Method for calculating exchange volume, program for calculating exchange volume, and method for exchanging culture medium

Abstract

To provide a method for calculating the amount of culture medium to be exchanged, a program for calculating the amount of culture medium to be exchanged, and a method for exchanging culture medium that enable effective culture medium exchange without discarding cells. [Solution] The exchange amount calculation method according to the present invention includes a measurement step of measuring a first distance from the edge of the condenser lens closest to the stage to the top surface of the stage, when the lens is in focus on the upper end of the cell located furthest to the top surface of the culture medium among a plurality of cells settled in a dish containing cells and culture medium; a first calculation step of calculating a first liquid level from the inner bottom surface of the dish to the upper end of the cell located furthest to the top surface of the culture medium by inputting the first distance into a linear equation; and a second calculation step of calculating a second liquid level, which is the liquid level of the culture medium to be exchanged, from the top surface of the culture medium to the upper end of the cell located furthest to the top surface of the culture medium among the cells settled in the dish, using the first liquid level.

Description

[Technical Field]

[0001] The present invention relates to a method for calculating the amount of exchange, a program for calculating the amount of exchange, and a method for exchanging culture medium. [Background technology]

[0002] In cell culture, for example, Patent Document 1 describes injecting the culture medium using an injection means such as a dropper, or separating the cells from the culture medium by centrifugation, and observing the cells collected at the tip of the corner of the container. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2020-103189 [Overview of the project] [Problems that the invention aims to solve]

[0004] By the way, in order to continue culturing cells, it is necessary to change the culture medium. When changing the medium, it is required to introduce new medium without discarding the cells. Thus, there has been a need for a technology that allows for changing the culture medium without discarding cells when culturing cells.

[0005] This invention has been made in view of these circumstances, and its purpose is to provide a method for calculating the amount of exchange, a program for calculating the amount of exchange, and a method for exchanging a culture medium that can perform effective culture medium exchange without discarding cells. [Means for solving the problem]

[0006] To solve the above-mentioned problems and achieve the objective, the method for calculating the amount of exchange according to one aspect of the present invention uses an upright microscope and focuses on the upper end of the cell located closest to the top surface of the culture medium among a plurality of cells settled in a dish containing cells and culture medium. The process includes: a measurement step of measuring a first distance from the stage-side end of the condenser lens within its effective diameter to the top surface of the stage; a first calculation step of calculating a first liquid level from the inner bottom surface of the dish to the top end of the cell located furthest towards the top surface of the culture medium by inputting the first distance into a linear equation; and a second calculation step of using the first liquid level to calculate a second liquid level, which is the liquid level of the culture medium to be replaced, from the top surface of the culture medium to the top end of the cell located furthest towards the top surface of the culture medium among the cells settled in the dish. The linear equation is generated using an upright microscope based on a second distance from the stage-side end of the condenser lens within its effective diameter to the top surface of the stage when the lens is in focus on the inner bottom surface of the dish containing the culture medium; a third distance from the stage-side end of the condenser lens within its effective diameter to the top surface of the stage when the lens is in focus on the top surface of the culture medium contained in the dish; and a third liquid level from the inner bottom surface of the dish to the top surface of the culture medium.

[0007] In one aspect of the present invention, the method for calculating the amount of exchange is such that the upright microscope is an upright phase-contrast microscope.

[0008] A method for calculating the amount of exchange according to one aspect of the present invention, in the above invention, the linear equation is an equation that represents a straight line passing through a plot on a two-dimensional orthogonal coordinate system that shows the relationship between the liquid level when the liquid level is focused on the inner bottom surface of the dish containing the culture medium, the distance from the stage-side end within the effective diameter of the condenser lens to the top surface of the stage and the liquid level from the inner bottom surface of the dish to the top surface of the culture medium, and plots the second and third distances and the third liquid level, respectively.

