EP-4741495-A1 - CELL DETACHMENT METHOD AND CELL DETACHMENT APPARATUS

EP4741495A1EP 4741495 A1EP4741495 A1EP 4741495A1EP-4741495-A1

Abstract

To provide a cell detachment method and cell detachment device capable of designing a vibration in accordance with a target cell by mechanically matching a culture substrate with a vibrating body, and detaching an adherent cell at a high detachment rate. A cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, wherein the cell detachment method detaches the cells from the culture surface by vibrating a vibrating body in a state where an acoustic transmission medium is provided between the culture substrate and the vibrating body, wherein the acoustic transmission medium comprises a first acoustic transmission medium and a second acoustic transmission medium, wherein the first acoustic transmission medium is a solid having elasticity at room temperature, and the second acoustic transmission medium is encapsulated within the first acoustic transmission medium and is liquid at room temperature or has a tanδ greater than that of the first acoustic transmission medium when vibration of frequency 0.1 Hz is applied to the second acoustic transmission medium at room temperature.

Inventors

TAKEMURA, KENJIRO
KURASHINA, Yuta
IMASHIRO, CHIKAHIRO
TSUBAKI, KEIICHIRO
FURUI, Takaaki
OGUCHI, Yuichiro
NOGUCHI, KAZUNORI

Assignees

Canon Kabushiki Kaisha

Dates

Publication Date: 20260513
Application Date: 20240717

Claims (15)

A cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, wherein the cell detachment method detaches the cells from the culture surface by vibrating a vibrating body in a state where an acoustic transmission medium is provided between the culture substrate and the vibrating body, wherein the acoustic transmission medium comprises a first acoustic transmission medium and a second acoustic transmission medium, wherein the first acoustic transmission medium is a solid having elasticity at room temperature, and the second acoustic transmission medium is encapsulated within the first acoustic transmission medium and is liquid at room temperature or has a tanδ greater than that of the first acoustic transmission medium when vibration of frequency 0.1 Hz is applied to the second acoustic transmission medium at room temperature.
The cell detachment method according to claim 1, wherein the first acoustic transmission medium has at least one protrusion that is convex in a direction facing the second acoustic transmission medium.
The cell detachment method according to claim 1, wherein the first acoustic transmission medium has at least one column connected across the second acoustic transmission medium.
The cell detachment method according to any one of claims 1 to 3, wherein the first acoustic transmission medium comprises at least one selected from the group consisting of a silicone rubber, a natural rubber, a urethane gel, and a gel material.
The cell detachment method according to any one of claims 1 to 4, wherein the second acoustic transmission medium is a solid having a tanδ of 1.0 or more when vibration with a frequency of 0.1 Hz is applied at room temperature.
The cell detachment method according to any one of claims 1 to 5, wherein the second acoustic transmission medium is a liquid at room temperature.
The cell detachment method according to any one of claims 1 to 6, wherein the second acoustic transmission medium comprises at least one selected from the group consisting of a liquid, a polymer solution, and a gel material.
The cell detachment method according to any one of claims 1 to 7, wherein the first acoustic transmission medium comprises a silicone rubber and the second acoustic transmission medium comprises water.
The cell detachment method according to any one of claims 1 to 8, wherein a surface of the acoustic transmission medium facing the vibrating body has a higher Young's modulus than a surface of the acoustic transmission medium facing the culture substrate.
The cell detachment method according to any one of claims 1 to 9, further comprising an encapsulation step wherein the second acoustic transmission medium is encapsulated within the first acoustic transmission medium.
The cell detachment method according to any one of claims 1 to 10, wherein the vibrating body generates vibrations in an ultrasonic band.
The cell detachment method according to any one of claims 1 to 11, wherein the cell detachment method comprises a pressing step of pressing the culture substrate and the vibrating body to bring them close together.
A cell detachment device for detaching cells attached to a culture surface of a culture substrate by transmitting vibrations generated by a vibrating body to the cells, comprising a vibrating body, an acoustic transmission medium, and a culture substrate installation portion in this order, and configured so that vibrations of the vibrating body are transmitted to the culture substrate via the acoustic transmission medium, wherein the acoustic transmission medium comprises a first acoustic transmission medium and a second acoustic transmission medium, wherein the first acoustic transmission medium is a solid having elasticity at room temperature, and the second acoustic transmission medium is encapsulated within the first acoustic transmission medium and is liquid at room temperature or has a tanδ greater than that of the first acoustic transmission medium when vibration of frequency 0.1 Hz is applied to the second acoustic transmission medium at room temperature.
The cell detachment device according to claim 13, wherein the vibrating body comprises an ultrasonic vibrator.
The cell detachment device according to claim 13 or 14, further comprising a pressing unit for pressing the culture substrate and the vibrating body to bring them close together.

