# US 12,760,261 B2 · Method for the adaptive evolution of living cells by continuous cell culture

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,760,261 B2
- Document ID: US12760261B2
- Status: Granted
- Kind: B2
- Application number: 17/926,217
- Filed: 2021-05-19
- Published: 2023-06-15
- Granted: 2026-10-13
- Earliest claimed: 2020-05-21
- Assignees: Lesaffre Et Compagnie
- Applicants: Lesaffre et Compagnie
- Inventors: Philippe Marliere; Julien Patrouix; Simon Trancart
- Web page: https://patentgrep.com/patent/US12760261B2

## CPC

- C12M 35/00: {Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion (machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87)}
- C12M 23/58: {Reaction vessels connected in series or in parallel (combinations of bioreactors with other apparatus, C12M43/00)}
- C12M 27/16: {Vibrating; Shaking; Tilting}
- C12M 29/24: {Recirculation of gas}
- 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

The present application relates to a method for adaptive evolution of living cells by continuous culture of said living cells.

## Claims

### Claim 1

A method for adaptive evolution of living cells, excluding human embryonic stem cells, by continuously culturing said living cells, wherein n culture vessels (RCi) are used, i ranging from 1 to n, where n≥2, said method comprising:
a) introducing at least one liquid culture medium and living cells into each of the n culture vessels (RCi);
b) in each of the n culture vessels (RCi), culturing said living cells according to a given selective regime, using predefined culture parameters, until a determined growth stage is reached in at least one of the n culture vessels (RCi), so as to obtain, in each of the n culture vessels (RCi), a suspension of living cells in said liquid culture medium;
c) transferring:
at least a portion of the suspension of living cells obtained in step b) from at least two of the n culture vessels (RCi) to at least n−1 mixing vessels (RMj), j ranging from 1 to n−1, so as to perform a destination transfer, or
at least a portion of the suspension of living cells obtained in step b) from at least one of the n culture vessels (RCi) to a different one of the n culture vessels (RCi) so as to perform a destination transfer;
d) homogenizing the transferred suspensions of the destination transfer of step c) to obtain a homogenized suspension of mixed living cells;
e) transferring at least a portion of the homogenized suspension obtained in step d) into at least two of the n culture vessels (RCi) so as to perform a return transfer;
f) repeating the preceding steps b) to e) at least once by varying RCi and RMj so that each of the at least two of the n culture vessels (RCi) is mixed at least twice; and
g) collecting after step f) living cells that have acquired a phenotype of interest from at least one of the n culture vessels (RCi).

### Claim 2 (depends on 1)

The method according to claim 1, wherein the living cells are eukaryotic cells or prokaryotic cells.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the selective regime of step b) comprises chemostat conditions, turbidostat conditions, medium swap, or iterated batch.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the predefined culture parameters of step b) comprise temperature, pH, cell density, culture medium composition, gas composition, exposure to electromagnetic radiation of a particular wavelength, exposure to a mutagenic agent, or a combination thereof.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the destination transfer of step c) is carried out by transferring at least a portion of the suspension of living cells obtained in step b) from at least two of the n culture vessels (RCi) to the at least n−1 mixing vessels (RMj).

### Claim 6 (depends on 1)

The method according to claim 1, wherein step c) is carried out by transferring at least a portion of the suspension of living cells obtained in step b) from at least one of the n culture vessels (RCi) to a different one of the n culture vessels (RCi) so as to perform a destination transfer.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the homogenizing of the transferred suspensions in step d) comprises agitation.

### Claim 8 (depends on 1)

The method according to claim 1, wherein step e) consists of transferring at least a portion of the homogenized suspension of mixed living cells obtained in step d) to at least two culture vessels (RCi).

### Claim 9 (depends on 8)

The method according to claim 8, wherein at least a portion of the suspension transferred in step e) corresponds to a fraction between 1 and 100% of the volume of said homogenized suspension of mixed living cells.