[0009] A program for calculating exchange volume according to one aspect of the present invention includes: a measurement step of measuring a first distance from the edge closest to the stage within the effective diameter of a condenser lens to the top surface of the stage, when the microscope is in focus on the upper end of the cell located furthest towards the top surface of the culture medium among a plurality of cells settled in a dish containing cells and culture medium; a first calculation step of calculating a first liquid level from the inner bottom surface of the dish to the upper end of the cell located furthest towards the top surface of the culture medium by inputting the first distance into a linear equation; and using the first liquid level, calculating from the top surface of the culture medium to the upper end of the cell located furthest towards the top surface of the culture medium among the cells settled in the dish. The computer is made to perform a second calculation step of calculating a second liquid level, which is the liquid level of the culture medium to be replaced, and is the second liquid level to the upper end of the cells located on the surface side. The linear equation is generated using an upright microscope, based on a second distance from the stage-side end within the effective diameter of the condenser lens, when focused on the inner bottom surface of the dish containing the culture medium, to the upper surface of the stage; a third distance from the stage-side end within the effective diameter of the condenser lens, when focused on the upper surface of the culture medium contained in the dish, to the upper surface of the stage; and a third liquid level from the inner bottom surface of the dish to the upper surface of the culture medium.

[0010] A method for changing a culture medium according to one aspect of the present invention involves discharging the culture medium from the top surface of the medium up to the second liquid level calculated by the exchange amount calculation method according to the above invention, and adding a new culture medium in an amount equivalent to the amount of medium that was discharged to the dish. [Effects of the Invention]

[0011] According to the exchange volume calculation method, exchange volume calculation program, and exchange volume calculation device of the present invention, it is possible to perform effective culture medium exchange without discarding cells. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a diagram illustrating the overview of a culture medium exchange according to one embodiment of the present invention. [Figure 2] Figure 2 is a diagram showing the configuration of an exchange amount calculation system according to an embodiment of the present invention. [Figure 3] Figure 3 is a diagram for explaining the configuration of a dish. [Figure 4] Figure 4 is a flowchart showing the flow of a method for deriving a liquid level calculation formula. [Figure 5] Figure 5 is a diagram (part 1) for explaining the derivation of the liquid level calculation formula. [Figure 6] Figure 6 is a diagram (part 2) for explaining the derivation of the liquid level calculation formula. [Figure 7] Figure 7 is a flowchart showing the flow of an exchange amount calculation method according to an embodiment of the present invention. [Figure 8] Figure 8 is a diagram for explaining an exchange amount calculation method according to an embodiment of the present invention. [Figure 9] Figure 9 is a diagram for explaining a test example of exchange amount calculation according to an embodiment of the present invention. [Figure 10] Figure 10 is a diagram for explaining a test example of exchange amount calculation according to an embodiment of the present invention, and shows the relationship of distances when the culture medium volume is 10 mL. [Figure 11] Figure 11 is a diagram for explaining a test example of exchange amount calculation according to an embodiment of the present invention, and shows the relationship of distances when the culture medium volume is 3 mL.

Embodiments for Carrying Out the Invention

[0013] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. In all the drawings of the following embodiment, the same or corresponding parts are denoted by the same reference numerals. Also, the present invention is not limited to the embodiment described below.

[0014] (Overview of Culture Medium Exchange) Figure 1 is a diagram illustrating an overview of a culture medium exchange according to one embodiment of the present invention. As shown in Figure 1(a), the dish 100 contains a plurality of cells 101 to be cultured and a culture medium 102 for culturing the cells 101.

[0015] When changing the culture medium after culturing in a specified environment, a portion of the culture medium 102 is drained from dish 100 (see Figure 1(b)), and the same amount of new culture medium 102 is added (see Figure 1(c)). In this case, the culture medium 102 is drained from dish 100 by, for example, using a pipette, inserting the tip of the pipette into the culture medium 102 and aspirating. The pipette insertion and aspiration operation may be performed manually by an operator or mechanically.

[0016] (Exchange volume calculation system) Figure 2 shows the configuration of an exchange volume calculation system according to one embodiment of the present invention. As shown in Figure 2, the exchange volume calculation system 1 comprises a microscope device 2, an imaging device 3, and a processing device 4. The exchange volume calculation system 1 is configured such that the imaging device 3 and the processing device 4 can input and output data from each other.