Description

[Technical Field] The present disclosure relates to a cell detachment method and a cell detachment device. [Background Art] In recent years, cell culture for culturing cells used in regenerative medicine and cells used for manufacturing biopharmaceuticals and the like has been actively conducted. Cell culture is broadly classified into adherent culture and suspension culture, of which adherent culture is suitable for various cell types and is widely used. However, when the cells are collected, it is necessary to perform enzyme treatment in order to detach the cells, and depending on the treatment method, the cells may be damaged. Therefore, cell detachment by enzyme treatment is also a cause of dispersion in cell quality. Therefore, detachment conditions for collecting cells at a high detachment rate without damaging the cells have been studied. In the case of cell detachment techniques using ultrasonic waves or acoustic radiation pressure, water is generally used as the acoustic transmission medium, and in this case, water tends to be particularly avoided in medical practice because of the strong concern about the propagation of bacteria. In Patent Literature 1, it is proposed that a patterning device for arranging cells in specific positions via a medium for transmitting acoustic radiation pressure to a culture substrate. Silicone rubber is described as an example of the acoustic transmission medium, but when the acoustic radiation pressure is transmitted to the culture substrate using this material, the cell detachment performance is not sufficient compared with water, and further improvement of the cell detachment performance and reduction of dispersion are required. Also in Patent Literature 2, a medical ultrasonic transmission device composed of a closed body is proposed. Although used for acoustic coupling between the ultrasonic probe and the target site, there is no description of using this ultrasonic transmission method to transmit ultrasonic vibrations and detach cells. [Citation List] [Patent Literature] PTL 1: Japanese Patent Laid-Open No. 2018-42534PTL 2: Japanese Patent No. 6325363 [Summary of Invention] [Technical Problem] The present disclosure has been made to solve the above problems, and provides a cell detachment method that enables vibration design in accordance with target cells and detachment of adherent cells at a high detachment rate. [Solution to Problem] The present disclosure provides a cell detachment method for detaching cells attached to a culture surface of a culture substrate from the culture surface, wherein the cell detachment method detaches the cells from the culture surface by vibrating a vibrating body in a state where an acoustic transmission medium is provided between the culture substrate and the vibrating body,wherein the acoustic transmission medium comprises a first acoustic transmission medium and a second acoustic transmission medium,wherein the first acoustic transmission medium is a solid having elasticity at room temperature,and the second acoustic transmission medium is encapsulated within the first acoustic transmission medium and is liquid at room temperature or has a tanδ greater than that of the first acoustic transmission medium when vibration of frequency 0.1 Hz is applied to the second acoustic transmission medium at room temperature. Also, the present disclosure provides a cell detachment device for detaching cells attached to a culture surface of a culture substrate by transmitting vibrations generated by a vibrating body to the cells, comprising a vibrating body,an acoustic transmission medium, anda culture substrate installation portion in this order,and configured so that vibrations of the vibrating body are transmitted to the culture substrate via the acoustic transmission medium,wherein the acoustic transmission medium comprises a first acoustic transmission medium and a second acoustic transmission medium,wherein the first acoustic transmission medium is a solid having elasticity at room temperature,and the second acoustic transmission medium is encapsulated within the first acoustic transmission medium and is liquid at room temperature or has a tanδ greater than that of the first acoustic transmission medium when vibration of frequency 0.1 Hz is applied to the second acoustic transmission medium at room temperature. [Advantageous Effects of Invention] According to the present disclosure, it is possible to provide a cell detachment method that enables vibration design in accordance with 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 showing an example of a configuration of a first embodiment of a cell detachment device according to the present disclosure.[Fig. 1B] Fig. 1B is a schematic cross-sectional view showing an example of the configuration of the first embodiment of the cell detachment device according to the present disclos