EP-4741489-A1 - CELL DETACHMENT METHOD AND CELL DETACHMENT DEVICE

EP4741489A1EP 4741489 A1EP4741489 A1EP 4741489A1EP-4741489-A1

Abstract

Provided are a cell detachment method and a cell detachment device that enable vibration design suitable for target cells and detachment of adherent cells at a high detachment rate. The cell detachment method of detaching cells adhering to a culture surface of a culture substrate from the culture surface includes a detachment step of detaching the cells from the culture surface by vibrating a vibrating body under a state in which a first acoustic transmission medium and a second acoustic transmission medium are arranged between the culture substrate and the vibrating body, and under a state in which at least one of the following (a) or (b) is satisfied: (a) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; or (b) the vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium. In the cell detachment method, the first acoustic transmission medium and the second acoustic transmission medium are different from each other, the first acoustic transmission medium being a solid at room temperature, the second acoustic transmission medium being a liquid or a solid with flowability at room temperature.

Inventors

TAKEMURA, KENJIRO
KURASHINA, Yuta
IMASHIRO, CHIKAHIRO
TSUBAKI, KEIICHIRO
FURUI ,Takaaki
NAKAMOTO, ATSUSHI
NOGUCHI, KAZUNORI

Assignees

Canon Kabushiki Kaisha

Dates

Publication Date: 20260513
Application Date: 20240716

Claims (20)

A cell detachment method of detaching cells adhering to a culture surface of a culture substrate from the culture surface, the cell detachment method comprising a detachment step of detaching the cells from the culture surface by vibrating a vibrating body under a state in which a first acoustic transmission medium and a second acoustic transmission medium are arranged between the culture substrate and the vibrating body, and under a state in which at least one of the following (a) or (b) is satisfied: (a) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; or (b) the vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium, wherein the first acoustic transmission medium and the second acoustic transmission medium are different from each other, the first acoustic transmission medium being a solid at room temperature, the second acoustic transmission medium being a liquid or a solid with flowability at room temperature.
The cell detachment method according to claim 1, wherein the detachment step includes detaching the cells from the culture surface by vibrating the vibrating body under a state in which the (a) is satisfied.
The cell detachment method according to claim 1 or 2, wherein the detachment step includes detaching the cells from the culture surface by vibrating the vibrating body under a state in which the (a) is satisfied, and under a state in which the vibrating body is in contact with the first acoustic transmission medium.
The cell detachment method according to claim 2 or 3, wherein the following expression is satisfied in the detachment step: 0.05≤S1/S1+S2, where S1 is an area of a portion in which the culture substrate is in contact with the first acoustic transmission medium, and S2 is an area of a portion in which the culture substrate is in contact with the second acoustic transmission medium.
The cell detachment method according to claim 1, wherein the detachment step includes detaching the cells from the culture surface by vibrating the vibrating body under a state in which the (b) is satisfied.
The cell detachment method according to claim 5, wherein the following expression is satisfied in the detachment step: 0.05≤S3/S3+S4, where S3 is an area of a portion in which the vibrating body is in contact with the first acoustic transmission medium, and S4 is an area of a portion in which the vibrating body is in contact with the second acoustic transmission medium.
The cell detachment method according to any one of claims 1 to 6, the following expression is satisfied: tanδ 1 <tanδ 2 , where tanδ 1 and tanδ 2 are a tanδ of the first acoustic transmission medium and a tanδ of the second acoustic transmission medium, respectively, obtained by dynamic viscoelasticity measurement at room temperature and a frequency of 1 Hz.
The cell detachment method according to any one of claims 1 to 7, wherein the second acoustic transmission medium is a solid having a tanδ of 1.0 or more obtained when a vibration having a frequency of 0.1 Hz is applied at room temperature.
The cell detachment method according to any one of claims 1 to 8, wherein the second acoustic transmission medium is a liquid at room temperature.
The cell detachment method according to any one of claims 1 to 9, wherein the second acoustic transmission medium is a liquid, and wherein a contact angle between the first acoustic transmission medium and the second acoustic transmission medium is 120° or less.
The cell detachment method according to claim 10, wherein the second acoustic transmission medium is a liquid, and wherein the contact angle between the first acoustic transmission medium and the second acoustic transmission medium is 90° or less.
The cell detachment method according to any one of claims 1 to 11, wherein the second acoustic transmission medium is a liquid, and wherein at least one of a contact angle at a contact surface between the culture substrate and the second acoustic transmission medium or a contact angle at a contact surface between the vibrating body and the second acoustic transmission medium is less than 40°.
The cell detachment method according to any one of claims 1 to 12, wherein the first acoustic transmission medium includes, in at least one of a surface facing the culture substrate or a surface facing the vibrating body, a continuous groove portion extending from a central portion to an outer peripheral portion of the at least one of the surfaces.
The cell detachment method according to any one of claims 1 to 13, wherein the detachment step includes setting a volume ratio of the second acoustic transmission medium to the first acoustic transmission medium to 0.05 or more and 2.0 or less.
The cell detachment method according to any one of claims 1 to 14, wherein the first acoustic transmission medium includes silicone rubber.
The cell detachment method according to any one of claims 1 to 15, wherein the first acoustic transmission medium includes a porous resin material.
The cell detachment method according to any one of claims 1 to 16, wherein the second acoustic transmission medium includes at least one type selected from the group consisting of water, physiological saline, ethanol, and diluted ethanol.
The cell detachment method according to any one of claims 1 to 17, wherein the detachment step includes a step of exciting vibration in an ultrasonic band in the vibrating body.
The cell detachment method according to any one of claims 1 to 18, further comprising an arrangement step of arranging the first acoustic transmission medium and the second acoustic transmission medium between the culture substrate and the vibrating body before the detachment step.
The cell detachment method according to claim 19, wherein the arrangement step includes arranging any one of the first acoustic transmission medium or the second acoustic transmission medium so as to be in contact with any one of the vibrating body or the culture substrate based on a state of vibration to be excited in the vibrating body in the detachment step.

