# US 9,434,924 B2 · Cell culture method and cell culture kit

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- Number: US 9,434,924 B2
- Document ID: US9434924B2
- Status: Granted
- Kind: B2
- Application number: 14/113,050
- Filed: 2012-04-20
- Published: 2014-03-27
- Granted: 2016-09-06
- Earliest claimed: 2011-04-21
- Assignees: Nipro Corporation
- Applicants: Yoshihiro Yoshikawa; Naomi Morikawa; Ryo Tomii
- Inventors: Yoshihiro Yoshikawa; Naomi Morikawa; Ryo Tomii
- Web page: https://patentgrep.com/patent/US9434924B2

## CPC

- C12N 5/0656: {Adult fibroblasts}
- C12M 23/14: {Bags}
- C12M 33/00: {Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus (chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00)}
- C12N 2509/10: Mechanical dissociation
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## Abstract

Provided is a means suitable for mass culture of adherent cells and which allows easy and safe collection of the cultured adherent cells. A cell culture kit has a culture bag having flexibility, such a level of hydrophilicity that the culture of adherent cells can be achieved, and permeability into a bag shape and which is filled with a culture medium; a dissociation solution bag filled with a dissociation solution containing a metal chelating agent or ornithine or a derivative thereof, and a solution sending circuit or a derivative thereof, and a solution sending circuit which connects the culture bag and the dissociation solution bag to each other and can send the dissociation solution from the dissociation solution bag to the culture bag.

## Claims

### Claim 1

A cell culture method of culturing adherent cells, comprising:
holding a liquid culture medium in a culture bag which is formed by shaping at least one sheet having flexibility and permeability into a bag shape, the sheet having an inner cell-contacting surface having hydrophilicity sufficient to promote a culture of cells that adhere to the inner cell-contacting surface, whereby upon sowing, the cells adhere directly to the inner cell-contacting surface having hydrophilicity;
incubating the cells;
discharging the culture medium from the culture bag, and then filling the culture bag with a dissociation solution which does not contain trypsin, the dissociation solution containing ornithine or a derivative of ornithine selected from N-[amino(hydroxyimino)methyl]-ornithine or a structural isomer thereof, N-acetyl-ornithine, N-acyl-ornithine, N-(L-carboxyethyl)-ornithine, N-succinyl-ornithine, N,N-dibenzoyl-ornithine, (difluoromethyl)ornithine and N-[(hydroxy) (imino)methyl]-ornithine, the dissociation solution filling the culture bag through a solution sending circuit which connects the culture bag and a bag holding the dissociation solution to each other;
bending the at least one sheet of the culture bag filled with the dissociation solution to dissociate the adherent cells from the inner cell-contacting surface; and
collecting the adherent cells dissociated from the inner cell-contacting surface.

### Claim 2 (depends on 1)

The cell culture method according to claim 1, wherein the dissociation solution further contains EDTA.

### Claim 3 (depends on 1)

The cell culture method according to claim 1, wherein in the step of bending the at least one sheet, the sheet is bent at an environmental temperature lower than an environmental temperature of the culture bag during said incubating.

### Claim 4 (depends on 1)

The cell culture method according to claim 1, wherein the sheet of the culture bag comprises a polyolefin resin layer on the inner cell-contacting surface that is imparted with hydrophilicity by subjecting the polyolefin resin layer to plasma treatment.

### Claim 5 (depends on 1)

The cell culture method according to claim 1, wherein the sheet of the culture bag comprises polyolefin resin layer that includes a cyclic polyolefin polymer on at least the inner cell-contacting surface that is imparted with hydrophilicity by subjecting the polyolefin resin layer to plasma treatment.

### Claim 6 (depends on 1)

The cell culture method according to claim 1, the inner cell-contacting surface is comprised of a synthetic resin layer having a cell-adhesive functional group.