A method for adaptively evolving living cells through continuous cell culture.

Abstract

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

Description

[Technical Field]

[0001] This invention relates to a method for adaptively evolving living cells through continuous cell culture of living cells. [Background technology]

[0002] Live cells are often used to produce, for example, food, animal feed, fragrances, cosmetics, fuels, chemicals, and health products. Live cells such as microalgae, fungi, yeasts, and bacteria have several advantages, including small cell size, short generation time, and relatively low culture costs. However, the conditions required for the efficient production of products are often different from the optimal conditions for the growth and survival of the cells used in industrial processes.

[0003] The manufacturing, decomposition, or recycling of products requires high-performance living cells obtained under artificial conditions, representing a compromise between the need for living cells and the conditions required for product manufacturing. Various approaches exist to improve the desired performance of living cells, including those involving genetic modification. However, several objections have been raised regarding genetically modified living cells and their use, including concerns about safety and their impact on other organisms, including humans. Furthermore, there is currently no model that can perfectly predict which genetic modification will yield living cells with the desired phenotype.

[0004] Therefore, alternative approaches are becoming increasingly important. These alternative approaches, for example, are based on non-genetically improving living cells by utilizing the potential for naturally occurring genetic mutations in microbial populations, in order to obtain industrially noteworthy variants without the knowledge of mutations necessary to achieve the desired phenotype, while adhering to regulations that prohibit the use of genetically modified organisms.

[0005] Genetic variation is constantly being generated, for example, through random mutation and sexual reproduction. Therefore, living cells in a population will have different degrees of adaptation to a given environment, which forms the basis of selection. Selection refers to the tendency for beneficial phenotypic traits to increase over time in a given population of cells, and these beneficial traits increase the likelihood of the cells surviving, mating, and / or reproducing. Conversely, the frequency of phenotypic traits that reduce the likelihood of the cells surviving, mating, and / or reproducing may decrease, or even be eliminated from the population. Thus, even as the environment changes, a population can adapt to each environment over time, accumulating advantageous, i.e., beneficial phenotypic traits and eliminating harmful, i.e., negatively impactful phenotypic traits, based on the genetic variation present within the population. The potential for genetic variation is utilized in artificial selection, for example, by selecting phenotypic traits desirable for intended uses, such as the efficient and commercial production of a product. However, implementing effective artificial selection is difficult.

[0006] In particular, if the selective pressure is too high, the genetic diversity that emerges through spontaneous mutation may be limited. Therefore, artificial selection faces the significant challenge of selecting the optimal selective pressure parameters to effectively achieve the target phenotype. Furthermore, in most cases, the selection of multiple phenotypic traits is required, such as phenotypic traits related to improved growth rate, adaptation to growth media with different compositions compared to the reference medium, adaptation to temperature, and adaptation to inhibitors present at specific concentrations and / or nutrients. To sequentially select multiple phenotypic traits, that is, to improve phenotypic traits one after another, it is necessary to go through intermediate stages specialized for a particular phenotypic trait, and from there, artificial selection to other traits is difficult, cumbersome, or impossible. Artificial selection can overcome this obstacle by improving several phenotypic traits in parallel, or simultaneously.

[0007] Under conditions optimized for low-cost and / or high-yield industrial production, there is a need for alternative solutions to efficiently and automatically select living cells in a suspended state that have acquired the desired phenotype, i.e., several traits simultaneously.

[0008] US2012 / 0263690 describes a method for artificially selecting a microorganism that has acquired a single phenotypic trait selected from among heat resistance, chemical resistance, and ultraviolet resistance, among other things.

[0009] International Application WO2009 / 112739 describes a method for selecting desired cells that transiently secrete an enzyme and grow in suspension, wherein the cells are maintained at a cell density value known to be a determining factor for the secretion of the desired enzyme.

[0010] Patent EP1135 describes a method for growing cells in a chemostat or turbidostat under a single selective condition.

[0011] The paper by Mans et al. (Mans et al. Current Opinion in Biotechnology, 2018, vol 50, pages 47 - 56) describes various methods for increasing the production of fuels and chemicals through the adaptive evolution of yeast strains. There are methods of sequentially transferring yeast from one container to another, or methods of continuously culturing in the same container. Furthermore, this paper points out that adaptive evolution generally occurs through a trade - off between the selected phenotypic trait and other physiological aspects of the organism. On the other hand, there remains a risk of selecting non - constitutive phenotypic traits, that is, traits that are induced by a selection pressure but cease to be expressed when the application of that selection pressure ceases. To avoid these difficulties, the paper by Mans et al. emphasizes the need for a dynamic evolution process in which the organism is exposed to different selective conditions. In particular, when transferring from one container to another, a strategy of alternately introducing media is explained. However, this strategy involves exploring one evolutionary pathway while sequentially changing the selective conditions.

[0012] International application WO2018 / 152442 describes an adaptive evolution method for liquid culture of microorganisms in a programmable continuous culture system, which involves exposing a microbial culture to a dynamic environment, exposing the culture to a stress ramp function superimposed on a culture fitness function, and then increasing the amount of stress applied to the microbial culture in response to an increase in the fitness of the microbial culture.

[0013] However, conventionally proposed methods are time-consuming, cumbersome to implement, and, most importantly, they fail to provide rapid and efficient access to microbial populations with complex phenotypes, particularly those requiring the simultaneous improvement of several phenotypic traits.

[0014] Therefore, it seems necessary to develop new methods for the adaptive evolution of living cells that enable the rapid and efficient acquisition of living cells that have acquired the desired phenotype. [Prior art documents] [Patent Documents]

[0015] [Patent Document 1] International Publication No. 2018 / 152442

[0016] [Non-Patent Document 1] Mans et al.Current Opinion in Biotechnology, 2018, vol 50, pages 47~56 [Non-Patent Document 2] Doring et al.ACS Synthetics Biology, 2018, vol 7(9), pages 2029~2036 [Overview of the project]

[0017] The present invention aims to provide a method for adaptively evolving living cells, excluding human embryonic stem cells, by continuous culture of the living cells in n culture vessels (RCi), wherein i is in the range of 1 to n, n ≥ 2, and the method is a) A step of introducing at least one liquid culture medium and living cells into each of the n culture vessels, b) In each of the n culture vessels, using predetermined culture parameters based on a given selective condition, the living cells are cultured in at least one of the n culture vessels until a determined growth stage is reached, and a suspension of the living cells in the liquid medium is obtained in each of the n culture vessels. c) A step of mixing at least a portion of the suspension of living cells from at least two culture vessels (RCi) obtained in step b) to obtain a mixed suspension of living cells. d) A step of homogenizing the mixed suspension of living cells obtained in step c) to obtain a homogenized suspension of mixed living cells. e) Distributing at least a portion of the homogenized suspension of mixed living cells obtained in step d) into at least two culture vessels (RCi), f) A process that repeats steps b) to e), g) A step of culturing viable cells that have acquired the desired phenotype in at least one of the n culture vessels for several cycles and then harvesting them. This method is characterized by including a feature that allows living cells to adapt and evolve.

[0018] The inventors have surprisingly demonstrated that the present method allows for the selection and collection of viable cells with a desired phenotype by applying selective conditions in parallel and multiple times. The present method is preferable because it saves time compared to the simple or sequential use of these selective conditions. In particular, by repeating steps b) through e), suspensions of viable cells taken from at least two different culture vessels can be sequentially mixed and redistributed into new culture vessels, facilitating the gradual selection of viable cells that respond best to the selective conditions applied during the culture cycle, thereby achieving a population of viable cells that have acquired the desired phenotype. Thus, the present method increases the number of evolutionary pathways that the suspension of viable cells can take, improving the probability and speed of the emergence of the desired phenotype.

[0019] This invention addresses, in particular, the need for efficient evolution of living cells, which is especially well-suited to suspension culture conditions and optimized for product production, degradation, or recycling.

[0020] The present invention is particularly based on obtaining live cells well adapted to target conditions by frequently mixing fractions of live cell suspensions from different culture vessels cultured in parallel under gradually increasing selective pressure based on different selective conditions and / or culture parameters.

[0021] For the purposes of this invention, “continuous culture” means the culture of living cells carried out in at least one liquid medium, wherein a portion of the medium is replaced in order to continue growing the living cells for a long period of time. Advantageously, the living cells are maintained for a number of generations not predetermined, advantageously more than 2 generations, advantageously more than 3 generations, advantageously more than 4 generations, advantageously more than 5 generations, advantageously more than 6 generations, advantageously more than 7 generations, advantageously more than 8 generations, advantageously more than 9 generations, advantageously more than 10 generations, advantageously more than 20 generations, advantageously more than 30 generations, advantageously more than 40 generations, advantageously more than 50 generations, advantageously more than 60 generations, advantageously more than 70 generations, advantageously more than 80 generations, advantageously more than 90 generations, advantageously more than 100 generations, advantageously more than 150 generations, advantageously more than 200 generations, advantageously more than 250 generations, advantageously more than 300 generations, advantageously more than 130 generations, advantageously more than 40 generations. It is advantageous for over 0 generations, advantageous for over 450 generations, advantageous for over 500 generations, advantageous for over 550 generations, advantageous for over 600 generations, advantageous for over 650 generations, advantageous for over 700 generations, advantageous for over 750 generations, advantageous for over 800 generations, advantageous for over 850 generations, advantageous for over 900 generations, advantageous for over 950 generations, advantageous for over 1000 generations, advantageous for over 5000 generations, advantageous for over 10000 generations, advantageous for over 15000 generations, advantageous for over 20000 generations, advantageous for over 25000 generations, advantageous for over 30000 generations, advantageous for over 35000 generations, advantageous for over 40000 generations, advantageous for over 45000 generations, and advantageous for over 50000 generations.

