Method for the adaptive evolution of living cells by continuous cell culture

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).