### Claim 10 (depends on 1)

The method according to claim 1, wherein, when repeating step b), the selective regime and/or the predefined culture parameters used during a culture cycle are the same as those used during the preceding culture cycle.

### Claim 11 (depends on 5)

The method according to claim 5, wherein steps c) to f) in combination comprise:
i) transferring at least a portion of the suspension of living cells obtained in step b) from a culture vessel (RCi) to a mixing vessel (RMj), so as to perform a destination transfer;
ii) homogenizing the suspension transferred from the culture vessel (RCi) to the mixing vessel (RMj), to obtain a homogenized suspension of mixed living cells;
iii) transferring at least part of the homogenized suspension obtained in step ii) from the mixing vessel (RMj) to the culture vessel (RCi) from which transfer was made in step i), so as to perform a return transfer, wherein part of the homogenized suspension that is not transferred remains in the mixing vessel (RMj) for any subsequent homogenizing step ii);
iv) repeating the preceding steps i) to iii) n−(i+1) times while incrementing i by one unit at each repetition so as to transfer all or part of the suspension from the culture vessel (RCi) to a mixing vessel RMj+1 so as to perform n−(i+1) further destination transfers and n−(i+1) further return transfers;
v) repeating steps i) to iv) n−(i+1) times while incrementing i by one at each repetition so that the culture vessel becomes RCi+1 after each end of step iv).

### Claim 12 (depends on 5)

The method according to claim 5, wherein each of said at least n−1 mixing vessel (RMj) is independent of the at least two n culture vessels (RCi) and is arranged to receive the contents of the n culture vessels.

### Claim 13 (depends on 12)

The method according to claim 12, wherein steps c) to e) are carried out as follows:
c) transferring at least a portion of the suspension of living cells obtained in step b) from at least two culture vessels (RCi) to the at least one of said n−1 mixing vessel (RMj) to obtain a suspension of mixed living cells;
d) homogenizing the mixed suspension of living cells obtained in step c) in the at least one of said n−1 mixing vessel (RMj), to obtain a homogenized suspension of mixed living cells; and
e) transferring at least a portion of the suspension obtained in step d) from the at least one of said n−1 mixing vessel (RMj) to each of the at least two culture vessels (RCi).

### Claim 14 (depends on 1)

The method according to claim 1, wherein the living cells are human cells, animal cells, or plant cells.

### Claim 15 (depends on 5)

The method according to claim 5, wherein at least one mixing vessel is independent of the at least two culture vessels.

### Claim 16 (depends on 7)

The method according to claim 7, wherein the agitation is performed using a mechanical agitator or an injection of a gas stream.

### Claim 17 (depends on 1)

The method according to claim 1, wherein, when repeating step b), the selective regime and/or the predefined culture parameter used during a culture cycle are different from those used during the preceding culture cycle.

### Claim 18 (depends on 1)

The method of claim 1, wherein the selective regime of step b) comprises chemostat conditions, turbidostat conditions, medium swap, or iterated batch, and wherein the predefined culture parameters of step b) comprise temperature, pH, cell density, culture medium composition, gas composition, exposure to electromagnetic radiation of a particular wavelength, exposure to a mutagenic agent, or a combination thereof.

### Claim 19 (depends on 5)

The method of claim 5, wherein the selective regime of step b) comprises chemostat conditions, turbidostat conditions, medium swap, or iterated batch, and wherein the predefined culture parameters of step b) comprise temperature, pH, cell density, culture medium composition, gas composition, exposure to electromagnetic radiation of a particular wavelength, exposure to a mutagenic agent, or a combination thereof.

### Claim 20 (depends on 11)

The method of claim 11, wherein the selective regime of step b) comprises chemostat conditions, turbidostat conditions, medium swap, or iterated batch, and wherein the predefined culture parameters of step b) comprise temperature, pH, cell density, culture medium composition, gas composition, exposure to electromagnetic radiation of a particular wavelength, exposure to a mutagenic agent, or a combination thereof.