US-12624323-B2 - Multilayer culture vessel

US12624323B2US 12624323 B2US12624323 B2US 12624323B2US-12624323-B2

Abstract

A multilayer culture vessel includes: an internal space that is divided by a boundary portion into a culture space on one side and a buffer space on the other side in a direction parallel to a bottom plate; at least one intermediate plate extending along the direction parallel to the bottom plate at least in the culture space and configured to divide the culture space into culture layers; wall portions at the boundary portion, the wall portions including one extending from the bottom plate in a direction toward a top plate and one extending from each of the at least one intermediate plate in a direction toward the top plate; communication portions formed at the boundary portion to bring the culture layers into communication with the buffer space; and a liquid supply/drainage port formed in the housing at a location facing the buffer space.

Inventors

Daichi Horii
Haruki Takeuchi

Assignees

SINFONIA TECHNOLOGY CO., LTD.

Dates

Publication Date: 20260512
Application Date: 20200615
Priority Date: 20190617

Claims (8)

1 . A multilayer culture vessel capable of culturing cells in a plurality of culture layers, the multilayer culture vessel comprising: a housing including a bottom plate, a top plate facing the bottom plate, and a plurality of side walls, which includes at least a first side wall and a second side wall opposite to the first side wall, connecting the bottom plate and the top plate; a boundary portion that divides an internal space of the housing into a culture space on one side of the boundary portion and a buffer space on the other side of the boundary portion in a first direction parallel to the bottom plate; at least one intermediate plate extending along the direction parallel to the bottom plate at least in the culture space and configured to divide the culture space into the plurality of culture layers; a plurality of wall portions at the boundary portion, the plurality of wall portions including one extending from the bottom plate in a direction toward the top plate and one extending from each of the at least one intermediate plate in a direction toward the top plate; a plurality of communication portions formed at the boundary portion to bring the culture layers into communication with the buffer space; a liquid supply/drainage port formed on the top plate of the housing at a location facing the buffer space; and an exhaust port that is formed, separately from the liquid supply/drainage port, on the top plate at a location facing the culture space, wherein the liquid supply/drainage port is arranged adjacent to the first side wall and the exhaust port is arranged adjacent to the second side wall, and wherein the first side wall and the second side wall are opposite to each other in a second direction perpendicular to the first direction.
2 . The multilayer culture vessel of claim 1 , further comprising an inclined plate extending from an upper end of the wall portion formed on the bottom plate toward the side wall that is on the other side of the boundary portion while being inclined toward the top plate.
3 . The multilayer culture vessel of claim 2 , wherein the at least one intermediate plate extends to the boundary portion in the culture space.
4 . The multilayer culture vessel of claim 2 , wherein the at least one intermediate plate extends along the direction parallel to the bottom plate in both the culture space and the buffer space to divide the buffer space into a plurality of buffer layers, and wherein a through-hole penetrating the at least one intermediate plate is formed in the buffer space.
5 . The multilayer culture vessel of claim 1 , wherein the at least one intermediate plate extends to the boundary portion in the culture space.
6 . The multilayer culture vessel of claim 1 , wherein the at least one intermediate plate extends along the direction parallel to the bottom plate in both the culture space and the buffer space to divide the buffer space into a plurality of buffer layers, and wherein a through-hole penetrating the at least one intermediate plate is formed in the buffer space.
7 . A cell culture method by using the multilayer culture vessel of claim 1 , wherein the plurality of side walls includes a first sidewall placed on a side of the culture space, a second sidewall opposite to the first sidewall and placed on a side of the buffer space, a third sidewall connected to the first sidewall and the second sidewall, and a fourth sidewall opposite to the third sidewall, wherein the cell culture method comprises: placing the multilayer culture vessel in a storage posture with the second sidewall located on the bottom side; supplying a peeling liquid to the buffer space via the liquid supply/drainage port; placing the multilayer culture vessel in a first posture with the third sidewall located on the bottom side; placing the multilayer culture vessel in a second posture with the first sidewall located on the bottom side; placing the multilayer culture vessel in a third posture with the bottom plate located on the bottom side; and placing the multilayer culture vessel in the storage posture or the first posture so that the cells in the plurality of culture layers are discharged via the liquid supply/drainage port.
