JP2010266261A - Apparatus and method for measuring oxygen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for measuring oxygen for performing a highly reliable evaluation of an oxygen consumption of biological samples such as cells attached to a carrier. <P>SOLUTION: The oxygen measuring apparatus includes a biological sample storage container for storing a biological sample attached to the carrier, a medium perfusion system for perfusing a medium to the biological sample storage container, and a flow cell which is disposed at a medium outlet of the biological sample storage container and in which an oxygen electrode is integrated. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an oxygen measuring device and an oxygen measuring method for measuring oxygen consumption of a biological sample such as a cell attached to a carrier.

  In recent years, active research has been conducted to measure respiratory activity, which is considered to be one of the indicators of the activity of biological samples such as cells, from the concentration of dissolved oxygen in a solution. For example, a study for measuring the amount of oxygen consumed by a biological sample in a solution from the concentration of dissolved oxygen in the solution.

  Currently, the oxygen concentration method in a solution generally used includes an oxygen electrode method using an oxygen electrode that generates a current proportional to the oxygen partial pressure, and an oxygen-sensitive fluorescent substance that emits fluorescence that is inversely related to the oxygen partial pressure. There are fluorescent methods used.

  Patent Document 1 discloses a container for storing a solution, a pair of electrodes provided in the container, and a sample guide for guiding a biological sample to one of the pair of electrodes arranged in the container. In the sample guide, a micro area surrounding one electrode and an introduction area for guiding the biological sample into the micro area are formed. Based on the micro current flowing through the pair of electrodes, the sample guide An oxygen measuring device is described in which a measurement amount relating to dissolved oxygen in the vicinity of a biological sample introduced into a micro region is measured.

When measuring the oxygen consumption of the fixing cells attached to the carrier using these methods, for example, a cell sample solution is prepared by the following procedure.
(1) Remove the old medium in the culture container by suction. (2) Wash the cell wall cells with a buffer (such as Hanks solution) and then remove the buffer by suction. (3) Put an enzyme solution such as trypsin solution in the culture container. Wet the cells thoroughly, put them in an incubator and leave them for about 5 to 10 minutes. (4) Add new medium, pipet, disperse the cells in the medium, centrifuge the cell solution, and aspirate the supernatant. Removal (5) Add fresh medium, stir to break up cell clumps and disperse well (6) Check the cell concentration of the solution and adjust to the required concentration

  Dispensing the cell sample solution thus prepared into a measurement container, and measuring the time-dependent change in the dissolved oxygen concentration in the liquid with an oxygen sensor (oxygen electrode method or fluorescence method) installed in the container, the oxygen consumption of the cell Get quantity.

  Except for cells that float in body fluids, such as blood cells, many cells perform their own cellular activities such as proliferation and differentiation while being attached to an appropriate carrier, and express their functions. In the measurement method described above, not only the cell activity is reduced by detaching the cell from the carrier, but also the oxygen consumption of the cell is evaluated in a state where the original cell function such as proliferation is impaired. In addition, the cell consumes oxygen and nutrients in the medium due to its activity, and produces harmful waste products such as ammonia and lactic acid. If the measurement container is not sealed, oxygen is replenished to some extent by diffusion and convection from the gas-liquid interface, but the cellular environment changes over time due to consumption of nutrients and production of waste products. It is inevitable that it gets worse.

  Therefore, the conventional measurement method evaluates the influence of substances related to the growth of fixed cells, or examines the influence of substances that require a long period of time before effects and harm appear, It is not necessarily suitable.

  For example, Patent Document 2 discloses a sample measurement method that achieves measurement of a sample by supplying the sample to an immobilized layer formed by immobilizing the target material and detecting the reaction between the target material and the sample with an electrode. A sample measurement method is described in which a sample is supplied while changing its concentration over time, and the sample is measured based on the amount of change in the output signal from the electrode. However, in the apparatus of Patent Document 2, since the immobilization layer obtained by immobilizing the target substance and the electrode are both housed in the same chamber (second chamber) of the measurement container, highly reliable evaluation cannot be obtained.

JP 2008-64661 A JP 2000-270838 A

  The present invention is an oxygen measuring apparatus and an oxygen measuring method that enable highly reliable evaluation of oxygen consumption of a biological sample such as a cell attached to a carrier.