[0017] Here, data input and output between the imaging device 3 and the processing device 4 can be performed via network communication through a network or cloud, or via contactless communication such as Bluetooth®. It is also possible to transfer data via USB (Universal Serial Bus) memory, CD (Compact Disc), DVD (Digital Versatile Disc), or BD (Blu-ray® Disc) discs. The network is composed of a combination of wired and wireless communication as appropriate, and consists of communication networks such as the Internet network and mobile phone network. The network may consist of one or more combinations of, for example, dedicated lines, public communication networks such as the Internet, such as LAN (Local Area Network), WAN (Wide Area Network), telephone communication networks and public lines such as mobile phones, and VPN (Virtual Private Network).

[0018] Microscope apparatus 2 consists of an upright phase-contrast microscope. Microscope apparatus 2 comprises a light source 21, an aperture member 22, a condenser lens 23, a stage 24, an objective lens 25, and a phase plate 26. The throttling member 22 has a ring-shaped opening. The phase plate 26 has a quarter-wave plate that shifts the phase of light by 1 / 4λ and a transparent portion provided around the wave plate.

[0019] In the microscope apparatus 2, the light emitted from the light source 21 is focused by the aperture member 22, and this light passes through the condenser lens 23 to the stage 24. The light that reaches the stage 24 is incident on the cells 101 (sample) in the dish 100 placed on the stage 24, and is split into transmitted light that has passed through the inside of the cells 101 and diffracted light that has been diffracted by the cells 101. Subsequently, the transmitted light and diffracted light each pass through the objective lens 25 and are incident on the phase plate 26. At this time, the transmitted light is focused by the objective lens 25 and passes through the quarter-wave plate, so its phase shifts by 1 / 4 and its brightness decreases. In contrast, the diffracted light passes through the transparent part of the phase plate 26 without phase shift and its brightness does not change. The transmitted light and diffracted light then reach the imaging plane of the imaging device 3 and are imaged by the imaging device 3.

[0020] The imaging device 3 is configured using an image sensor such as a CCD (Charge Coupled Device) or CMOS (Complementary Metal Oxide Semiconductor). The imaging device 3 outputs the captured image to the processing device 4. Furthermore, the imaging device 3 can operate, for example, in response to instructions input from the processing device 4.

[0021] The processing unit 4 comprises a communication unit 41, a measurement unit 42, a formula generation unit 43, a calculation unit 44, an input / output unit 45, a control unit 46, and a storage unit 47.

[0022] The communication unit 41 is, for example, a LAN interface board, a wired communication circuit for wired communication, or a wireless communication circuit for wireless communication. The LAN interface board, wired communication circuit, or wireless communication circuit is connected to a network. The communication unit 41, acting as both a transmitter and receiver, is connected to the network and communicates with the imaging device 3.

[0023] The measurement unit 42 measures the distance between the condenser lens 23 and the stage 24, the distance from the bottom of the dish to the surface of the liquid contained in the dish (liquid level), and so on. The measurements performed by the measurement unit 42 include obtaining distances by reading encoders or the like provided on the microscope device 2, and obtaining numerical values ​​input by the operator via the input / output unit 45.

[0024] The formula generation unit 43 generates a formula for calculating the distance from the upper end of the cell located on the uppermost side relative to the culture medium among the cells precipitated in the dish to the inner bottom surface of the dish.

[0025] The calculation unit 44 uses the formula generated by the formula generation unit 43 to calculate the volume of liquid from the top surface of the culture medium to the top of the cell located on the uppermost side.

[0026] The input / output unit 45 can be composed of, for example, a touch panel display or a speaker microphone. The input / output unit 45 as an input means may include an interface that receives various information transmitted from an external server via the communication unit and outputs it to the control unit 46. The input / output unit 45 also includes a user interface such as a keyboard, input buttons, levers, a touch panel for manual input superimposed on a display such as an LCD, or a microphone for voice recognition. The input / output unit 45 is configured to allow predetermined information to be input to the control unit 46 by an operator or other person operating it. The input / output unit 45 as an output means displays predetermined images on a display monitor, displays characters or graphics on the screen of a touch panel display, or outputs sound from a speaker, according to the control unit 46. In other words, the input / output unit 45 is configured to be able to notify the outside of predetermined information. The input unit and output unit of the input / output unit 45 may be configured as separate units.

