Cell culture apparatus and cell culture method

Patent metadata

Field	Value
Publication number	US-11141708-B2
Application number	US-201615562447-A
Country	US
Kind code	B2
Filing date	Mar 8, 2016
Priority date	Apr 3, 2015
Publication date	Oct 12, 2021
Grant date	Oct 12, 2021

Abstract

A cell culture apparatus, includes a connected culture container including n number of units disposed in parallel along a second direction that is a different direction from a first direction where each of the n number of the units is constituted of m number of culture chambers and one or more communication-channels, the m number of the culture chambers each having a cell-holding portion that holds seeded cells, the m number of the culture chambers storing liquid culture media, the m number of the culture chambers being disposed in parallel along the first direction, the communication-channels communicating the m number of the culture chambers with each other; and a plurality of pneumatic pipes communicating same-row-disposed culture chambers that are disposed on a same row along the second direction with each other in the connected culture container.

First claim

The invention claimed is:

1. A cell culture apparatus, comprising:
a connected culture container comprising n number of units, where each of the n number of the units consists of m number of culture chambers and one or more communication-channels, wherein each of the culture chambers comprises a cell-holding portion that holds seeded cells,
wherein the m number of the culture chambers are disposed in parallel along a first direction and communicating with each other by the one or more communicating-channels,
wherein each of the culture chambers is configured to store liquid culture media,
wherein the n number of units are disposed in parallel along a second direction that is different from the first direction,
wherein m is an integer of two or more, and n is an integer of two or more,
a plurality of pneumatic pipes communicating same-row-disposed culture chambers that are disposed on a same row along the second direction with each other in the connected culture container, the pneumatic pipes being configured to pressurize the same-row-disposed culture chambers at the same time or open the same-row-disposed culture chambers to atmospheric pressure at the same time, the pneumatic pipes being configured to send the liquid culture media to each of the culture chambers through the communication-channels using pressure differences in each of the culture chambers, and
further comprising a Laplace valve configured at portions connected to the one or more communication-channels and the culture chambers, to prevent flows of air using interface tension to the communication-channels from the culture chambers.

2. The cell culture apparatus according to claim 1 , further comprising:
a backward flow prevention mechanism configured to control flow directions from the communication-channels toward the m number of the culture chambers, the backward flow prevention mechanism being provided at an end of the communication-channels or in the communication-channels,
wherein the backward flow prevention mechanism is
a check valve,
a backward flow prevention Laplace valve,
a division portion dividing the culture chamber with a portion provided with a channel introduction opening of a communication-channel of the one or more communication-channels and a portion provided with the LaPlace valve, or
a first channel introduction opening of a communication-channel of the one or more communication-channels disposed at a higher location than an inlet of the other communication-channels.

3. The cell culture apparatus according to claim 1 ,
wherein each of the m number of the culture chamber comprises a chamber structure formed in a container-shaped tank main body storing liquid culture media and a lid portion configured to open an opening-portion of the tank main body, or to close air-tightly the opening-portion of the tank main body.

4. The cell culture apparatus according to claim 3 , further comprising:
a lid portion-pressing portion configured to hold the lid portion so as to be pressed toward the tank main body,
wherein the lid portion-pressing portion comprises a base body portion that supports the tank main body and a pressing member that presses the lid portion toward the tank main body supported by the base body portion and
further comprising a pressing member configured to hold the lid portion so as to be pressed toward the tank main body,
wherein the pressing member is
a clasp, or
a base body portion that supports the tank main body and a pressing member that presses the lid portion toward the tank main body supported by the base body portion.

5. The cell culture apparatus according to claim 4 ,
wherein the tank main body comprises a wall portion and a bottom plate including the communication-channels.

6. The cell culture apparatus according to claim 5 , further comprising:
a wall portion-pressing portion that holds the wall portion so as to be pressed toward the bottom plate,
wherein the wall portion-pressing portion comprises a pressing member that presses the wall portion toward the base body portion and the bottom plate supported by the base body portion.

7. The cell culture apparatus according to claim 1 ,
wherein, when a width of a microchannel constituting the Laplace valve is represented by w L , a depth of the microchannel is represented by h L , the interface tension is represented by γ, and a Laplace pressure of the Laplace valve is represented by ΔP Lap ,
the Laplace pressure is expressed by an expression of ΔP Lap =2γ(1/w L +1/h L ), and
the microchannel is constituted so that the Laplace pressure ΔP Lap becomes larger than a pressure applied to the connected culture container.

8. The cell culture apparatus according to claim 1 , further comprising:
a resistance channel portion having a channel cross-sectional area that is 1/10 or less of a channel cross-sectional area of a communication-channel in a part of the communication-channels in order to adjust flow rates.

9. A cell culture method, comprising:
preparing the cell culture apparatus according to claim 1 ,
a first step of opening a lid of the connected culture container and seeding and adhering cells in each of the culture chambers in the cell culture apparatus;
a second step of filling first row-culture chambers in the first direction with a liquid culture medium and closing the lid in the cell culture apparatus where the first row-culture chambers are disposed on a same row along the second direction;
a third step of pressurizing an inside of the first row-culture chambers and opening second row-culture chambers in the first direction which are disposed on a same row along the second direction to atmospheric pressure, thereby sending the liquid culture medium to the second row-culture chambers from the first row-culture chambers through the communication-channels;
a fourth step of sequentially pressurizing the culture chambers and opening the culture chambers to atmospheric pressure in ascending order of row numbers in the first direction, thereby sending the liquid culture medium from the m-1 th row-culture chambers in the first direction to the m th row-culture chambers through the communication-channels;
a fifth step of pressurizing an inside of the m th row-culture chambers and opening an inside of the first row-culture chambers to atmospheric pressure, thereby sending the liquid culture medium from the m th row-culture chambers to the first row-culture chambers through the communication-channels; and
a sixth step of repeating the third step through the fifth step, thereby circulating the liquid culture medium in each of the culture chambers in the cell culture apparatus.

Assignees

• Aist

Inventors

• Sugiura Shinji
• Satoh Taku
• Kanamori Toshiyuki
• SHIN Kazumi

Classifications

• B01F33/30

  Micromixers · CPC title

• B01L2300/0874

  Three dimensional network · CPC title

• B01L3/502738 Primary

  characterised by integrated valves (throttle valves in microfluidic sample containers B01L3/502746) · CPC title

• C12M29/10

  Perfusion · CPC title

• B01L2400/0688

  surface tension valves, capillary stop, capillary break · CPC title