  The present invention relates to a biological sample storage container for storing a biological sample attached to a carrier, a culture medium perfusion system for perfusing a culture medium to the biological sample storage container, and an oxygen electrode installed at the culture medium outlet of the biological sample storage container Is an oxygen measuring device.

  In the oxygen measuring device, it is preferable that a bypass path for connecting a culture medium inlet side pipe and a culture medium outlet side pipe of the biological sample storage container is provided.

  The present invention is also an oxygen measurement method in which a culture medium is perfused into a biological sample storage container that stores a biological sample attached to a carrier, and a dissolved oxygen concentration of the culture medium after flowing out of the biological sample storage container is measured.

  Further, in the oxygen measurement method, the dissolved oxygen concentration of the medium not passing through the biological sample storage container is measured to obtain a reference dissolved oxygen concentration, and the medium after flowing out of the biological sample storage container with respect to the reference dissolved oxygen concentration It is preferable to measure the amount of oxygen consumed by the biological sample by determining the amount of change in the dissolved oxygen concentration.

  In the present invention, a biological sample storage container for storing a biological sample attached to a carrier, a culture medium perfusion system for perfusing a culture medium to the biological sample storage container, and an oxygen electrode installed at the culture medium outlet of the biological sample storage container are incorporated. By providing such a flow cell, it is possible to provide an oxygen measuring device that enables highly reliable evaluation of oxygen consumption of biological samples such as cells.

  In the present invention, the culture medium is perfused into a biological sample storage container that stores the biological sample attached to the carrier, and the dissolved oxygen concentration of the culture medium after flowing out from the biological sample storage container is measured, so that a biological material such as a cell is obtained. It is possible to provide an oxygen measuring method that enables highly reliable evaluation such as oxygen consumption of a sample.

It is a schematic structure figure showing an example of the oxygen measuring device concerning the embodiment of the present invention. It is a figure which shows the measurement result of the oxygen consumption of the cell (SDR-M) by the oxygen electrode in the Example of this invention.

  Embodiments of the present invention will be described below. This embodiment is an example for carrying out the present invention, and the present invention is not limited to this embodiment.

  The oxygen measuring device 1 according to the embodiment of the present invention measures a dissolved oxygen concentration in a culture medium by respiration (oxygen consumption) of a biological sample such as a cell while perfusing the culture medium, thereby providing a highly reliable biological sample. The measurement of oxygen consumption by means of is realized. An outline of an example of the oxygen measuring apparatus according to the present embodiment is shown in FIG. 1 and the configuration thereof will be described. The oxygen measuring apparatus 1 includes a biological sample storage container 10 that stores a biological sample such as a cell attached to a carrier, a culture medium perfusion system 12 that perfuses a culture medium to the biological sample storage container 10, and a flow cell in which an oxygen electrode 14 is incorporated. 16 and a microammeter 32 and a recording / display device 34 capable of measuring and recording the response current value output from the oxygen electrode 14 over time. The medium perfusion system 12 includes a medium container 18 and a liquid feed pump 20.

  In the oxygen measuring apparatus 1, the culture medium container 18 is connected to the inflow side of the liquid feed pump 20 by a pipe or the like, and the inlet of the biological sample storage container 10 is connected to the discharge side of the liquid feed pump 20 by a pipe or the like. The outlet of the biological sample storage container 10 is connected to the inlet of the flow cell 16 by piping or the like, and the outlet 22 of the flow cell 16 is connected to the container 22 for storing the medium discharged from the flow cell 16 by piping or the like. An oxygen electrode 14 is incorporated in the flow cell 16. The pipe on the inlet side and the pipe on the outlet side of the biological sample storage container 10 are connected by a bypass pipe 26 via three-way valves 24a and 24b whose flow paths can be switched. The medium container 18 is provided with a stirrer such as a stirrer 28, and a controller 30 that controls the stirrer is connected to the stirrer by electrical connection or the like. A microammeter 32 is connected to the oxygen electrode 14 by electrical connection or the like, and a recording / display device 34 is connected to the microammeter 32 by electrical connection or the like.