[0027] The control unit 46 specifically includes a processor having hardware such as a CPU (Central Processing Unit), a DSP (Digital Signal Processor), and an FPGA (Field-Programmable Gate Array), as well as a main memory unit such as RAM (Random Access Memory) and ROM (Read Only Memory) (none of which are shown).

[0028] The storage unit 47 is composed of a storage medium selected from volatile memory such as RAM, non-volatile memory such as ROM, EPROM (Erasable Programmable ROM), hard disk drive (HDD), and removable media. The removable media can be, for example, a USB memory stick, or a disk recording medium such as a CD, DVD, or BD. Alternatively, the storage unit 47 may be configured using a computer-readable recording medium such as an externally insertable memory card.

[0029] The memory unit 47 can store various programs such as the operating system (OS) for executing the operation of the processing unit 4, applications for calculating exchange amounts, various tables, and various databases. These various programs can also be recorded on computer-readable recording media such as hard disks, flash memory, CD-ROMs, DVD-ROMs, and flexible disks and widely distributed.

[0030] (Calculation process for exchange amount) Next, the method for calculating the exchange amount performed by the processing device 4 configured as described above will be explained with reference to Figures 3 to 7. First, the generation of the formulas generated by the processing unit 4 will be explained with reference to Figures 3 to 6. Figure 3 is a diagram illustrating the configuration of the dish. The equation (linear equation) generated in this embodiment determines the position of the cell at the uppermost liquid level, which is the measurement target. It is created by measuring the distance from the lens to the stage at liquid levels above the cell (surface layer) and below the cell (bottom of the dish), and using the measured values ​​within that range. Then, the actual cell position is calculated by interpolation using this linear equation.

[0031] Dish 200, used for formula generation, has the same shape as the cell culture dish (dish 100), and a mark 201 for focusing is provided on its inner bottom surface (see Figure 3). Dish 200 contains the same amount of culture medium 102 as used for cell culture (see Figure 5). Note that the mark 201 is not limited to the mark shown in Figure 3, but may be any other letter, shape, symbol, etc., as long as it allows for the identification of the state in focus on the inner bottom surface of dish 200.

[0032] Figure 4 is a flowchart showing the flow of the derivation method for the liquid level calculation formula. Figures 5 and 6 are diagrams illustrating the derivation of the liquid level calculation formula. The measuring unit 42 first measures the distance D from the end of the condenser lens 23 closest to the stage 24 within the effective diameter of the condenser lens 23 when it is in focus on the mark 201 of the dish 200 in the microscope device 2, to the upper surface of the stage 24 (the outer bottom surface of the dish 200). 10 Measure the (second distance) (Step S1). At this time, the distance (liquid level) from the inner bottom surface of dish 200 to the top surface of culture medium 102 is set to zero.

[0033] Furthermore, the measuring unit 42 measures the distance D from the end of the condenser lens 23 closest to the stage 24 within its effective diameter when it is focused on the upper surface of the culture medium contained in the dish 200, to the upper surface of the stage 24 (the outer bottom surface of the dish 200) in the microscope device 2. 11 Measure the (third distance) (Step S2).

[0034] Furthermore, the measuring unit 42 measures the distance (liquid level) D from the inner bottom surface of the dish 200 to the top surface of the culture medium 102 at this time. L10Measure the (third liquid level) (step S3).

[0035] The formula generation unit 43 uses the combinations of the distance and the liquid level measured by the measurement unit 42 (D 10 , 0), (D 11 , D L10 ) to generate a linear equation (step S4). As shown in FIG. 6, the formula generation unit 43 is a two-dimensional rectangular coordinate system showing the relationship between the distance (mm) from the end closest to the stage side within the effective diameter of the condenser lens to the bottom surface of the dish (the upper surface of the stage 24) and the distance (liquid level) (mm) from the inner bottom surface of the dish to the upper surface of the culture medium. In this coordinate system, plot (D 10 , 0), (D 11 , D L10 ) respectively, and generate a linear equation representing the straight line L1 passing through these plots. In this way, a linear equation for calculating the liquid level is generated.

