CN-116323902-B - Cell culture system and method for detecting proliferation of cells

CN116323902BCN 116323902 BCN116323902 BCN 116323902BCN-116323902-B

Abstract

The present invention provides a cell culture system capable of detecting the proliferation of cells in a culture vessel at a desired time during culture when the cells are cultured in a stationary state by using a culture vessel made of a gas permeable member. A cell culture system of the present invention is a cell culture system for culturing cells in a stationary state by using a culture vessel (20) at least a part of which is made of a gas permeable member, and comprises an oxygen concentration sensor (21) for measuring the oxygen concentration in the vicinity of a culture surface in the culture vessel (20), and a proliferation detection unit (631) for detecting the proliferation of cells based on the oxygen permeability of the gas permeable member, the measurement value of the oxygen concentration sensor, the oxygen concentration around the culture vessel (20), and the oxygen consumption per 1 cell.

Inventors

Kozai Osamu
TANAKA TAKASHI
TOTANI TAKAHIKO
Matsuya Yousuke
NISHIYAMA MASAHARU

Assignees

东洋制罐集团控股株式会社

Dates

Publication Date: 20260505
Application Date: 20211111
Priority Date: 20201113

Claims (6)

1. A cell culture system, characterized in that, The cell culture system cultures cells in a stationary state using at least a portion of a culture vessel made of a gas permeable member, The cell culture system has: an oxygen concentration sensor for measuring the oxygen concentration on the culture surface in the culture vessel, and A proliferation detection unit that detects proliferation of the cells based on the oxygen permeability of the gas permeable member, the measurement value of the oxygen concentration sensor, the oxygen concentration around the culture container, and the oxygen consumption per 1 cell, The proliferation detection unit detects the proliferation of the cells in culture in a stationary state, The oxygen concentration around the culture vessel is the oxygen concentration in the incubator in which the culture vessel is housed or the oxygen concentration in the atmosphere, The proliferation detection unit calculates the number of cells per unit area as the proliferation of the cells using the following formula (1): c=gx (M/100-D/100S. Formula (1) The number of cells per unit area is expressed in units of one/cm 2 , G the oxygen permeability of the breathable member, in units of mg/(cm 2 . Hr. Atm), M is the oxygen concentration around the culture vessel in units of, The unit of the measured value of the oxygen concentration sensor is, S, oxygen consumption per unit time of 1 cell, which is in mg/(hr·number).
2. The cell culture system of claim 1, wherein the cell culture system comprises a plurality of cells, The proliferation detection unit uses an estimated value of the oxygen concentration in the culture vessel at a desired time as a measurement value of the oxygen concentration sensor, The estimated value of the oxygen concentration is obtained based on a ratio of a slope of a change in an ascending peak of the oxygen concentration when a new medium is added to the culture vessel a plurality of times to a slope of a change in the oxygen concentration when the culture vessel is stabilized.
3. A cell culture system according to claim 1 or 2, wherein, The oxygen concentration sensor is fluorescent.
4. A cell culture system according to claim 1 or 2, wherein, The oxygen concentration sensor is fixed to a culture surface in the culture vessel using an open-cell resin member.
5. The cell culture system of claim 4, wherein the cell culture system comprises a plurality of cells, The culture vessel and the open-cell resin member comprise any one selected from the group consisting of polyethylene, polypropylene, a copolymer of ethylene and an alpha-olefin, a copolymer of ethylene and vinyl acetate, and an ionomer obtained using a copolymer of ethylene and acrylic acid and/or methacrylic acid and a metal ion.
6. A method for detecting proliferation of cells in cell culture, characterized by comprising the steps of, Is a method for detecting the proliferation of cells in cell culture in which cells are cultured in a stationary state by using a culture vessel at least a part of which is made of a gas permeable member, In the detection method, the oxygen concentration on the culture surface in the culture vessel is measured by an oxygen concentration sensor arranged on the culture surface in the culture vessel, Calculating the number of cells per unit area based on the oxygen permeability of the permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption per 1 cell, thereby detecting the proliferation of the cells in culture in a stationary state, The oxygen concentration around the culture vessel is the oxygen concentration in the incubator in which the culture vessel is housed or the oxygen concentration in the atmosphere, The proliferation detection unit calculates the number of cells per unit area as the proliferation of the cells using the following formula (1): c=gx (M/100-D/100S. Formula (1) The number of cells per unit area is expressed in units of one/cm 2 , G the oxygen permeability of the breathable member, in units of mg/(cm 2 . Hr. Atm), M is the oxygen concentration around the culture vessel in units of, The unit of the measured value of the oxygen concentration sensor is, S, oxygen consumption per unit time of 1 cell, which is in mg/(hr·number).