8 . The method of claim 7 , wherein a time period for which the multilayer culture vessel is in the third posture is longer than a time period for which the multilayer culture vessel is in the second posture.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national stage application of International Patent Application No. PCT/JP2020/023460, filed on Jun. 15, 2020, which claims priority to Japan Patent Application No. 2019-112230, filed on Jun. 17, 2019, both of which are hereby incorporated by reference in their entireties. TECHNICAL FIELD The present disclosure relates to a multilayer culture vessel capable of culturing cells in multiple culture layers. BACKGROUND There are several types of cell culture methods. The most common method is static culture in which cells are cultured while adhering to a culture vessel. Specifically, cells are brought into close contact with a bottom surface of a culture vessel such as a petri dish or a culture flask, a medium is added to a culture layer formed by the bottom surface and a side surface of the culture vessel, and cell culture is performed under predetermined environmental conditions. As the culture progresses, cells proliferate along the bottom surface of the culture vessel. Further, in recent years, in order to perform static culture for a large amount of cells, there has been developed a multilayer culture vessel that has a plurality of adherence surfaces to which cells can adhere and includes a plurality of culture layers formed therein (see FIG. 6). In the static culture, when performing subculture in which proliferated cells are transferred to a new culture vessel or when recovering cultured cells, it is necessary to peel the cells from the adherence surfaces with a peeling liquid. In particular, when cell peeling is performed on the multilayer culture vessel, it is required to supply a large amount of peeling liquid, and it is necessary to distribute the supplied peeling liquid to the respective culture layers inside the culture vessel by a predetermined procedure. For example, as shown in FIG. 6, a conventional multilayer culture vessel 201 includes a rectangular parallelepiped housing composed of a bottom plate 202, a top plate 203, and a side wall 204, a plurality of intermediate plates 205 configured to divide the internal space of the housing into a plurality of culture layers 221, a liquid supply/drainage part 206 configured to supply and drain a liquid to and from the multilayer culture vessel 201, and an exhaust port 207 configured to discharge a gas inside the housing. The liquid supply/drainage part 206 has a cylindrical shape in which the top plate 203 side is opened and which penetrates the plurality of intermediate plates 205 from the top plate 203 side to the bottom plate 202 side. A plurality of communication portions 214 in communication with the respective culture layers 221 is formed on the cylindrical side surface of the liquid supply/drainage part 206. Further, as shown in FIG. 6, the opening portion of the liquid supply/drainage part 206 is arranged at the corner of the top plate 203, and the exhaust port 207 is arranged at the corner adjacent to the arrangement position of the liquid supply/drainage part 206. The procedure for supplying a peeling liquid 30 to the conventional multilayer culture vessel 201 and peeling the cells will be described below with reference to FIGS. 7A to 7D. The hatched portion in FIGS. 7A to 7D is a region where the peeling liquid 30 exists. First, as shown in FIG. 7A, the multilayer culture vessel 201 is arranged so that the liquid supply/drainage part 206 is located on the bottom side with the exhaust port 207 located on the top side and the bottom plate 202 is perpendicular to a horizontal mounting surface (hereinafter referred to as first posture), and the medium inside the multilayer culture vessel 201 is drained from the liquid supply/drainage part 206. Subsequently, in the state in which the multilayer culture vessel 201 is arranged at the first posture, a predetermined amount of the peeling liquid 30 is supplied from the liquid supply/drainage part 206 to the inside of the multilayer culture vessel 201. After the peeling liquid 30 is supplied, as shown in FIG. 7B, the multilayer culture vessel 201 is re-arranged so that the liquid supply/drainage part 206 and the exhaust port 207 are located on the top side while the bottom plate 202 is kept perpendicular to the mounting surface (hereinafter referred to as second posture). As a result, the peeling liquid 30 flows into the respective culture layers 221 substantially evenly via the communication portions 214. Next, as shown in FIG. 7C, the multilayer culture vessel 201 is re-arranged so that the bottom plate 202 comes into contact with the mounting surface and the liquid supply/drainage part 206 and the exhaust port 207 are located on the top side (third posture). As a result, the peeling liquid 30 spreads uniformly along the bottom plate 202 and the intermediate plates 205, and the entire cells adhering to the bottom plate 202 and the intermediate plates 205 are immersed in the peeling liquid 30. Then, by leaving the multilayer culture vessel