[0022] The culture medium, or its components (diluents), may be replaced permanently, regularly, or periodically. The medium may be replaced with respect to one or more components of its composition, or with respect to the entire mixture of these components. The medium is replaced in such a way that at least 0.01% of the cells in culture are retained. Advantageously, at least 0.1% of the cells in culture are in a suspension state, advantageously at least 1%, advantageously at least 2%, advantageously at least 3%, advantageously at least 4%, advantageously at least 5%, advantageously at least 6%, advantageously at least 7%, advantageously at least 8%, advantageously at least 9%, advantageously at least 10%, advantageously at least 11%, advantageously at least 12%, advantageously at least 13%, advantageously at least 14%, advantageously at least 15%, advantageously at least 16%, advantageously at least 17%, advantageously at least 18%, advantageously at least 19%, advantageously at least 20%, advantageously at least 21%, advantageously at least 22%, advantageously at least 23%, advantageously at least 24%, advantageously at least 25%, advantageously at least 26%, advantageously at least 27%, advantageously at least 28%, advantageously at least 29%, advantageously at least 30%, advantageously at least 31%, advantageously at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%,To have an advantage is at least 66%, to have an advantage is at least 67%, to have an advantage is at least 68%, to have an advantage is at least 69%, to have an advantage is at least 70%, to have an advantage is at least 71%, to have an advantage is at least 72%, to have an advantage is at least 73%, to have an advantage is at least 74%, to have an advantage is at least 75%, to have an advantage is at least 76%, to have an advantage is at least 77%, to have an advantage is at least 78%, to have an advantage is at least 79%, to have an advantage is at least 80%, to have an advantage is at least 81%, to have an advantage is at least 82%, to have an advantage is at least 83% The culture medium is refreshed so that, advantageously, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the cultured cells are preserved.

[0023] For the purposes of this invention, "living cells that have acquired a desired phenotype" or "cell mutants" means daughter cells that do not possess the same physiological characteristics as parent cells grown under the same conditions.

[0024] For the purposes of this invention, a “method for adaptive evolution” is a process in which a population of living cells is exposed to selective conditions, accumulating favorable physiological changes to facilitate the acquisition of a desired phenotype. These physiological changes may be caused by genetic recombination (point mutation, loss or acquisition of genetic material) or epigenetic modification, or by stress or other factors that can have a permanent effect on the behavior of living cells in culture.

[0025] The present invention does not require these physiological changes to be predetermined. Rather, the object of the present invention is to promote the emergence of living cells that have acquired a desired phenotype without prior knowledge of the genetic and physiological modifications leading to that phenotype, and then to collect the living cells that have acquired the desired phenotype, which have a competitive advantage over other cells, such as being particularly tolerant of stress, growing under given conditions, growing faster under given conditions, making better use of the culture medium, or giving other characteristics that meet industrial standards.

[0026] For the purposes of this invention, "selective pressure" means stress caused by solubilized or gaseous toxic compounds, stress caused by a deficiency of solubilized or gaseous essential compounds, stress caused by rising temperature, stress caused by falling temperature, stress caused by rising pH, stress caused by falling pH, exposure to electromagnetic waves of specific wavelengths, particularly ultraviolet light, exposure to infrared light, exposure to electromagnetic waves of wavelengths lethal to cells, exposure to mutagenic substances, or a combination of these stresses, which can lead to, for example, decreased productivity due to an increase in the doubling time of living cells, decreased yield due to a decrease in the production of living cells, or the demise of living cells.

[0027] For the purposes of this invention, “cell,” “living cell,” or “population of living cells” means one or more small biological entities consisting of membrane-bound cytoplasm and possessing the ability to regenerate autonomously. Advantageously, living cells may be eukaryotes or prokaryotes, humans, animals, or plants, with the exception of human embryonic stem cells. Microorganisms are considered to be cells. Advantageously, living cells are selected from mammalian cells, insect cells, bacteria, yeasts, microalgae, plant cells, fungi, and microorganisms. For the purposes of this invention, “microalgae” means single-celled algae. Advantageously, “living cell” means eukaryotes or prokaryotes, humans, animals, or plants, with the exception of human embryonic stem cells, that may be infected with viruses, phages, parasites, and / or have one or more desired plasmids incorporated into them.

[0028] For the purposes of this invention, "culture vessel," "culture chamber," or "culture tank" means a vessel for culturing living cells in a culture medium based on a given selective condition, and in particular, a manner in which culture conditions are set based on operating rules based on given culture parameters until a given growth stage is reached. In certain embodiments, the culture vessel is recyclable. In certain embodiments, the culture vessel is disposable.

[0029] For the purposes of this invention, "suspension" means a liquid culture medium containing living cells.

[0030] According to the present invention, step c), which consists of mixing at least a portion of the suspension of living cells from at least two culture vessels (Ri) obtained in step b), can be carried out by using one of the at least two culture vessels as a mixing vessel, or by enabling a mixing vessel independent of the at least two culture vessels to contain all or part of the contents of the at least two culture vessels.

[0031] For the purposes of this invention, “mixing vessel” or “mixing tank” means a vessel in which the contents of at least two culture vessels are mixed. Therefore, the mixing vessel contains at least a suspension from the first culture vessel and a suspension from the second culture vessel. Advantageously, the method of this invention may include the same number of mixing vessels as the number of culture vessels. Advantageously, the number of mixing vessels may be varied in the range of 1 to n. In a particular embodiment, the number of mixing vessels is at least 2. In a particularly advantageous embodiment, the number of mixing vessels is equal to 3. In another particularly advantageous embodiment, the number of mixing vessels is equal to 4. In another particularly advantageous embodiment, the number of mixing vessels is equal to 5. In another particularly advantageous embodiment, the number of mixing vessels is equal to 6. In another particularly advantageous embodiment, the number of mixing vessels is equal to 7. In another particularly advantageous embodiment, the number of mixing vessels is equal to 8. In another particularly advantageous embodiment, the number of mixing vessels is equal to 9. In another particularly advantageous embodiment, the number of mixing vessels is equal to 10.

[0032] In certain embodiments, the mixing container is recyclable. In certain embodiments, the mixing container is disposable.

[0033] In step a), at least 1 volume of liquid medium and a specific amount of live cells are introduced into each of the n culture vessels. Advantageously, the specific amount of live cells is at least 1 live cell, at least 10 live cells, advantageously at least 20 live cells, advantageously at least 30 live cells, advantageously at least 40 live cells, advantageously at least 50 live cells, advantageously at least 60 live cells, advantageously at least 70 live cells, advantageously at least 80 live cells, advantageously at least 90 live cells, advantageously at least 100 live cells, advantageously at least 200 live cells, advantageously at least 300 live cells, advantageously at least 400 live cells, advantageously at least 500 live cells, advantageously at least 600 live cells, advantageously at least 700 live cells, advantageously at least 800 live cells, advantageously at least 900 live cells, advantageously at least 1000 live cells, advantageously at least 10 4 live cells, advantageously at least 10 5 live cells, advantageously at least 10 6 live cells, advantageously at least 10 7 live cells, advantageously at least 10 8 live cells, advantageously at least 10 9 live cells, advantageously at least 10 10 live cells, advantageously at least 10 11 live cells, advantageously at least 10 12 1]live cells, meaning at least 10

[0034] In a particular embodiment of the invention, at least one medium contains nutrients essential for the growth of live cells. A person skilled in the art will know at least one medium to be used, and in particular the way of adapting its composition, based on the type of live cells. Advantageously, the at least one medium is a fresh and sterile medium.

[0035] In a particular embodiment of the present invention, at least one culture medium and live cells are introduced into each of the n culture vessels via a supply means, such as a pump and valve connected by a line, which allows the culture vessel to be connected to each of the n culture vessels.

[0036] In step b), the determined growth stage can be reached at the end of a predetermined period, which is related to cell density, or physical / chemical indicators measurable in the culture medium, such as pH, fluorescence, radioactivity, concentration of generated molecules, concentration of consumed molecules such as nutrients, presence of toxic substances, dilution ratio, number of stress medium injections, and elapsed time between two medium injections.

[0037] This "determined growth stage" may be based on the target phenotype, or on a typical growth curve established in advance from experimental or literature data.