[0036] FIG. 7 is a flowchart showing the flow of the exchange amount calculation method according to an embodiment of the present invention. FIG. 8 is a diagram for explaining the exchange amount calculation method according to an embodiment of the present invention. The calculation unit 44 calculates the discharge amount of the culture medium using this linear equation.

[0037] First, the measuring unit 42 measures the distance D1 (the first distance) from the end closest to the stage 24 side within the effective diameter of the condenser lens 23 to the upper surface of the stage 24 (the outer bottom surface of the dish 200) in a state where the measuring unit 42 is focused on the upper end of the cell located closest to the upper surface of the culture medium among the cells precipitated in the dish (step S11: measurement step, see FIG. 8).

[0038] The calculation unit 44 inputs the distance D1 into the linear equation, thereby calculating the distance (liquid level) D L1 (the first liquid level) from the inner bottom surface of the dish 100 to the upper end of the cell located closest to the upper surface of the culture medium (step S12: the first calculation step).

[0039] After that, the calculation unit 44 calculates the inner diameter of the dish 100, the volume of the culture solution, and the liquid level D L1Using this method, measure the liquid level D from the top surface of the culture medium to the upper end of the cell located closest to the top surface of the culture medium among the cells settled in the dish. L2 The second liquid level is calculated (Step S13: Second calculation step). The liquid level calculated here is the liquid level to be discharged. Using this liquid level and the inner diameter of dish 100, the amount of liquid to be discharged can be obtained. Note that the liquid level from the inner bottom surface of dish 100 to the top surface of the culture medium (for example, liquid level D) L3 If (assuming) is known, then this liquid level D L3 And, liquid level D L1 The difference (D L3 -D L1 The liquid level to be discharged may be calculated by ( ).

[0040] The worker, at this liquid level D L2 Based on this, the culture medium is drained from dish 100, and the same amount of culture medium is added to replace it. When draining the culture medium, as described above, a pipette is placed on the top surface of the culture medium, and while aspirating the culture medium with the pipette, the tip of the pipette is brought to liquid level D L2 Move the pipette down to aspirate the culture medium, or move the tip of the pipette from the top surface of the culture medium to liquid level D. L2 The medium is then inserted into the culture medium and the medium is aspirated or drained.

[0041] Next, a test example for calculating the exchange volume in this embodiment will be described with reference to Figures 9 to 11. Figure 9 is a diagram illustrating a test example for calculating the exchange volume according to one embodiment of the present invention. In this test, a dish 300 was filled with culture medium 301 and test pieces T1 to T5 in place of cells cultured in the medium. The culture medium 301 was tested in 10 mL and 30 mL quantities, respectively.

[0042] Test specimens T1 to T4 are plate-shaped with different longitudinal lengths, and are placed in dish 300 with one end aligned along their longitudinal direction. Test specimens T1, T2, T3, and T4 are stacked in that order from the inner bottom surface of dish 300. Test specimen T5 is a thin plate-shaped specimen floating on the surface of the culture medium. Test specimens T3 and T4 are exposed outside the culture medium when the culture medium volume is 10 mL, and are located inside the culture medium when the culture medium volume is 30 mL.

[0043] In this study, the focusing positions (marks) of the phase-contrast microscope were defined as follows: position A on the inner bottom surface of dish 300, position B on the top surface of test piece T1, position C on the top surface of test piece T2, position D on the top surface of test piece T3, position E on the top surface of test piece T4, and position F on the top surface of test piece T5. The distance (mm) from the inner bottom surface of dish 300 to each position (mark) and the distance (mm) from the edge of the condenser lens closest to the stage within the effective diameter when the focus is at each position to the top surface of the stage were measured. Table 1 shows the measurement results when the culture medium was 10 mL, and Table 2 shows the measurement results when the culture medium was 30 mL. Note that the average of three measurements is shown for each test piece.

[0044] [Table 1]

[0045] [Table 2]

[0046] Figure 10 is a diagram illustrating a test example for calculating the exchange volume according to one embodiment of the present invention, showing the relationship of distances when the culture medium volume is 10 mL. Figure 11 is a diagram illustrating a test example for calculating the exchange volume according to one embodiment of the present invention, showing the relationship of distances when the culture medium volume is 30 mL. Figure 10 plots the measurement results shown in Table 1. Figure 11 plots the measurement results shown in Table 2.

