of cell division age
- the cell division age represents the time from the last division to the present.
- FIG. Lineage branching represents cell division, and breaks in the lineage indicate that the cells have been washed away.
- Example 11 A cell culture device having the same cell culture substrate, PDMS-made liquid feeding pad, syringe (liquid feeding pump), and waste liquid tank as in Example 3 was placed on the stage of an inverted electric microscope, and cells in the culture groove The time lapse was observed.
- the Escherichia coli of Example 3 was replaced with mouse ES cells, and an undifferentiated maintenance-free serum-free medium ESF7 was continuously flowed by a syringe pump at a flow rate of 2 ml / h instead of the M9 minimal medium for Escherichia coli culture.
- the surface of the glass substrate was coated with collagen or E-cadherin-Fc in order to promote adhesion to mouse ES cells.
- the mouse ES cells in the culture groove were subjected to time-lapse observation at 15-minute intervals using an inverted electric microscope with a 20 ⁇ magnification objective lens.
- mouse ES cells were stably held and cultured in the culture groove L by the groove shape and semipermeable membrane of the cell culture substrate.
- the culture groove L By supplying the culture solution to the mouse ES cells through the semipermeable membrane, the culture groove L is maintained in a stable and uniform environment, and the mouse ES cells present at both ends of the culture groove are appropriately discharged. It was confirmed that the number of mouse ES cells in the culture groove L can be maintained at a predetermined number by being washed away into the groove, and the cells can be continuously cultured and observed. It was also confirmed that the mother cells were prevented from remaining in the culture groove L, and the mouse ES cells in the culture groove L could be cultured for a long time without a change in physiological state accompanying aging.
- a culture groove L having a groove depth L1 smaller than the cell size was formed on the surface of the glass substrate to examine whether E.
- the culture groove L (L1: groove depth 0.5 ⁇ m, L2: groove width 2.0 ⁇ m, length 30 ⁇ m, 50), discharge groove M (M1: groove depth 17 ⁇ m, M2: groove) A width of 60 ⁇ m, a length of 5000 ⁇ m, and 20 pieces) were provided in a lattice shape.
- the cell culture substrate was covered with a semipermeable membrane in the same manner as in Example 2, the culture solution was supplied to the cell culture substrate with the same apparatus configuration as in Example 3, and the cells were observed.
- the cell culture substrate was covered with a semipermeable membrane in the same manner as in Example 2, the culture solution was supplied to the cell culture substrate with the same apparatus configuration as in Example 3, and the cells were observed.
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Abstract
Description
<実施例1>細胞培養基板の作成
大腸菌を連続的に培養するための細胞培養基板として、ガラス基板(60mm(長さ)×24mm(幅)×0.17mm(厚さ))に、培養用溝L(溝深さL1:1.0μm、溝幅L2:3.0μm、長さ30μm、50本)、排出用溝M(M1:溝深さ17μm、M2:溝幅60μm、長さ5000μm、20本)を格子状に設けた(図1参照)。
<実施例2>半透膜による被覆
実施例1で作成した細胞培養基板の表面をビオチンで修飾した。一方、半透膜として幅1mm×長さ1mmのセルロース製半透膜を使用し、表面をストレプトアビジンで修飾した。そして、細胞培養基板の表面の培養用溝L、排出用溝Mの上方から大腸菌を含む培養液を1μl滴下した後、細胞培養基板の培養用溝Lおよび排出用溝Mの上方の中央部分の一部の領域を半透膜でシールした。