[0038] In certain embodiments, cell density can be measured by optical measurement.

[0039] In particularly advantageous embodiments, specific values ​​of measurable physical / chemical indicators are set, such as a threshold cell density, a specific dilution ratio, the number of injections of stress medium, the elapsed time between two injections of medium, a specific pH, a specific temperature, a specific gas composition for agitating the suspension, a specific fluorescence, a specific radioactivity, electromagnetic waves or radioactive radiation at a specific intensity and frequency, a specific concentration of generated molecules, a specific nutrient concentration, a specific growth factor concentration, or a specific toxic substance concentration.

[0040] In this method, after seeding in the culture medium, the viable cells grow to a set value. This critical value is reached in at least one culture vessel, and all or part of the culture, i.e., the suspension consisting of the culture medium and viable cells, is transferred to at least one mixing vessel.

[0041] In another specific embodiment, the determined growth stage may be related to a predetermined duration. The predetermined duration may be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, or 1 The incubation period can be fixed at 15 minutes, advantageously 120 minutes, advantageously 3 hours, advantageously 4 hours, advantageously 5 hours, advantageously 6 hours, advantageously 7 hours, advantageously 8 hours, advantageously 9 hours, advantageously 10 hours, advantageously 11 hours, advantageously 12 hours, advantageously 13 hours, advantageously 14 hours, advantageously 15 hours, advantageously 16 hours, advantageously 17 hours, advantageously 18 hours, advantageously 19 hours, advantageously 20 hours, advantageously 21 hours, advantageously 22 hours, advantageously 23 hours, or advantageously 24 hours, but this list is not limiting.

[0042] Advantageously, the determined growth stages described above are: 1 minute after culturing, advantageously 2 minutes after culturing, advantageously 3 minutes after culturing, advantageously 4 minutes after culturing, advantageously 5 minutes after culturing, advantageously 6 minutes after culturing, advantageously 7 minutes after culturing, advantageously 8 minutes after culturing, advantageously 9 minutes after culturing, advantageously 10 minutes after culturing, advantageously 15 minutes after culturing, advantageously 20 minutes after culturing, advantageously 25 minutes after culturing, advantageously 30 minutes after culturing, advantageously 35 minutes after culturing, advantageously 40 minutes after culturing, advantageously 45 minutes after culturing, advantageously 50 minutes after culturing, advantageously 55 minutes after culturing, advantageously 60 minutes after culturing, advantageously 65 minutes after culturing, advantageously 70 minutes after culturing, advantageously 75 minutes after culturing, advantageously 80 minutes after culturing, advantageously 85 minutes after culturing, advantageously 90 minutes after culturing, advantageously 95 minutes after culturing, advantageously 100 minutes after culturing. It can reach 5 minutes, 110 minutes to an advantage, 115 minutes to an advantage, 120 minutes to an advantage, 3 hours to an advantage, 4 hours to an advantage, 5 hours to an advantage, 6 hours to an advantage, 7 hours to an advantage, 8 hours to an advantage, 9 hours to an advantage, 10 hours to an advantage, 11 hours to an advantage, 12 hours to an advantage, 13 hours to an advantage, 14 hours to an advantage, 15 hours to an advantage, 16 hours to an advantage, 17 hours to an advantage, 18 hours to an advantage, 19 hours to an advantage, 20 hours to an advantage, 21 hours to an advantage, 22 hours to an advantage, 23 hours to an advantage, and 24 hours to an advantage, but this list is not exhaustive.

[0043] In this method, after seeding the culture medium, the living cells grow for a predetermined period. When this culture time is reached, all or part of the culture, i.e., the suspension consisting of the culture medium and living cells, is transferred to at least one mixing vessel.

[0044] In a particular embodiment of the method of the present invention, in step b), living cells are cultured in a selective condition, the selective condition being selected from a chemostat, a turbidostat, a medium swap, and a repeating batch.

[0045] For the purposes of this invention, “chemostat conditions” or “chemostat” means a type of cell culture in which a single medium, preferably sterile, containing at least one essential nutrient in limited quantities is used to dilute cells, and the flow rate of the medium is constant. Advantageously, the medium is supplied to the culture vessel continuously, i.e., without interruption, at a predetermined rate, to dilute the cells while keeping the volume of the vessel constant. Alternatively, a predetermined amount of medium is supplied to the culture vessel at regular intervals to dilute the cells.

[0046] For the purposes of this invention, "constant interval" means every 30 seconds, preferably every 35 seconds, preferably every 40 seconds, preferably every 45 seconds, preferably every 50 seconds, preferably every 55 seconds, preferably every 1 minute, preferably every 2 minutes, preferably every 3 minutes, preferably every 4 minutes, preferably every 5 minutes, preferably every 10 minutes, preferably every 15 minutes, preferably every 20 minutes, preferably every 25 minutes, and preferably every 30 minutes.

[0047] For the purposes of this invention, “turbidstat conditions” or “turbidstat” means a type of cell culture in which a single medium, preferably sterile, containing an excess of all essential nutrients is used to dilute the cells, and the flow rate of the medium is constant. Advantageously, the medium is supplied to the culture vessel continuously, i.e., without interruption, at a variable rate, to dilute the cells while keeping the volume of the vessel constant and maintaining the turbidity at the threshold. Alternatively, the cultured cell density is measured at regular intervals in at least one culture vessel and compared to the threshold. If the measured cell density is less than the threshold, the culture continues until the next interval is completed. If the measured cell density is greater than the threshold, a predetermined amount of medium is supplied to the culture vessel to dilute the cells.

[0048] For the purposes of this invention, "culture medium swap" refers to a type of cell culture condition in which two different, preferably sterile, culture media, called tolerance medium and stress medium, are used, the volume of the culture vessel is kept constant, and the media is added semi-continuously to dilute the cells. In this case, the cultured cell density is measured at regular intervals in at least one culture vessel and compared to the threshold. If the measured cell density is less than the threshold, a predetermined amount of tolerance medium is sent to the culture vessel. If the measured cell density is greater than the threshold, a predetermined amount of stress medium is sent to the culture vessel. Culture medium swap can be performed as described in Doring et al. (ACS Synthetics Biology, 2018, vol 7(9), pages 2029~2036).

[0049] In certain embodiments of the present invention, at least one culture medium may be a so-called "acceptable" medium or a so-called "stress" medium. An "acceptable medium" is a medium that is perfectly suited to cell growth, containing nutrients and growth factors essential for cell growth, and not containing or presenting in non-lethal amounts to the cells any toxic substances that cause cell death, such as cell growth inhibitors. A "stress medium" is a medium that contains nutrients essential for cell growth, but contains toxic substances in amounts that cause cell death, such as cell growth inhibitors, or does not contain growth factors essential for cell growth, or presents toxic substances in amounts that are not negligible, or lacks growth factors essential for cell growth, or is not a preferred substrate for the cells.

[0050] For the purposes of this invention, “repeated batch” means a type of cell culture condition in which cells are diluted with a culture medium, preferably sterile. In this case, cells grow in at least one culture vessel without diluting the medium until a predetermined cell density is reached. Once the cell density is reached, the suspension is maintained for a predetermined culture time. After the time has elapsed since the cell density threshold was reached in the culture vessel, a fixed amount of sterile medium is delivered to the culture vessel. In an alternative embodiment, after the time has elapsed since the cell density threshold was reached in the culture vessel, a portion of the suspension from the culture vessel is delivered to at least one different culture vessel that already contains growth medium and is preferably sterile.

[0051] In certain embodiments, in step b), the living cells are cultured under chemostat-selective conditions.

[0052] In certain embodiments, in step b), the living cells are cultured under turbidostat-selective conditions.

[0053] In certain embodiments, in step b), the living cells are cultured under a medium-swap selective condition.

[0054] In certain embodiments, in step b), the living cells are cultured under repeated batch-selective conditions.

[0055] In a particularly advantageous embodiment, the selective conditions used in step b) of the method of the present invention may be the same for all n culture vessels.

[0056] In a particularly advantageous embodiment, the selective conditions used in step b) of the method of the present invention may differ from each other in each culture vessel. In a particular embodiment of the present invention, different selective conditions can be used in culture step b) among n culture vessels. Advantageously, two different selective conditions can be used among n culture vessels, advantageously three selective conditions, advantageously four selective conditions, or advantageously five selective conditions.

[0057] In a particular embodiment of the method of the present invention, in step b), living cells are cultured in at least one of n culture vessels under the selective conditions defined above, using predefined culture parameters, until a determined growth stage is reached, to obtain a suspension. In a particular embodiment of the method of the present invention, the predefined culture parameters in step b) are selected from dilution ratio, temperature, pH, cell density, culture medium composition, gas flow composition, exposure to electromagnetic waves of a specific wavelength, exposure to mutagenic substances, or a combination thereof.