Description

[Technical Field] The present disclosure relates to a cell detachment method and a cell detachment device. [Background Art] In recent years, cell culturing in which cells to be used in regenerative medicine and cells to be used in the manufacture of biopharmaceuticals and the like are cultured has been actively performed. Cell culturing is roughly classified into adhesion culture and suspension culture, and of those, adhesion culture is a culturing method suitable for various cell types and is widely used. However, at the time of collecting cells, it is required to perform enzyme treatment to detach the cells, and in some treatment methods, the cells may be damaged. Thus, cell detachment through enzyme treatment is also a cause of variation in cell quality. For that reason, studies have been made on detachment conditions for collecting cells at a high detachment rate without damaging the cells. In Patent Literature 1, there is proposed a patterning device that arranges cells at specific positions through a medium that transmits acoustic radiation pressure to a culture substrate. However, a liquid such as water is used as the acoustic transmission medium, and hence there is a risk of contamination due to the liquid of the vibration transmission medium entering the inside of the culture substrate when vibration is applied, depending on the amount of the acoustic transmission medium used. In addition, when silicone rubber is used as the acoustic transmission medium, vibration generated by a vibration generation source may not be successfully transmitted to the culture substrate, or the vibration may change. [Citation List] [Patent Literature] PTL 1: Japanese Patent Laid-Open No. 2018-42534 [Summary of Invention] [Technical Problem] The present disclosure has been made to address the above-mentioned problems, and is directed to providing a cell detachment method and a cell detachment device that enable vibration design suitable for target cells and detachment of adherent cells at a high detachment rate. [Solution to Problem] According to the present disclosure, there is provided a cell detachment method of detaching cells adhering to a culture surface of a culture substrate from the culture surface, the cell detachment method including a detachment step of detaching the cells from the culture surface by vibrating a vibrating body under a state in which a first acoustic transmission medium and a second acoustic transmission medium are arranged between the culture substrate and the vibrating body, and under a state in which at least one of the following (a) or (b) is satisfied: (a) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; or(b) the vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium, wherein the first acoustic transmission medium and the second acoustic transmission medium are different from each other, the first acoustic transmission medium being a solid at room temperature, the second acoustic transmission medium being a liquid or a solid with flowability at room temperature. According to the present disclosure, the following cell detachment device is also provided. A cell detachment device for detaching cells from a culture surface of a culture substrate by transmitting vibration generated by a vibrating body to the cells provided on the culture surface, the cell detachment device including: a vibrating body;a first acoustic transmission medium; anda culture substrate placement portion, in the stated order, andthe cell detachment device further including supply unit for supplying a second acoustic transmission medium,wherein the supply unit is configured to supply the second acoustic transmission medium so that at least one of the following (a) or (b) is satisfied: (a) the culture substrate is in contact with the first acoustic transmission medium and the second acoustic transmission medium; or(b) the vibrating body is in contact with the first acoustic transmission medium and the second acoustic transmission medium. [Advantageous Effects of Invention] According to the present disclosure, it is possible to provide the cell detachment method that enables vibration design suitable for target cells and detachment of adherent cells at a high detachment rate. [Brief Description of Drawings] [Fig. 1A] Fig. 1A is a schematic cross-sectional view for illustrating an example of a configuration of a cell detachment device according to a first embodiment of the present disclosure.[Fig. 1B] Fig. 1B is a schematic cross-sectional view for illustrating an example of the configuration of the cell detachment device according to the first embodiment of the present disclosure.[Fig. 1C] Fig. 1C is a schematic cross-sectional view for illustrating an example of the configuration of the cell detachment device according to the first embodiment of the present disclosure.[Fig. 2A] Fig. 2