<実施例3>細胞の培養・観察
本発明の細胞培養装置によって、細胞を連続的に培養・観察した。具体的には、両面テープである四角枠形状をしたフレームシール(バイオラッド社製:SLF-0201)を培養用溝および排出用溝を取り囲む形で細胞培養基板上にシールし、さらにフレームシール上面にPDMS製の送液パッドを貼付けた。PDMS製の送液パッドに取付けてある2本のシリコン製チューブのうち1本をシリンジ(送液ポンプ)に連結し、もう1本を廃液タンク内に導入した(図3参照)。シリンジにより、2ml/hの流速で、0.2%重量比のグルコースを栄養源として含む、大腸菌培養用M9最小培地を連続的に流し続けた。この細胞培養装置を、倒立型電動顕微鏡のステージ上に配置することによって図6に示すような細胞培養長期観察装置を構成し、培養用溝内の細胞を倍率100倍位相差対物レンズを用いて、2分間隔でタイムラプス観察した。
<実施例4>タンパク質発現量の頻度分布
実施例3と同様の細胞培養基板、PDMS製の送液パッド、シリンジ(送液ポンプ)、および廃液タンクを有する細胞培養装置を倒立型電動顕微鏡のステージ上に配置し、培養用溝内の蛍光タンパク質GFPを発現する大腸菌をタイムラプス観察した。シリンジにより、2ml/hの流速で、0.2%重量比のグルコースを栄養源として含む、大腸菌培養用M9最小培地を連続的に流し続けた。観察時の培養温度は37℃に維持し、培養用溝内の細胞を1分間隔で5000分にわたってタイムラプス観察した。
<実施例5>細胞サイズの頻度分布
実施例4のタイムラプス観察において、細胞サイズの頻度分布を測定した。その結果を図10に示す。
<実施例6>成長率の頻度分布
実施例4のタイムラプス観察において、成長率の頻度分布を測定した。1細胞の成長率は分裂から分裂までの細胞サイズの変化を指数関数C×2ktでフィッティングし、その指数kを各細胞の成長率とした。ただし、Cは定数、tは時間を表す。その結果を図11に示す。
<実施例7>世代時間分布
実施例4のタイムラプス観察において、世代時間の頻度分布を測定した。世代時間とは、各細胞が分裂から次の分裂にまで要する時間である。その結果を図12に示す。
<実施例8>タンパク質発現量の自己相関関数
実施例4のタイムラプス観察において、タンパク質発現量の自己相関関数を測定した。ある時点tでのタンパク質発現量をx(t)とすると、時間t離れた時点との自己相関A(t)は次式を計算することで求めた。
<実施例9>細胞分裂齢の分布
実施例4のタイムラプス観察において、細胞分裂齢の頻度分布を測定した。細胞分裂齢とは、直前の分裂から現在までの時間を表す。その結果を図14に示す。
<実施例10>ひとつの観察用溝内で観察される細胞系譜
実施例4のタイムラプス観察において、ひとつの観察用溝内で観察された細胞系譜を測定した。その結果を図15に示す。系列の枝分かれは細胞分裂を表し、系列の途切れているところは細胞が洗い流されたことを示している。
<実施例11>
実施例3と同様の細胞培養基板、PDMS製の送液パッド、シリンジ(送液ポンプ)、および廃液タンクを有する細胞培養装置を倒立型電動顕微鏡のステージ上に配置し、培養用溝内の細胞をタイムラプス観察した。実施例3の大腸菌をマウスES細胞に代えて、シリンジポンプにより2ml/hの流速で、大腸菌培養用M9最小培地に代えて未分化維持用無血清培地ESF7を連続的に流し続けた。前記ガラス基板表面は、マウスES細胞のへの接着を促進するため、コラーゲンもしくは、Eカドヘリン-Fcをコートした。培養用溝内のマウスES細胞は、倒立型電動顕微鏡で倍率20倍位相差対物レンズを用い、タイムラプス観察を15分間隔で行った。
<比較例1>
ガラス基板の表面に、溝深さL1が細胞のサイズよりも小さい培養用溝Lを形成し、大腸菌の培養が可能であるか検討した。具体的には、培養用溝L(L1:溝深さ0.5μm、L2:溝幅2.0μm、長さ30μm、50本)、排出用溝M(M1:溝深さ17μm、M2:溝幅60μm、長さ5000μm、20本)を格子状に設けた。
<比較例2>
ガラス基板の表面に、溝深さL1が細胞サイズ(厚さ)の2倍を超えて大きい培養用溝Lを形成し、大腸菌の培養が可能であるか検討した。具体的には、培養用溝L(L1:溝深さ2.0μm、L2:溝幅2.0μm、長さ30μm、50本)、排出用溝M(M1:溝深さ17μm、M2:溝幅60μm、長さ5000μm、20本)を格子状に設けた。
1a 細胞培養長期観察装置
2 細胞培養基板
3 半透膜
4 供給手段
5 顕微観察手段
41 送液ポンプ
42 送液パッド
43 廃液タンク
44 泡トラップ用溝
51a 明視野観察光源
51b 蛍光観察用光源
52a 自動シャッター
52b 自動シャッター
53 コンデンサーレンズ
54 ダイクロイックミラー
55 対物レンズ
56 XYステージ
57 カメラ
58 コンピュータ
L 培養用溝
M 排出用溝
A 開口部
S フレームシール
Claims (9)
- 細胞培養基板と、半透膜と、培養液の供給手段とを有する細胞培養装置であって、
細胞培養基板は、表面に、細胞を保持培養するための細い培養用溝と、この培養用溝内に保持培養された細胞を排出するための太い排出用溝を有するとともに、前記培養用溝の両端は、排出用溝に接続し、排出用溝は、培養用溝より太く、深いものであり、
半透膜は、細胞培養基板の培養用溝および排出用溝を覆うように使用されるものであり、
培養液の供給手段は、半透膜によって覆われた細胞培養基板に、培養液を連続的に供給できるものであることを特徴とする細胞培養装置。 - 前記半透膜は、ビオチン-アビジン結合を介して細胞培養基板を被覆可能とされていることを特徴とする請求項1に記載の細胞培養装置。
- 前記培養液の供給手段は、送液パッドを有することを特徴とする請求項1に記載の細胞培養装置。
- 請求項1から3のいずれか一項に記載の細胞培養装置と、細胞培養基板上の細胞を観察可能な顕微観察手段とを備えることを特徴とする細胞培養長期観察装置。
- 顕微観察手段は、倒立型顕微鏡であることを特徴とする請求項4に記載の細胞培養長期観察装置。
- 請求項1に記載の細胞培養装置によることを特徴とする細胞長期培養方法。
- 所望の細胞を細胞培養基板の培養用溝内に保持する工程と、
半透膜で、細胞培養基板の培養用溝および排出用溝を覆う工程と、
供給手段によって培養液を連続的に細胞培養基板へ送り、細胞培養基板の培養用溝内に保持された細胞へ半透膜を介して培養液の供給を行うとともに、培養用溝の両端と接続する排出用溝を流れる培養液によって、培養用溝内の細胞の一部を排出用溝へ排出する工程と、
を含む請求項6に記載の細胞長期培養方法。 - 請求項4に記載の細胞培養長期観察装置によることを特徴とする細胞培養長期観察方法。
- 所望の細胞を細胞培養基板の培養用溝内に保持する工程と、
半透膜で、細胞培養基板の培養用溝および排出用溝を覆う工程と、
供給手段によって培養液を連続的に細胞培養基板へ送り、細胞培養基板の培養用溝内に保持された細胞へ半透膜を介して培養液の供給を行うとともに、培養用溝の両端と接続する排出用溝を流れる培養液によって、培養用溝内の細胞の一部を排出用溝へ排出する工程と、
顕微観察手段によって細胞培養基板上の細胞を観察する工程と、
を含む請求項8に記載の細胞培養長期観察方法。
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