[0058] According to the present invention, "different selective conditions" means either selective conditions that have the same properties but different intensities, or selective conditions that have different properties regardless of their intensity. For example, if the selective condition is pH, it may take different values ​​for each culture vessel. According to another embodiment, if living cells are exposed to a specific selective condition, such as the effect of pH, in a given culture vessel, and living cells are exposed to another specific selective condition, such as the effect of temperature, in another culture vessel, then the selective conditions are different.

[0059] For the purposes of this invention, "exposure to electromagnetic waves of a specific wavelength" means exposure to electromagnetic waves of visible wavelengths (longer than 400 nm and shorter than 800 nm), exposure to ultraviolet rays (shorter than 400 nm), exposure to infrared rays (longer than 800 nm), and exposure to electromagnetic waves of wavelengths that cause cell death.

[0060] For the purposes of this invention, "mutagenic substance" means a chemical substance that causes insertional, deletional, or substitutional mutations in the genome of a cell. Advantageously, the mutagenic substance can be selected from alkylating agents such as N-nitroso-N-ethylurea (also known as N-ethyl-N-nitrosourea (ENU)) or ethyl methanesulfonate (also known as ethyl methanesulfonate (EMS)), intercalating agents such as proflavin or acridine orange, and reactive oxygen species such as free radicals, oxygen ions, and peroxides.

[0061] In particularly advantageous embodiments, the selective conditions used in step b) of the method of the present invention may be the same in all n culture vessels.

[0062] In particularly advantageous embodiments, the selective conditions used in step b) of the method of the present invention may differ from each other in each of the culture vessels.

[0063] In a particularly advantageous embodiment, the culture parameters used in step b) of the present invention may be the same in all n culture vessels.

[0064] In particularly advantageous embodiments, the culture parameters used in step b) of the method of the present invention may differ from each other in each culture vessel.

[0065] For the remainder of this explanation, all culture vessels (RCi) will be denoted as follows: - T0i, the initial temperature used in step b) of the culture vessel RCi, - pH(i,k), initial pH used in step b) of culture vessel RCi, - DO(i,k), initial cell density used in step b) of culture vessel RCi, - Composition of at least one initial medium used in step b) of MC(i, k) and culture vessel RCi, - G(i,k), composition of the initial gas flow used in step b) of culture vessel RCi, - λ(i,k), exposure to electromagnetic waves of a specific initial wavelength used in step b) of the culture vessel RCi, - Td(i, k), initial dilution ratio used in step b) of culture vessel RCi, and - Initial exposure to mutagenic material used in step b) of AM(i, k) and culture vessel RCi.

[0066] Advantageously, in step b), the cells are cultured in all n culture vessels in at least one medium having a temperature T0i, a pH equal to pH(i,k), a cell density DOk(i,k), and a composition MC(i,k), in the presence of a gas with a dilution ratio Td(i,k) and composition G(i,k).

[0067] According to certain embodiments, the culture method can provide a gas flow being injected into the suspension under pressure by a gas supply device, for example, in the form of an aeration rod introduced into the culture vessel. This injection of gas flow allows for aeration of the suspension, homogenization of the suspension (agitation by bubbling), and maintenance of a constant gas pressure within the culture vessel. Advantageously, this method allows a sterile gas flow to pass through the culture vessel. This gas flow may consist of a gas selected from air, nitrogen (N2), carbon monoxide (CO), carbon dioxide (CO2), methane (CH4), hydrogen sulfide (H2S), oxygen (O2), nitrous oxide (N2O), hydrogen (H2), or mixtures thereof.

[0068] In certain embodiments, the culture method may provide means for mechanically agitating the suspension in a culture vessel.

[0069] In step c), as soon as the growth stage determined in step b) is reached, all or part of the suspension contained in at least one culture vessel is mixed with all or part of the suspension contained in at least one other culture vessel. As described above, the mixing of the suspensions can be carried out in one of the at least two culture vessels, which functions as a mixing vessel, the contents of which are sufficient to accommodate all or part of the suspension coming from at least one other culture vessel, or in at least one independent mixing vessel having an internal volume to accommodate all or part of each suspension from the at least two culture vessels.

[0070] In a particular embodiment of the present invention, all or part of the suspensions contained in at least three culture vessels, preferably all or part of the suspensions contained in at least four culture vessels, or preferably all or part of the suspensions contained in at least n culture vessels, are transferred to at least one mixing vessel.

[0071] In a particular embodiment of the present invention, in step c), only portions of each suspension contained in at least two culture vessels are mixed together. Advantageously, for the purposes of the present invention, “part of the suspension” is at least 1% of the suspension contained in the culture vessel, advantageously at least 2% of the suspension contained in the culture vessel, advantageously at least 3%, advantageously at least 4%, advantageously at least 5%, advantageously at least 6%, advantageously at least 7%, advantageously at least 8%, advantageously at least 9%, advantageously at least 10%, advantageously at least 11%, advantageously at least 12%, advantageously at least 13%, advantageously at least 14%, advantageously at least 15%, advantageously at least 16%, advantageously at least 17%, advantageously at least 18%, advantageously at least 19%, advantageously at least 20%, advantageously at least 21%, advantageously at least 22%, advantageously at least 23%, advantageously at least 24%, advantageously at least 25%, advantageously at least 26%, advantageously at least 27%, advantageously at least 28%, advantageously at least 29%, advantageously at least 30%, advantageously at least 31%, advantageously at least 32%, advantageously at least 33% To the advantage is at least 34%, to the favor is at least 35%, to the favor is at least 36%, to the favor is at least 37%, to the favor is at least 38%, to the favor is at least 39%, to the favor is at least 40%, to the favor is at least 41%, to the favor is at least 42%, to the favor is at least 43%, to the favor is at least 44%, to the favor is at least 45%, to the favor is at least 46%, to the favor is at least 47%, to the favor is at least 48%, to the favor is at least 49%, to the favor is at least 50%, to the favor is at least 51%, To have an advantage is at least 52%, to have an advantage is at least 53%, to have an advantage is at least 54%, to have an advantage is at least 55%, to have an advantage is at least 56%, to have an advantage is at least 57%, to have an advantage is at least 58%, to have an advantage is at least 59%, to have an advantage is at least 60%, to have an advantage is at least 61%, to have an advantage is at least 62%, to have an advantage is at least 63%, to have an advantage is at least 64%, to have an advantage is at least 65%, to have an advantage is at least 66%, to have an advantage is at least 67%, to have an advantage is at least 68%, to have an advantage is at least 69%,This is understood to mean at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

[0072] In another embodiment of the present invention, step c) is performed using a mixing vessel, and at least a portion of the suspension obtained in step b) is transferred from at least two culture vessels to at least one mixing vessel. In this embodiment, the fractions of the suspension transferred from each culture vessel to at least one mixing vessel may be the same or different.

[0073] In an advantageous embodiment of the present invention, at least one mixing vessel is an arranged culture vessel whose internal volume is sufficient to accommodate the contents of at least two culture vessels. Advantageously, at least one mixing vessel is a culture vessel arranged to accept the contents of at least three culture vessels, advantageously at least four culture vessels, or advantageously at least n culture vessels. Advantageously, “contents of at least one culture vessel” means the suspension obtained in step b) that is present in at least one culture vessel. Advantageously, “contents of at least two culture vessels” means the suspension obtained in step b) that is present in at least two culture vessels.

[0074] In an advantageous embodiment of the present invention, at least one mixing vessel is a single vessel, independent of the set of culture vessels, and is arranged to receive the contents of the set of culture vessels.

[0075] In a particular embodiment of the present invention, if step c) includes the operation of transferring the suspension to at least one mixing vessel, the transfer of all or part of the suspension from each of the at least two culture vessels to at least one mixing vessel is carried out by a transfer means, which can consist in particular of one or more pumps, one or more valves, one or more gas flow injectors, one or more pipetting robots, or a combination thereof.

[0076] Advantageously, the pump may be mechanically driven or, advantageously, automatically controlled electrically or electronically using control means. Advantageously, the pump may be a peristaltic pump driven by a control device, advantageously driven by an automaton or computer.

[0077] Advantageously, the valve can be driven mechanically or, advantageously, automatically, electrically or electronically using control means. Advantageously, the valve can be a solenoid valve driven by a control device, advantageously driven by an automaton or computer.

[0078] Advantageously, the gas flow can be injected from, for example, a gas supply device located in the culture vessel, or from an external gas source.

[0079] In certain embodiments of the present invention, if step c) includes a transfer operation, the transfer is carried out using one or more pumps and one or more valves.

[0080] In another specific embodiment of the present invention, if step c) includes a transfer operation, the transfer is carried out using one or more valves and one or more gas streams, for example, gas streams generated by a pressure difference.

[0081] In certain embodiments of the present invention, if step c) includes a transfer operation, the transfer is performed using one or more pipette robots.

[0082] Step d) of this method consists of homogenizing the mixture obtained from mixing all or part of the suspensions of living cells from at least two culture vessels in step c). According to the present invention, the homogenization step yields a suspension of living cells in which the distribution of cells from each of the at least two culture vessels in the volume of the mixed vessel is random.

[0083] In certain embodiments of the present invention, the homogenization step d) is carried out in whole or in part by stirring means selected from mechanical stirring and injection of a gas stream.