  Since the dissolved oxygen concentration in the liquid and the response current of the oxygen electrode are temperature dependent, in order to avoid temperature correction (calibration), measurement devices such as the microammeter 32 and the recording / display device 34 are excluded. The culture medium container 18, the biological sample storage container 10, the flow cell 16, the oxygen electrode 14 and the like are preferably installed in an environment that can be maintained at a constant temperature condition. In FIG. 1, the culture medium container 18, the stirrer 28, the biological sample storage container 10, the flow cell 16, the oxygen electrode 14, and the container 22 are stored in a thermostatic chamber 36. Temperature sensors 38a, 38b, and 38c are installed in the medium container 18, the thermostat 36, and the flow cell 16, respectively.

  The biological sample storage container 10 includes a storage unit that stores a biological sample such as a cell attached to a carrier, an inlet unit and an outlet unit that communicate with the storage unit, and allows a liquid medium to flow into the storage unit. Anything is possible. If the liquid volume of the biological sample storage container 10 is large, the rate of change of the dissolved oxygen concentration due to respiratory metabolism of cells and the like is slowed down. Therefore, the liquid volume of the biological sample storage container 10 is increased in order to increase the responsiveness to changes in the dissolved oxygen concentration. Is preferably as small as possible. The biological sample storage container 10 may have an observation window using a transparent material such as glass or resin in the storage unit.

  The flow cell 16 has a flow path, an inlet part and an outlet part that communicate with the flow path, and can incorporate a liquid medium so that the oxygen electrode 14 can be detected in the flow path. Anything that can do. In order to increase the responsiveness of the oxygen electrode 14 to changes in the dissolved oxygen concentration, the liquid capacity of the flow cell 16 is preferably as small as possible.

  As shown in FIG. 1, the biological sample storage container 10 and the flow cell 16 may be connected to each other by piping or the like, but the biological sample storage container 10 and the culture medium from the biological sample storage container 10 flow in. However, it may have a structure (one-dot chain line portion in FIG. 1) in which the flow cell 16 incorporating the oxygen electrode 14 is integrated. A structure in which the biological sample storage container 10 and the flow cell 16 are integrated is preferable because the response speed is high and leakage of oxygen and the like from piping and connecting parts can be avoided.

  In order to increase the response speed of the oxygen electrode 14 to changes in the dissolved oxygen concentration, it is preferable to reduce the capacity of the pipe between the biological sample storage container 10 and the flow cell 16 so that the medium flows smoothly. .

  The oxygen electrode 14 is not particularly limited as long as it generates a current proportional to the oxygen partial pressure. However, even in a medium perfusion system having a small liquid contact area and a small flow rate (for example, about 2 mL / hr). What can obtain a stable response current value is preferable. As such an electrode, for example, a microcathode oxygen electrode in which an electrode portion (cathode) for detecting oxygen is configured to have an extremely small area and self-oxygen consumption is extremely small.

  The liquid feed pump 20 preferably has a small flow rate (for example, about 2 mL / hr) and good flow rate accuracy and reproducibility.

  A method for measuring oxygen consumption by a biological sample such as a cell using the oxygen measuring device 1 according to this embodiment will be described.

<Preparation of biological sample to be measured>
A biological sample such as a cell to be measured attached to a carrier such as a glass plate is accommodated in the biological sample container 10 together with the carrier.

  Examples of biological samples to be measured include cells. Examples of the carrier to which the biological sample is attached include glass and resin. For example, cells obtained by monolayer culture with a glass plate may be used, but any culture form may be used as long as it can be incorporated into a perfusion system. By using a carrier having a known surface area, the oxygen consumption per cell number can be determined as described later.

<Warming and degassing of medium>
A medium is accommodated in the medium container 18. The medium is a liquid containing components suitable for the survival of the biological sample. At low temperatures, the gas saturation concentration in the liquid is high, and in the refrigerated medium, nearly twice as much gas (oxygen, nitrogen, etc.) may be dissolved as compared to the medium at around 37 ° C. used for culture. If the medium is fed without sufficiently degassing, bubbles may be generated due to a temperature rise in the middle of the feeding, which may hinder measurement of the feeding and dissolved oxygen concentration. Therefore, it is preferable that the medium in the medium container 18 is heated and controlled to a predetermined temperature (for example, around 37 ° C.) by the thermostatic bath 36 while being stirred by a stirring device such as a stirrer 28 as necessary. The medium may be separately heated using a thermostatic water tank or the like before being accommodated in the medium container 18. Moreover, it is better to keep the culture medium container 18 open without sealing so that sufficient gas exchange is possible at the gas-liquid interface. The stirring device such as the stirrer 28 is controlled by the control unit 30. The temperature of the medium in the medium container 18 and the temperature in the thermostatic chamber 36 are monitored by the temperature sensors 38a and 38b, recorded as data by the recording / display device 34 as necessary, and displayed as a graph or the like. .