[0047] As shown in Figures 10 and 11, the distance from the edge of the condenser lens closest to the stage within its effective diameter to the top surface of the stage, when focused at each position, changes linearly with respect to the distance from the inner bottom surface of the dish 300 to each position, regardless of the volume of the culture medium. Therefore, the liquid level can be calculated by expressing the measurement results as a linear equation.

[0048] Furthermore, in Figures 10 and 11, for each capacitance, plots located between the ends of the plots, L2 and L3, tend to show smaller values. Therefore, the distance from the edge closest to the stage within the effective diameter of the condenser lens to the top surface of the stage (liquid level), calculated by a linear equation when the focus is at each position, is calculated to be higher than the actual value. As a result, the discharged liquid level (liquid volume) is slightly smaller than the measured value. In this way, it can be said that the misdelivery of cells due to excessive discharge can be suppressed by calculating a slightly smaller liquid volume as the discharged volume.

[0049] According to the embodiment described above, the distance D from the end of the condenser lens 23 closest to the stage 24 within the effective diameter when in focus on the mark 201 of the dish 200 to the upper surface of the stage 24 (the outer bottom surface of the dish 200) is 10 The distance D is calculated from the end of the condenser lens 23 closest to the stage 24 within its effective diameter when focused on the upper surface of the culture medium contained in the dish 200, to the upper surface of the stage 24 (the outer bottom surface of the dish 200). 11 The distance (liquid level) D is from the inner bottom surface of dish 200 to the top surface of culture medium 102. L10 Using a linear equation based on the above, the liquid level D is calculated from the top surface of the culture medium to the upper end of the cell located closest to the top surface of the culture medium among the cells settled in the dish. L2 This value was calculated and used as the liquid level of the culture medium to be drained. According to this embodiment, by performing the culture medium exchange based on the liquid level of the culture medium to be drained calculated as described above, an effective culture medium exchange can be performed without discarding cells.

[0050] (Recording medium) In the embodiment described above, a program that causes the processing method executed by the processing device 4 to be executed can be recorded on a recording medium that can be read by a computer or other machine or device such as a wearable device (hereinafter referred to as "computer, etc."). By having the computer, etc. read and execute the program on this recording medium, the computer, etc. functions as a mobile device control device. Here, a recording medium that can be read by a computer, etc. refers to a non-temporary recording medium that stores information such as data and programs by electrical, magnetic, optical, mechanical, or chemical action and can be read by a computer, etc. Examples of such recording media that can be removed from a computer, etc. include flexible disks, magneto-optical disks, CD-ROMs, CD-R / Ws, DVDs, BDs, DATs, magnetic tapes, and memory cards such as flash memory. In addition, recording media that are fixed to a computer, etc. include hard disks and ROMs. Furthermore, an SSD can be used as both a recording medium that can be removed from a computer, etc. and a recording medium that is fixed to a computer, etc.

[0051] Although one embodiment of the present invention has been described in detail above, the present invention is not limited to the above-described embodiment, and various modifications are possible based on the technical idea of ​​the present invention. For example, the numerical values ​​given in the above-described embodiment are merely examples, and different numerical values ​​may be used as needed, and the present invention is not limited by the description and drawings that constitute part of the disclosure of the present invention in this embodiment. For example, in the above-described embodiment, an example of determining the liquid level for changing the culture medium was described, but it can be used not only for dishes and culture media, but also for calculating the amount of liquid to be changed when changing liquids, etc., while leaving the contents intact.

[0052] Furthermore, in this invention, when measuring the liquid level from the inner bottom surface of the dish and the distance from the condenser lens, it is necessary to use the same microscope device and to ensure that the observation magnification and measurement conditions for the type of dish are consistent.

[0053] Furthermore, although one embodiment described an example of using a phase-contrast microscope in an upright microscope, other observation modes such as bright-field or dark-field microscopes may be used if distance and liquid level measurement is possible.