[0084] In certain embodiments of the present invention, the homogenization step d) is carried out in whole or in part by the injection of a gas stream, which is injected under pressure into the container containing the mixed cell suspension of step c) by a gas supply device. This injection of the gas stream allows for homogenization of the suspension mixture by bubbling agitation (e.g., using the principle of airlift). Advantageously, this gas stream may consist of a gas selected from air, nitrogen (N2), carbon monoxide (CO), carbon dioxide (CO2), methane (CH4), hydrogen sulfide (H2S), oxygen (O2), nitrous oxide (N2O), hydrogen (H2), or a mixture of these gases, depending on the selected culture parameters.

[0085] Preferably, step e) of this method comprises transferring at least a portion of the homogenized suspension of mixed living cells obtained in step d) to at least two culture vessels (RCi).

[0086] In a particularly advantageous embodiment of the present invention, at least a portion of the suspension transferred in step e) corresponds to a fraction between 1 and 100% of the homogenized suspension of mixed living cells.

[0087] Advantageously, the fraction of the volume of the homogenized suspension of mixed living cells transferred to step e) is at least 1% of the total volume of the homogenized suspension, advantageously at least 2%, advantageously at least 3%, advantageously at least 4%, advantageously at least 5%, advantageously at least 6%, advantageously at least 7%, advantageously at least 8%, advantageously at least 9%, advantageously at least 10%, advantageously at least 11%, advantageously at least 12%, advantageously at least 13%, advantageously at least 14%, advantageously at least 15%, advantageously At least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, and at least a small Both 35%, at least 36% to favor, at least 37% to favor, at least 38% to favor, at least 39% to favor, at least 40% to favor, at least 41% to favor, at least 42% to favor, at least 43% to favor, at least 44% to favor, at least 45% to favor, at least 46% to favor, at least 47% to favor, at least 48% to favor, at least 49% to favor, at least 50% to favor, at least 51% to favor, at least 52% to favor, at least 53% to favor, at least 5 4%, at least 55% to favor, at least 56% to favor, at least 57% to favor, at least 58% to favor, at least 59% to favor, at least 60% to favor, at least 61% to favor, at least 62% to favor, at least 63% to favor, at least 64% to favor, at least 65% to favor, at least 66% to favor, at least 67% to favor, at least 68% to favor, at least 69% to favor, at least 70% to favor, at least 71% to favor, at least 72% to favor, at least 73% to favor,The advantages are at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 100%.

[0088] In another embodiment of step e) of the method of the present invention, when at least a portion of the suspension obtained in step d) is transferred to n culture vessels, the fractions of the homogenized suspension of mixed living cells transferred to each of the n culture vessels may be the same or different.

[0089] In a particular embodiment of the present invention, the method comprises n culture vessels (RCi) where i is in the range of 1 to n and n is equal to at least 2, and j is in the range of 1 to n-1 and at least n-1 mixing vessels (RMj), wherein each of the at least n-1 mixing vessels is arranged to receive the contents of at least two culture vessels, and the method comprises steps c) to e), i) Transferring all or part of the suspension obtained in step b) from a culture vessel called a culture start vessel (RCi) to a mixing vessel called a destination vessel (RMj) is performed to carry out the transfer to the destination. ii) In the destination container (RMj), homogenize the suspension in the culture initiation container (RCi) with the suspension in the destination container (RCj) to obtain a homogenized suspension of mixed living cells. iii) Transfer at least a portion of the suspension obtained in step ii) from the destination container (RMj) to the initial culture container (RCi), and perform a return transfer. iv) The method is further characterized by repeating steps i) to iii) above, while varying RCi and RMj, so that all suspensions are mixed together in pairs at least once.

[0090] In a particularly advantageous embodiment, n is 3. In another particularly advantageous embodiment, n is 4. In yet another particularly advantageous embodiment, n is 5. In yet another particularly advantageous embodiment, n is 6. In yet another particularly advantageous embodiment, n is 7. In yet another particularly advantageous embodiment, n is 8. In yet another particularly advantageous embodiment, n is 9. In yet another particularly advantageous embodiment, n is 10.

[0091] Advantageously, the present invention includes three culture vessels, a first culture vessel RC1, a second culture vessel RC2, and a third culture vessel RC3, the three culture vessels function sequentially as mixing vessels RM1, RM2, and RM3, and each culture vessel is arranged to receive the contents of the other two culture vessels, and the method comprises steps c) to e), i) Transfer the suspension of living cells from the first culture vessel RC1 to the second culture vessel RC2, which also functions as the first mixing vessel RM1, and then transfer it to the destination. ii) Homogenize the suspensions from the first culture vessel RC1 and the second culture vessel RC2 in the second culture vessel RC2, which functions as the first mixing vessel RM1 (RC2=RM1), to obtain a homogenized suspension of mixed viable cells. iii) Transfer at least a portion of the suspension from the first mixing container RM1 to the first culture container RC1, and perform a return transfer. iv) Transfer the suspension from the third culture vessel RC3 to the first culture vessel RC1, which also functions as the second mixing vessel RM2, and then transfer it to the destination. v) Homogenize the suspensions from the third culture vessel RC3 and the first culture vessel RC1 in the first culture vessel RC1, which functions as the second mixing vessel RM2 (RC1=RM2), to obtain a homogenized suspension of mixed live cells. vi) Transfer at least a portion of the suspension from the second mixing container RM2 to the third culture container RC3, and perform a return transfer. vii) Transfer the suspension from the second culture vessel RC2 to the third culture vessel RC3, which also functions as the third mixing vessel RM3, and then transfer it to the destination. viii) Homogenize the suspensions from the second culture vessel RC2 and the third culture vessel RC3 in the third culture vessel RC3, which functions as the third mixing vessel RM3 (RC3=RM3), to obtain a homogenized suspension of mixed living cells. ix) This is characterized by performing a return transfer by transferring at least a portion of the suspension from the third mixing container RM3 to the second culture container RC2.

[0092] In another specific embodiment of the present invention, the method comprises n culture vessels (RCi) where i is in the range of 1 to n and n is equal to at least 2, and j is in the range of 1 to n-1 and at least n-1 mixing vessels (RMj), each of which at least n-1 mixing vessels is a culture vessel (RCi) arranged to receive the contents of at least two culture vessels, and steps c) to e) are i) Transferring all or part of the suspension of living cells obtained in step b) from a culture vessel called a culture initiation vessel (RCi) to a mixing vessel called a destination vessel (RMj) is performed to carry out the transfer to the destination. ii) In the destination container (RMj), homogenize the suspension from the culture initiation container (RCi) with the suspension in the destination container (RMj) to obtain a homogenized suspension of mixed live cells. iii) Transfer at least a portion of the suspension obtained in step ii) from the destination container (RMj) to the starting culture container (RCi), and perform the return transfer. iv) Transfer all or part of the suspension from the culture starter container (RCi) to the transfer destination container RMj+1, and then perform n-(i+1) transfers to the culture destination and n-(i+1) return transfers, by increasing j by 1 unit with each iteration, and repeating steps i) to iii) n-(i+1) times. The process is characterized by repeating steps i) through iv) n-(i+1) times, with i increasing by 1 in each iteration, so that the number of culture starting vessels becomes RCi+1 at the end of step v).

[0093] In a particularly advantageous embodiment, n is 3. In another particularly advantageous embodiment, n is 4. In yet another particularly advantageous embodiment, n is 5. In yet another particularly advantageous embodiment, n is 6. In yet another particularly advantageous embodiment, n is 7. In yet another particularly advantageous embodiment, n is 8. In yet another particularly advantageous embodiment, n is 9. In yet another particularly advantageous embodiment, n is 10.

[0094] Advantageously, the present invention includes three culture vessels, namely a first culture vessel RC1, a second culture vessel RC2, and a third culture vessel RC3, wherein the second and third culture vessels (RC2 and RC3) function sequentially as mixing vessels RM1 and RM2, and each culture vessel RC2 and RC3 is arranged to receive the contents of the two culture vessels, and the method is such that steps c) to e) i) Transfer the suspension of viable cells obtained in step b) from the first culture vessel RC1 to the second culture vessel RC2, which also functions as the first mixing vessel RM1, thereby performing a transfer to the new culture location. ii) Homogenize the suspensions from the first culture vessel RC1 and the second culture vessel RC2 in the culture vessel RC2, which functions as the first mixing vessel RM1, to obtain a homogenized suspension of mixed living cells. iii) Transfer at least a portion of the suspension from the first mixing container RM1 to the first culture container RC1 and perform a return transfer. iv) Transfer the suspension from the first culture vessel RC1 to the third culture vessel RC3, which also functions as the second mixing vessel RM2, and perform the culture destination transfer. v) Homogenize the suspensions from the first culture vessel RC1 and the third culture vessel RC3 in the third culture vessel, which functions as the second mixing vessel RM2, to obtain a homogenized suspension of mixed living cells. vi) Transfer at least a portion of the suspension from the second mixing container RM2 to the first culture container RC1, and perform a return transfer. vii) Transfer the suspension from the second culture vessel RC2 to the third culture vessel RC3, which also functions as the second mixing vessel RM2, and perform the culture destination transfer. viii) Homogenizing the suspensions from the second culture vessel RC2 and the third culture vessel RC3 in the third culture vessel RC3, which functions as the second mixing vessel RM2. ix) The procedure is characterized by transferring at least a portion of the suspension from the second mixing container RM2 to the second culture container RC2, and then performing a return transfer.