<Measurement of standard dissolved oxygen concentration current value>
First, the reference dissolved oxygen concentration current value (A) is measured. In a state where the three-way valves 24 a and 24 b are switched to a path passing through the bypass pipe 26, the medium in the medium container 18 is fed to the flow cell 16 by the liquid feeding pump 20. For example, in Example 1, which will be described later, the liquid feeding flow rate is 1 to 3 mL / hr, but the oxygen consumption rate of cells and the like varies greatly depending on the species and is proportional to the number of cells. What is necessary is just to set suitably by the kind of biological sample, such as, the number, etc. If necessary, the piping system may be purged by increasing the liquid feed flow rate. The response current value of the oxygen electrode 14 generated in proportion (proportional) to the dissolved oxygen concentration contained in the medium flowing through the flow path of the flow cell 16 is measured by the microammeter 32 and recorded by the recording / display device 34 as necessary. ,indicate. The temperature in the flow cell 16 is monitored by the temperature sensor 38c, and recorded and displayed by the recording / display device 34 as necessary. Even if the temperature of the culture medium reaches a predetermined temperature (for example, around 37 ° C.), the dissolved oxygen concentration does not always decrease to the saturation concentration of the temperature, so the current value by the oxygen electrode 14 and the temperature sensor 38a. , 38b, 38c until the temperature is sufficiently stabilized. When the measurement is complete, proceed to the next step. The measurement of the reference dissolved oxygen concentration current value may be performed after the measurement of the dissolved oxygen concentration current value in the next step.

<Measurement of dissolved oxygen concentration current value>
Next, a dissolved oxygen concentration current value (B) that reflects oxygen consumption of the culture medium by a biological sample such as a cell is measured. In a state where the three-way valves 24 a and 24 b are switched to the path passing through the biological sample storage container 10, the medium in the medium container 18 is fed to the flow cell 16 by the liquid feed pump 20. The response current value of the oxygen electrode 14 generated in proportion (proportional) to the dissolved oxygen concentration contained in the medium flowing through the flow path of the flow cell 16 is measured by the microammeter 32 and recorded by the recording / display device 34 as necessary. ,indicate. Measurement is performed until the current value by the oxygen electrode 14 and the temperature by the temperature sensors 38a, 38b, and 38c are sufficiently stabilized. When the measurement is complete, proceed to the next step.

<Measurement (confirmation) of reference dissolved oxygen concentration current value>
For confirmation, the standard dissolved oxygen concentration current value (C) is measured again. In a state where the three-way valves 24 a and 24 b are switched to a path passing through the bypass pipe 26, the medium in the medium container 18 is fed to the flow cell 16 by the liquid feeding pump 20. If necessary, the piping system may be purged in order to increase the flow rate of the liquid and expel the medium passing through the biological sample storage container 10. The response current value of the oxygen electrode 14 generated in proportion (proportional) to the dissolved oxygen concentration contained in the medium flowing through the flow path of the flow cell 16 is measured by the microammeter 32 and recorded by the recording / display device 34 as necessary. ,indicate. Measurement is performed until the current value by the oxygen electrode 14 and the temperature by the temperature sensors 38a, 38b, and 38c are sufficiently stabilized.

  If the reference dissolved oxygen concentration current value (A) and the reference dissolved oxygen concentration current value (C) do not match, contamination of the flow path, etc., or the temperature during measurement of the reference dissolved oxygen concentration current value (A) It is possible that the saturated oxygen concentration was not reached. In the measurement of the dissolved oxygen concentration current value (B), a medium having a high dissolved oxygen concentration that has been saturated with oxygen at a temperature of about room temperature may flow in the initial stage of the measurement. The current value may be higher than the current value (A) or the reference dissolved oxygen concentration current value (C).