[0054] Furthermore, in one embodiment, the terms "part" as described above can be replaced with "circuit" or the like. For example, the control unit can be replaced with a control circuit.

[0055] Further effects and modifications can be readily derived by those skilled in the art. Broader aspects of this disclosure are not limited to the specific details and representative embodiments expressed and described above. Therefore, various modifications are possible without departing from the spirit or scope of the overall concept of the invention as defined by the appended claims and their equivalents. [Explanation of symbols]

[0056] 1. Exchange Volume Calculation System 2. Microscope apparatus 3. Imaging device 4 Processing Unit 21 Light source 22 Restriction member 23. Condenser lens 24 stages 25 Objective lens 26 Phase plate 41 Communications Department 42 Measurement section 43 Expression generator 44 Calculation Section 45 Input / output section 46 Control Unit 47 Memory section 100, 200, 300 dishes 101 cells 102 culture medium

Claims

1. A measurement step in which, using an upright microscope, the upper end of the condenser lens is in focus on the upper end of the cell located closest to the top surface of the culture medium among multiple cells precipitated in a dish containing cells and culture medium, and the first distance from the edge closest to the stage within the effective diameter of the condenser lens to the top surface of the stage, A first calculation step involves inputting the first distance into a linear equation to calculate a first liquid level from the inner bottom surface of the dish to the upper end of the cell located closest to the top surface of the culture medium, A second calculation step involves using the first liquid level to calculate a second liquid level, which is the liquid level of the medium to be replaced, and is the level from the top surface of the culture medium to the upper end of the cell located closest to the top surface of the culture medium among the cells settled in the dish. Includes, The aforementioned linear equation is generated using an upright microscope based on a second distance from the stage-side end within the effective diameter of the condenser lens, when focused on the inner bottom surface of the dish containing the culture medium, to the upper surface of the stage; a third distance from the stage-side end within the effective diameter of the condenser lens, when focused on the upper surface of the culture medium contained in the dish, to the upper surface of the stage; and a third liquid level from the inner bottom surface of the dish to the upper surface of the culture medium. Exchange amount calculation method.

2. The aforementioned upright microscope is an upright phase-contrast microscope. The method for calculating the amount of replacement according to claim 1.

3. The aforementioned linear equation is an equation that represents a straight line passing through a two-dimensional orthogonal coordinate system that plots the second and third distances, as well as the third liquid level, on a two-dimensional orthogonal coordinate system that shows the relationship between the liquid level when the lens is focused on the inner bottom surface of the dish containing the culture medium, the distance from the stage-side end of the condenser lens within the effective diameter to the top surface of the stage, and the liquid level from the inner bottom surface of the dish to the top surface of the culture medium. The method for calculating the amount of replacement according to claim 1.

4. A measurement step in which, using an upright microscope, the upper end of the condenser lens is in focus on the upper end of the cell located closest to the top surface of the culture medium among multiple cells precipitated in a dish containing cells and culture medium, and the first distance from the edge closest to the stage within the effective diameter of the condenser lens to the top surface of the stage, A first calculation step involves inputting the first distance into a linear equation to calculate a first liquid level from the inner bottom surface of the dish to the upper end of the cell located closest to the top surface of the culture medium, A second calculation step involves using the first liquid level to calculate a second liquid level, which is the liquid level of the medium to be replaced, and is the level from the top surface of the culture medium to the upper end of the cell located closest to the top surface of the culture medium among the cells settled in the dish. Have the computer run it, The aforementioned linear equation is generated using an upright microscope based on a second distance from the stage-side end within the effective diameter of the condenser lens, when focused on the inner bottom surface of the dish containing the culture medium, to the upper surface of the stage; a third distance from the stage-side end within the effective diameter of the condenser lens, when focused on the upper surface of the culture medium contained in the dish, to the upper surface of the stage; and a third liquid level from the inner bottom surface of the dish to the upper surface of the culture medium. Exchange volume calculation program.

5. According to the second liquid level calculated by the exchange volume calculation method described in claim 1, the culture medium is discharged from the top surface of the culture medium up to the second liquid level. Add a new amount of culture medium to the dish that is equivalent to the amount of medium that was drained. Method for changing the culture medium.

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