Description

Cell culture system and method for detecting proliferation of cells Technical Field The present invention relates to a cell culture technique, and more particularly to a cell culture system for detecting proliferation of cells when the cells are cultured in a stationary state using a culture vessel made of a gas-permeable member. Background In recent years, in the fields of production of pharmaceuticals, gene therapy, regenerative medicine, immunotherapy, and the like, it has been demanded to efficiently culture a large amount of cells, tissues, and the like in an artificial environment. Under such circumstances, it has been proposed to automatically culture a large amount of cells in a closed system using a bag-like culture vessel made of a gas-permeable member. In the mass culture of cells, it is important to detect the proliferation of cells and confirm whether or not the cells are properly cultured. In order to detect the proliferation of the cells, it is preferable to confirm the number of cells in the culture vessel at a desired time during the culture. The following 3 methods are examples of a method for confirming the number of cells in a culture vessel. (1) A part of the medium in the culture vessel is collected (sampled), and the number of cells is measured using a counting plate or the like. (2) The number of cells was measured by performing image processing on a photograph of cells in the culture vessel taken by a microscope and a camera. (3) The cell number was measured based on the decrease in dissolved oxygen in the culture vessel. Prior art literature Patent literature Patent document 1 Japanese patent laid-open No. 63-15150 Patent document 2 Japanese patent laid-open No. 6-121667 Disclosure of Invention Problems to be solved by the invention However, in the method (1), since a part of the culture vessel needs to be opened in order to sample the culture medium, there is a problem in that the risk of contamination increases. In addition, in the case of culturing adherent cells, there is a problem that only a part of the cells cannot be peeled off during the culture, and the number of cells cannot be measured at a desired timing. In the method (2), although the measurement can be performed when the culture density is small in the case of culturing suspension cells, there is a problem that the measurement becomes difficult because the cells are stacked when the cell density becomes high after the middle stage of the culture. In addition, since a microscope, a camera, and an image processing apparatus are required in this method, there is a problem in that the structure of the cell culture system becomes complicated. In the method (3), since it is necessary to measure the reduction in dissolved oxygen in the culture vessel, it is necessary to form a closed state so that the gas does not move from the inside of the culture vessel to the outside or the gas does not flow into the culture vessel from the outside. Or, when a space exists on the medium in the culture vessel, it is necessary to block the dissolution of oxygen into the medium by replacing the space with nitrogen gas or the like, and the amount of dissolved oxygen consumed by the cells in the medium is measured. However, in the case of performing cell culture using a culture vessel made of a gas permeable member, it is not possible to detect only a change in the concentration of dissolved oxygen consumed by cells in the culture medium, and it is necessary to measure the inside of the culture vessel with stirring, so that there is a problem in that stationary culture cannot be performed. Patent document 1 discloses a method of measuring the amount of dissolved oxygen in a sample solution containing microorganisms in a state where the sample solution is sealed in a container, but this measurement method needs to be performed using a culture container having no air permeability. Patent document 2 discloses a cell culture apparatus that calculates a time variation amount of oxygen amount associated with the number of cells in a culture vessel based on the oxygen amount measured by an oxygen amount measuring means that measures the amount of oxygen in a culture medium dissolved in the culture vessel. However, this cell culture apparatus requires a closed vessel in which gas does not flow from the outside into the culture vessel, and in order to change from a normal culture state to a state in which measurement is possible, it is necessary to replace the space above the medium in the culture vessel with nitrogen gas to block dissolution of oxygen into the medium, and by such an operation, the consumption of dissolved oxygen in the medium is measured after changing the culture conditions. The inventions described in these patent documents are not inventions that can be suitably used in the case of culturing cells in a stationary state using a culture vessel made of a gas permeable member. The present invention has been made in view of the above