  The reference dissolved oxygen concentration current values (A) and (C) and the dissolved oxygen concentration current value (B) are measured on the upstream and downstream sides of the biological sample container 10 without providing the bypass pipe 26, respectively. Measurement may be performed by providing a flow cell in which is incorporated. By measuring in common with one oxygen electrode, the measurement variation due to the variation between electrodes is eliminated, contamination by germs caused by oxygen electrode and flow cell that is not sufficiently sterilized, and elution substances from the electrode and flow cell It is preferable to measure by switching the path by providing a bypass pipe 26 because the medium can be flowed to the biological sample storage container 10 by reducing the possibility of being affected by the above.

<Calculation of oxygen consumption>
The dissolved oxygen concentration in the medium changes according to the respiration of the biological sample, that is, according to the oxygen consumption by the biological sample. Therefore, for example, the respiratory activity of a biological sample can be evaluated from the dissolved oxygen concentration.

  Change amount of oxygen electrode response current value due to passing through biological sample in biological sample storage container 10 (change amount of dissolved oxygen concentration current value (B) with respect to reference dissolved oxygen concentration current value (A), ie, biological sample ( Here, the change amount of the oxygen amount after passing through the biological sample (here, the change amount of oxygen after passing through the biological sample storage vessel 10) with respect to the oxygen amount supplied to the biological sample storage vessel 10) is obtained. The amount of change is, for example, a change rate or a difference. For example, the supply rate of oxygen to the change rate of the dissolved oxygen concentration current value (B) with respect to the reference dissolved oxygen concentration current value (A), that is, the change rate of the oxygen amount after passing through the biological sample with respect to the oxygen amount supplied to the biological sample. Multiply the amount to determine the oxygen consumption of the biological sample. The amount of oxygen supplied is determined by (saturated oxygen concentration) x (liquid feed flow rate) from the oxygen partial pressure of the gas in contact with the medium, the saturated oxygen concentration of the medium determined by the measurement temperature, salt concentration, etc., and the liquid feed flow rate. Calculated. Moreover, the oxygen consumption amount can be calculated by dividing the oxygen consumption amount by the number of cells in the biological sample storage container 10.

  The obtained oxygen consumption includes oxygen consumption by the oxygen electrode 14 itself. The oxygen consumption of the electrode can be calculated according to Faraday's law. As described above, the oxygen consumption by the oxygen electrode 14 itself can be almost ignored by using a micro-cathode oxygen electrode or the like that consumes very little oxygen as the oxygen electrode 14.

  Since it is not necessary to peel biological samples such as fixing cells from the carrier by the oxygen measuring device and the oxygen measuring method according to the present embodiment, the cells and the like while maintaining the activity and function of the biological sample close to the original ones. Respiratory oxygen consumption, which is one of the indices indicating the activity of, can be measured. Since the biological sample can be handled together with the carrier, the preparation work for the measurement is simplified. Moreover, there is little damage and loss of a biological sample, and it is easy to use repeatedly.

  In addition, since the culture medium is supplied to the biological sample in a fresh state by perfusion, the activity of cells and the like can be maintained for a long period of time, and long-term oxygen consumption kinetics can be measured. It is possible to evaluate the effects on the cells by weak substances. By perfusion, it is possible to easily control the concentration and time of reagents and additives in the medium.

  Moreover, the oxygen consumption per cell number can be evaluated by utilizing a carrier having a known surface area such as a glass plate as the cell carrier.

  Furthermore, by using a transparent material such as glass or resin as an observation window in the biological sample storage container 10, it is possible to perform observation of cells with an optical microscope or the like as well as measurement of oxygen consumption.

  From the above, the oxygen measuring device and the oxygen measuring method according to the present embodiment enable highly reliable evaluation of the oxygen consumption of the biological sample attached to the carrier. Of course, a measurement amount related to oxygen other than the oxygen consumption by the biological sample (for example, the amount of oxygen in the fluid in replacement fluid, blood transfusion, dialysis, etc.) may be measured.

  Hereinafter, although an example and a comparative example are given and the present invention is explained more concretely in detail, the present invention is not limited to the following examples.