[0095] In another specific embodiment of the present invention, the method of the present invention uses n culture vessels (RCi), where i is in the range of 1 to n and n is equal to at least 2, and also uses at least one mixing vessel, the at least one mixing vessel being a single vessel independent of the set of culture vessels and arranged to receive the contents of the n culture vessels, and steps c) to e) are, c) Transfer all or part of the suspension of living cells obtained in step b) from at least two culture vessels (RCi) to at least one mixing vessel (RM) to obtain a mixed suspension of living cells. d) Homogenize the mixed suspension of living cells obtained in step c) in at least one mixing container (RM) to obtain a homogenized mixed suspension of living cells. e) This is characterized by transferring at least a portion of the suspension obtained in step d) from the at least one mixing vessel (RM) to each of the at least two culture vessels (RCi).

[0096] In another particularly advantageous embodiment, n is 3. In another particularly advantageous embodiment, n is 4. In another particularly advantageous embodiment, n is 5. In another particularly advantageous embodiment, n is 6. In another particularly advantageous embodiment, n is 7. In another particularly advantageous embodiment, n is 8. In another particularly advantageous embodiment, n is 9. In another particularly advantageous embodiment, n is 10.

[0097] The process can be more easily carried out and the speed of mixing step d) can be increased by using a single container that is independent of the set of culture vessels and is positioned to receive the contents of the n culture vessels.

[0098] In a particularly advantageous embodiment of the present invention, at least a portion of the suspension transferred to each of the at least two culture vessels in step e) corresponds to a fraction of the volume of the homogenized suspension of mixed living cells, wherein the fraction is between 1 and 100% of the total volume of the suspension.

[0099] Advantageously, the fraction of the homogenized suspension of mixed living cells transported in step e) is at least 1%, advantageously at least 2%, advantageously at least 3%, advantageously at least 4%, advantageously at least 5%, advantageously at least 6%, advantageously at least 7%, advantageously at least 8%, advantageously at least 9%, advantageously at least 10%, advantageously at least 11%, advantageously at least 12%, advantageously at least 13%, advantageously at least 14%, advantageously at least 15%, advantageously at least 16%, advantageously at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36 %, at least 37% advantageous, at least 38% advantageous, at least 39% advantageous, at least 40% advantageous, at least 41% advantageous, at least 42% advantageous, at least 43% advantageous, at least 44% advantageous, at least 45% advantageous, at least 46% advantageous, at least 47% advantageous, at least 48% advantageous, at least 49% advantageous, at least 50% advantageous, at least 51% advantageous, at least 52% advantageous, at least 53% advantageous, at least 54% advantageous, at least 55% advantageous , at least 56% to the advantage, at least 57% to the advantage, at least 58% to the advantage, at least 59% to the advantage, at least 60% to the advantage, at least 61% to the advantage, at least 62% to the advantage, at least 63% to the advantage, at least 64% to the advantage, at least 65% to the advantage, at least 656% to the advantage, at least 67% to the advantage, at least 68% to the advantage, at least 69% to the advantage, at least 70% to the advantage, at least 71% to the advantage, at least 72% to the advantage, at least 73% to the advantage, at least 74% to the advantage,The advantages are at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 100%.

[0100] In another embodiment of step e) of the method of the present invention, when at least a portion of the suspension obtained in step d) is transferred from the at least one mixing container to the n culture vessels, the fractions of the homogenized suspension of mixed living cells transferred to each of the n culture vessels may be the same or different.

[0101] For example, if four culture vessels are used, in transfer step e), a fraction of 25% of the total volume of the homogenized suspension of mixed live cells can be distributed to each of the four culture vessels.

[0102] For example, if four culture vessels are used, in transfer step e), 10% of the total volume of the homogenized suspension of living cells contained in the mixing vessel can be transferred to the first culture vessel, 20% to the second culture vessel, 30% to the third culture vessel, and 5% to the fourth culture vessel.

[0103] For example, if four culture vessels are used, in transfer step e), 0% of the total volume of the homogenized suspension of living cells contained in the mixing vessel can be transferred to the first culture vessel, 20% of the total volume to the second culture vessel, 30% of the total volume to the third culture vessel, and 5% of the total volume to the fourth culture vessel.

[0104] The aforementioned example is not restrictive and serves only to illustrate process e).

[0105] Step f) of the above method consists of repeating steps b) to e). For the purposes of the present invention, "culture cycle" means repeating steps b) to e) of this method.

[0106] In certain embodiments of the present invention, when step b) is repeated, the selective conditions and / or culture parameters used during the culture cycle may be the same as or different from those used in the preceding culture cycle. Advantageously, the selective conditions and / or culture parameters used may be the same or different from one culture cycle to the next.

[0107] In certain embodiments of the present invention, the selective conditions selected for each culture vessel may be the same or different for each culture cycle. Advantageously, the selective conditions are selected from the group including chemostats, turbidostats, medium swaps, and repeat batches, but this list is not limiting.

[0108] In particularly advantageous embodiments of the present invention, the selective conditions in a given culture vessel may be the same in each subsequent cycle. For example, a chemostat selective condition may be applied to a given culture vessel during one culture cycle, and then the same selective condition may be applied to the next culture cycle in the same culture vessel. For example, a turbidostat selective condition may be applied to a given culture vessel during one culture cycle, and then the same selective condition may be applied to the next culture cycle in the same culture vessel. The above examples are not limiting and can be applied to all selective conditions.

[0109] In a particularly advantageous embodiment, the selective conditions within a given culture vessel may be changed from one cycle to another. For example, in a given culture vessel, a chemostat-selective condition may be applied in one culture cycle, and then the selective culture conditions of the same culture vessel may be changed to a turbidostat-selective condition in the next culture cycle. For example, in a given culture vessel, a chemostat-selective condition may be applied in one culture cycle, and then the selective culture conditions of the same culture vessel may be changed to a turbidostat-selective condition in the next culture cycle, and then the selective culture conditions of the same culture vessel may be changed to a chemostat-selective condition in the next culture cycle. For example, in a given culture vessel, a chemostat-selective condition may be applied in one culture cycle, and then the selective culture conditions of the same culture vessel may be changed to a turbidostat-selective condition in the next culture cycle, and then the selective culture conditions of the same culture vessel may be changed to a medium-swap-selective condition in the next culture cycle. The above examples are not limiting and apply to all selective culture conditions.

[0110] In certain embodiments of the present invention, when step b) is repeated, the culture parameters used may be the same or different for each culture vessel from one culture cycle to another. Advantageously, the culture parameters are selected from the group consisting of dilution ratio, temperature, pH, medium composition, gas flow composition, and combinations thereof, and the parameters are potentially identical from one cycle to another. Advantageously, the culture parameters are selected from the group consisting of dilution ratio, temperature, pH, medium composition, gas flow composition, and combinations thereof, and the parameters are potentially changed from one cycle to another. Advantageously, it is possible to change only one of these culture parameters, i.e., only the temperature, only the pH, only the medium composition, or only the gas flow composition. Advantageously, these culture parameters can be changed simultaneously. Advantageously, at least two culture parameters, at least three culture parameters, and at least four culture parameters can be changed simultaneously.

[0111] In a particularly advantageous embodiment of the present invention, in the second culture cycle, the initial temperature T0i of each culture vessel RCi may increase by a value ΔT compared to the first culture cycle, and thereafter, in each new culture cycle, the temperature increase of ΔT may be received for each culture cycle. Advantageously, the value ΔT may be 0.1°C, advantageously 0.2°C, advantageously 0.3°C, advantageously 0.4°C, advantageously 0.5°C, advantageously 0.6°C, advantageously 0.7°C, advantageously 0.8°C, advantageously 0.9°C, advantageously 1.0°C, advantageously 1.1°C, advantageously 1.2°C, advantageously 1.3°C, advantageously 1.4°C, advantageously 1.5°C, advantageously 1.6°C, advantageously 1.7°C, advantageously 1.8°C, advantageously 1.9°C, or advantageously 2.0°C. The favorable values ​​are 2.1°C, 2.2°C, 2.3°C, 2.4°C, 2.5°C, 2.6°C, 2.7°C, 2.8°C, 2.9°C, 3.0°C, 3.1°C, 3.2°C, 3.3°C, 3.4°C, 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C, and 4.0°C. Favouritely, the ΔT value may be the same or different in each new cycle.