<Example 1>
The oxygen consumption of the cells was measured using an oxygen measuring device having the following configuration.
[Oxygen concentration measurement flow cell]
Flow cell: Liquid capacity 0.6mL
Oxygen electrode: 1302 type micro cathode electrode (manufactured by Strathkelvin, oxygen self-consumption: 93 fmol / min (typical, response current 0.6 nA))
Built-in temperature sensor: Platinum resistance thermometer Pt100classA (± 0.15 + 0.002t ° C)
[Oxygen electrode power supply]
Silver oxide battery (The voltage applied to the electrode was adjusted to 0.65 V by resistance)
[Biological sample container]
Inner diameter: 25mm (for φ22 glass plate)
Liquid capacity when glass plate is accommodated: 0.4 mL
[Feed pump]
Pump: Tubing pump (AC-2120, manufactured by atto)
Flow rate: 0.1 to 1000 mL / hr
[Liquid feeding piping]
Liquid feeding tube: Tygon R3603
[Temperature sensor]
Constant temperature bath: Platinum resistance thermometer Pt100classA (± 0.15 + 0.002t ° C.)
Medium container: T-type thermocouple [microammeter]
Micro ammeter TR8652 (manufactured by advantest, current measurement accuracy: 1 pA)
[Recording device]
Data logger NR-500 (manufactured by keyence)

The following biological samples and media were used.
[Biological sample]
Periodontal ligament-derived epithelial cells (SDR-M), φ22 glass plate monolayer culture (culture days: 7 to 90 days, cell count: 2E + 5 (estimated value when confluent (growth saturation))
[Culture medium]
DMEM / F12 medium Serum 15%
Medium flow rate: 2 mL / hr

  Periodontal ligament-derived epithelial cells cultured on a φ22 glass plate (SDR-M, culture days: 7, 8, 15, 22, 3 months) The oxygen electrode response current at the time of cell measurement FIG. 2 shows the result of normalization (used as the reference dissolved oxygen concentration current value in this example). The rate of change of the oxygen electrode current value, that is, the change of the dissolved oxygen concentration was about −3% to −6%. The saturated oxygen concentration of the 37 ° C. medium is about 6.7%, and when the medium flow rate is 2 mL / hr (0.033 mL / min), the oxygen supply amount to the cells (biological sample storage container) is about 0.7 nmol / min. Therefore, the oxygen consumption amount of each measurement result is 0.21 to 0.42 nmol / min, and the number of cells at the time of confluence (growth saturation state) with a φ22 glass plate is estimated to be 200,000, so SDR-M The oxygen consumption rate of the cells is calculated as 1 to 2 fmol / min / cell.

  Thus, it was possible to measure the oxygen consumption without interfering with the original activity and function of the cells.

  DESCRIPTION OF SYMBOLS 1 Oxygen measuring apparatus, 10 biological sample storage container, 12 culture medium perfusion system, 14 oxygen electrode, 16 flow cell, 18 culture medium container, 20 liquid feeding pump, 22 containers, 24a, 24b three-way valve, 26 bypass piping, 28 stirrer, 30 control Part, 32 microammeter, 34 recording / display device, 36 thermostat, 38a, 38b, 38c temperature sensor.

Claims (4)

A biological sample storage container for storing the biological sample attached to the carrier;
A medium perfusion system for perfusing a medium to the biological sample storage container;
A flow cell installed at the culture medium outlet of the biological sample storage container and incorporating an oxygen electrode;
An oxygen measuring device comprising: The oxygen measuring device according to claim 1,
An oxygen measuring apparatus comprising a bypass path for connecting a culture medium inlet side pipe and a culture medium outlet side pipe of the biological sample storage container.   A method for measuring oxygen, comprising: perfusing a culture medium into a biological sample storage container storing a biological sample attached to a carrier, and measuring a dissolved oxygen concentration of the culture medium after flowing out of the biological sample storage container. The oxygen measuring method according to claim 3,
A reference dissolved oxygen concentration is determined by measuring a dissolved oxygen concentration of the medium not passing through the biological sample storage container, and a change amount of the dissolved oxygen concentration of the medium after flowing out of the biological sample storage container with respect to the reference dissolved oxygen concentration is determined. An oxygen measuring method characterized by measuring the oxygen consumption by the biological sample by obtaining.

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