[0112] In a particularly advantageous embodiment of the present invention, in the second culture cycle, the initial temperature T0i of each culture vessel RCi may decrease by a value ΔT compared to the first culture cycle, and thereafter, in each new culture cycle, the temperature decrease of ΔT may be received for each culture cycle. Advantageously, the value ΔT may be -0.1°C, -0.2°C, -0.3°C, -0.4°C, -0.5°C, -0.6°C, -0.7°C, -0.8°C, -0.9°C, -1.0°C, -1.1°C, -1.2°C, -1.3°C, -1.4°C, -1.5°C, -1.6°C, -1.7°C, -1.8°C, -1.9°C, or -2.0°C. The favorable values ​​are -2.1°C, -2.2°C, -2.3°C, -2.4°C, -2.5°C, -2.6°C, -2.7°C, -2.8°C, -2.9°C, -3.0°C, -3.1°C, -3.2°C, -3.3°C, -3.4°C, -3.5°C, -3.6°C, -3.7°C, -3.8°C, -3.9°C, and -4.0°C. The favorable values ​​are -2.1°C, -2.2°C, -2.3°C, -2.4°C, -2.5°C, -2.5°C, -2.6°C, -2.8°C, -2.9°C, and -3.9°C. The favorable values ​​are -3.0°C, -3.1°C, -3.2°C, -3.3°C, -3.4°C, -3.5°C, -3.6°C, -3.7°C, -3.8°C, -3.9°C, and -4.01°C, -3.2°C, -3.3°C, -3.4°C, -3

[0113] In another particularly advantageous embodiment of the present invention, in the second culture cycle, the initial pH(i,k) may increase by a value ΔpH compared to the first culture cycle, and thereafter, in each new culture cycle, the temperature increase in ΔpH may be received for each culture cycle. Advantageously, the value ΔpH is 0.1 pH units, advantageously 0.2 pH units, advantageously 0.3 pH units, advantageously 0.4 pH units, or advantageously 0.5 pH units. Advantageously, the ΔpH value may be the same or different in each new cycle.

[0114] In another particularly advantageous embodiment of the present invention, in the second culture cycle, the initial pH(i,k) may decrease by a value ΔpH compared to the first culture cycle, and thereafter, in each new culture cycle, the temperature decrease in ΔpH may be received for each culture cycle. Advantageously, the value ΔpH is -0.1 pH units, advantageously -0.2 pH units, advantageously -0.3 pH units, advantageously -0.4 pH units, or advantageously -0.5 pH units. Advantageously, the ΔpH value may be the same or different in each new cycle.

[0115] In another particularly advantageous embodiment of the present invention, in the second culture cycle, the composition of the initial medium MC(i,k), or the mediums MC(i,k)-tolerant and MC(i,k)-stress, can be modified for each of the culture vessels RCi compared to the first culture cycle, and thereafter further modifications can be made for each new culture cycle. Advantageously, the modification of one or more medium compositions may consist of increasing the content of toxic agents such as growth inhibitors. Advantageously, the modification of medium compositions may consist of decreasing the content of growth factors essential for cell growth. Advantageously, the modification of medium compositions may consist of replacing one substrate with another. Advantageously, the modification of medium compositions may consist of replacing tolerant medium with stress medium. Advantageously, the modification of medium compositions may consist of replacing rich medium with minimal medium. Advantageously, the modification of medium compositions may consist of replacing minimal medium with rich medium. Advantageously, the modification of medium compositions may consist of replacing synthetic medium (also called normal medium) with compound medium (also called non-normal medium). Advantageously, changes to the culture medium composition may consist of replacing a compound medium (also called a non-normal medium) with a synthetic medium (also called a normal medium). Advantageously, the culture medium composition may be the same or different for each new cycle.

[0116] In a particularly advantageous embodiment of the present invention, in the second culture cycle, the dilution ratio Td(i,k) of each culture vessel RCi may increase by a value ΔTd compared to the first culture cycle, and thereafter, in each new culture cycle, the increase in ΔTd may be received. Advantageously, the value ΔTd is 0.01h. -1 , advantageous 0.02h -1 , advantageous 0.03h -1 , advantageous 0.04h -1 , advantageous 0.05h -1 , advantageous 0.06h -1 , advantageous 0.07h -1 0.08h is advantageous -1 , advantageous 0.09h -1 , advantageous 0.10h -1 , advantageous 0.15h -1 , advantageous 0.20h -1 , advantageous 0.02h -1 , advantageous 0.02h -1 , advantageous 0.02h -1 , advantageous 0.25h -1 , advantageous 0.30h -1 0.35h -1 , advantageous 0.40h -1 , advantageous 0.45h -1 , advantageous 0.50h -1 , advantageous 0.55h -1 , advantageous 0.60h -1 , advantageous 0.65h -1 , advantageous 0.70h -1 , advantageous 0.75h -1 , advantageous 0.80h -1 0.85h -1 , advantageous 0.90h -1 , advantageous 0.95h -1 , advantageous 1.00h -1 , advantageous 1.05h -1 , advantageous 1.10h -1 , advantageous 1.15h -1 , advantageous 1.20h -1 , advantageous 1.25h -1 , advantageous 1.30h -1 1.35h -1 , advantageous 1.40h-1 , advantageous 1.45h -1 , advantageous 1.50h -1 Therefore, advantageously, the ΔTd value may be the same or different in each new cycle.

[0117] In another particularly advantageous embodiment of the present invention, in the second culture cycle, the composition of the initial gas flow Gi of each culture vessel RCi may be changed compared to the first culture cycle, and thereafter, the gas flow composition can be further changed in each new culture cycle. The change in gas flow composition may consist of increasing or decreasing the content of one or more gases constituting the flow, the gases being selected from air, nitrogen (N2), carbon monoxide (CO), carbon dioxide (CO2), methane (CH4), hydrogen sulfide (H2S), oxygen (O2), nitrous oxide (N2O), hydrogen (H2), or mixtures thereof. Advantageously, the gas flow composition may be the same or different in each new cycle.

[0118] In another particularly advantageous embodiment of the present invention, in a second culture cycle, the suspension in each culture vessel RCi can be maintained at a temperature T0 + ΔT and a pH pHk + ΔpH for one culture cycle, and thereafter, in each new culture cycle, the temperature can be increased by ΔT and the pH by ΔpH for each of the n culture vessels.

[0119] The above examples are not limiting and apply equally to changes in a single culture parameter or to simultaneous changes in several culture parameters.

[0120] In a particular embodiment of the present invention, when step b) is repeated, the selective conditions and / or culture parameters used can be changed from one culture cycle to another, and such changes apply to all n culture vessels. Whatever changes are made, they apply to all n culture vessels.

[0121] In another specific embodiment of the present invention, when step b) is repeated, the selective conditions and / or culture parameters used may be changed from one culture cycle to another, and such changes may not apply to all n culture vessels.

[0122] In another specific embodiment of the present invention, when step b) is repeated, the selective condition can be changed from one culture cycle to another, and the change is not applicable to all n culture vessels.

[0123] For example, in step b), during one culture cycle, culture vessel RCi can undergo chemostat-selective conditions, while the remaining n-1 culture vessels can undergo turbidstat-selective conditions, and thereafter, the selective conditions can be changed for each new culture cycle.

[0124] For example, in step b), during one culture cycle, culture vessel RC1 may be subjected to chemostat-selective conditions, culture vessel RC2 to turbidstat-selective conditions, and the remaining n-2 culture vessels to be subjected to medium-swap-selective conditions, and thereafter, the selective conditions can be changed for each new culture cycle.

[0125] The above examples are not limited and apply to all selective culture conditions.

[0126] In certain advantageous embodiments of the present invention, when step b) is repeated, the culture parameters used can be changed from one culture cycle to another, and such changes are not applied to all n culture vessels.

[0127] For example, in step b), during one culture cycle, culture vessel RC1 can be maintained at a temperature of T0 + 0.1°C, and the remaining n-1 culture vessels can be maintained at the initial temperature T0. Thereafter, in each new culture cycle, the temperature of each of the n culture vessels can be increased by 0.1°C. In this case, during the second cycle, culture vessel RC1 will be maintained at a temperature of T0 + 0.2°C, and the remaining n-1 culture vessels will be maintained at a temperature of T0 + 0.1°C. The process is similar thereafter.

[0128] As another example, in step b), during one culture cycle, culture vessel RC1 can be maintained at a temperature of T0 + 0.2°C, culture vessel RC2 can be maintained at a temperature of T0 + 0.1°C, and the remaining n-2 culture vessels can be maintained at the initial temperature T0. Thereafter, in each new culture cycle, the temperature of each of the n culture vessels can be increased by 0.1°C for each culture cycle. In this case, during the second cycle, culture vessel RC1 will be maintained at a temperature of T0 + 0.3°C, culture vessel RC2 will be maintained at a temperature of T0 + 0.2°C, and the remaining n-2 culture vessels will be maintained at a temperature of T0 + 0.1°C. The process is then similar thereafter.

[0129] The above examples are not limited and apply equally to changes in a single culture parameter and simultaneous changes in several culture parameters.

[0130] In a particular embodiment of the present invention, steps b) through e) are repeated as many times as necessary until living cells with the desired phenotype appear.

[0131] In certain embodiments of the present invention, selective conditions and / or culture parameters can be automatically adapted in response to observable / measurable criteria for culture in a preceding cycle. For example, selective pressure criteria such as temperature or concentration of cell growth inhibitory molecules may be changed in the current cycle if the cell growth rate in a preceding cycle was below / above a predetermined threshold. Alternatively, after several cycles of evolution have been performed to adapt the suspension of live cells to a target concentration of the inhibitory molecule (medium swap mode), culture parameters can be changed to increase the cell growth rate (turbidostat mode).

[0132] Step g) of this method consists of recovering viable cells that have acquired the desired phenotype after several cycles of culture in at least one of the n culture vessels.

[0133] Advantageously, the desired phenotype is acquired through evolutionary adaptation, particularly the accumulation of beneficial mutations over several generations in living cells, whether spontaneous or not, such as after exposure to ultraviolet light, after exposure to one or more mutagenic substances, or after genetic modification that results in a mutation rate higher than the organism's natural mutation rate.

[0134] In certain embodiments of the present invention, the recovery step g) is performed using a sampling means, which can be selected from a sterile syringe, a sterile micropipette, or any other means for sampling living cells that have acquired a desired phenotype.

[0135] Advantageously, living cells that have acquired the desired phenotype are obtained after 2 cycles, advantageously after 3 cycles, advantageously after 4 cycles, advantageously after 5 cycles, advantageously after 6 cycles, advantageously after 7 cycles, advantageously after 8 cycles, advantageously after 9 cycles, advantageously after 10 cycles, advantageously after 20 cycles, advantageously after 30 cycles, advantageously after 40 cycles, advantageously after 50 cycles, advantageously after 60 cycles, advantageously after 70 cycles, advantageously after 80 cycles, advantageously after 90 cycles, advantageously after 100 cycles, advantageously after 150 cycles, advantageously after 200 cycles, advantageously after 250 cycles, advantageously after 300 cycles, advantageously after 350 cycles, advantageously after 400 cycles, advantageously after 4 Harvesting is advantageously performed after 50 cycles, 500 cycles, 550 cycles, 600 cycles, 650 cycles, 700 cycles, 750 cycles, 800 cycles, 850 cycles, 900 cycles, 950 cycles, 1000 cycles, 5000 cycles, 10000 cycles, 15000 cycles, 20000 cycles, 25000 cycles, 30000 cycles, 35000 cycles, 40000 cycles, 45000 cycles, and 50000 culture cycles.

[0136] In certain embodiments of the present invention, the method may optionally further include at least one sterilization step for n culture vessels and at least one mixing vessel. Advantageously, the at least one sterilization step is performed as soon as at least one culture vessel and / or at least one mixing vessel is empty. Advantageously, the at least one sterilization step is performed on the culture vessels after step c). Advantageously, the at least one sterilization step is performed on at least one mixing vessel after step e). In one particular embodiment, the at least one sterilization step is performed by adding a sterilizing solution selected from a solution containing a weak base such as ammonia, a strong base such as caustic alkali (NaOH) or potassium (KOH), a strong acid such as sulfuric acid or hydrochloric acid, a weak acid such as acetic acid, an oxidizing agent such as hydrogen peroxide and sodium hypochlorite, a solvent such as ethanol and isopropanol, or a combination thereof. Advantageously, in the case of the above combinations of solutions, the solutions can be used simultaneously or sequentially, i.e., sequentially. Advantageously, the sterilization solution is a caustic alkali solution, preferably at least 0.1 M, and more preferably greater than 1 M. Advantageously, the sterilization process can be carried out, for example, as described in Patent EP1135460, namely by temporarily transferring the entire volume of the live cell suspension from the culture or mixing vessel to a storage vessel, then bringing the entire inner surface of the culture or mixing vessel into contact with the sterilization solution, followed by a rinsing solution. The live cell suspension is then returned to the sterilized initial culture or mixing vessel.

[0137] Advantageously, the sterilization process allows for the removal of biofilms on the one hand, and for the restoration of at least one culture vessel and at least one mixing vessel to a nominal sterile state corresponding to their respective initial states on the other hand.

[0138] In certain embodiments of the present invention, the method may optionally further include at least one washing step for n culture vessels and at least one mixing vessel. Advantageously, the at least one washing step is performed after at least one sterilization step. In certain embodiments, the at least one washing step is performed by adding a washing solution capable of neutralizing the sterilizer. Advantageously, the washing solution is selected from acidic solutions such as acetic acid solutions and sulfuric acid solutions, and cleaning solutions, particularly solutions containing surfactants.

[0139] In certain embodiments of the present invention, the method may optionally further include at least one rinsing step for n culture vessels and at least one mixing vessel. Advantageously, at least one rinsing step is performed after at least one washing step. In certain embodiments, at least one rinsing step is performed by adding a rinsing solution that allows for the removal of any remaining washing solution. Advantageously, the rinsing solution is water, preferably sterile water.

[0140] In certain embodiments of the present invention, the method may optionally further include at least one ultraviolet exposure step and / or a mutagenic substance exposure step. Advantageously, at least one ultraviolet exposure step and / or a mutagenic substance exposure step may be performed at any point in the method of the present invention. In particularly advantageous embodiments of the present invention, the ultraviolet exposure step may be performed between steps c) and d) of the method of the present invention, or between steps d) and e) of the method of the present invention.

[0141] In another particularly advantageous embodiment of the present invention, the step of exposure to a mutagenic substance is performed between steps c) and d) of the method of the present invention, or between steps d) and e) of the method of the present invention. Advantageously, the mutagenic substance can be selected from alkylating agents such as N-nitroso-N-ethylurea (also known as N-ethyl-N-nitrosourea (ENU)) or ethylmethanesulfonic acid (also known as ethylmethanesulfonate (EMS)), insertors such as proflavin or acridine orange, and reactive oxygen species, particularly free radicals, oxygen ions, and peroxides.

[0142] The method of the present invention can be carried out in an apparatus for continuous culture of living cells for the evolutionary adaptation of the living cells, and the apparatus consists of the following: - There are n culture vessels, each culture vessel is arranged to receive culture medium and living cells. - At least one mixing vessel, and - At least one sterile fluid supply device including at least one culture vessel, the sterile fluid supply device is connected to the n culture vessels and the at least one mixing vessel via a main supply line.

[0143] Advantageously, the circulation of fluids in the sterile fluid supply device, i.e., the circulation of gases, sterilizing solutions, washing solutions, and rinsing solutions, and the circulation of culture media, is achieved through the use of pumps and valves. The pumps and valves can be operated, for example, mechanically, or electrically or electronically, advantageously, automatically using control means not shown.

[0144] In certain embodiments, the sterile fluid supply device includes at least one external gas source, at least one sterile fluid tank, at least one washing fluid tank, at least one rinsing fluid tank, and at least one culture medium tank. In certain embodiments, the sterile fluid supply device further includes a collection means, preferably a syringe, for introducing live cells into each of the n culture vessels and enabling the recovery of live cells that have acquired the desired phenotype. In alternative embodiments, live cells that have acquired the desired phenotype are recovered by a fluid connection means to other analytical instruments.

[0145] Advantageously, each of the n culture vessels may further include at least one gas supply device. The at least one gas supply device may enable the injection of a pressurized gas stream into the culture vessel, thereby supplying the gas necessary for the growth of the suspension and homogenizing (bubbling) the suspension. Advantageously, the at least one gas supply device may enable the injection of a pressurized gas stream, called a transfer stream, into the culture vessel, thereby increasing the pressure within the culture vessel and pushing the suspension into the mixing vessel like a syringe plunger.

[0146] Alternatively, in addition to at least one gas supply device, each of the n culture vessels may further include at least one transfer gas supply device. In this case, the transfer flow comes alone and directly from at least one transfer gas supply device, and the aeration flow and agitation flow come alone from at least one gas supply device. The use of the transfer flow increases the pressure in the culture vessel, pushing the suspension into the mixing vessel like a plunger in a syringe.

[0147] Advantageously, each of the n culture vessels may further include at least one supply line. The at least one supply line allows for filling the culture vessel with culture medium from at least one culture tank, and also for filling the culture vessel with sterile solution, washing solution, and rinsing solution from the corresponding tank.

[0148] The supply line also allows for the transfer of all or part of the suspension from the culture vessel to the mixing vessel by pressurizing the culture vessel and emptying it. At least one supply line also allows for the filling of the culture vessel when transferring all or part of the suspension from the mixing vessel to the culture vessel. The supply line also allows for the flow of the contents of the culture vessel into the waste tank to empty it during sterilization, washing, and rinsing operations.

[0149] The supply valve controls access to the supply line and allows for filling or emptying of culture vessels.

[0150] Advantageously, each of the n culture vessels may further include at least one dispensing device which allows for the release of gas, one or more culture media, and sterile solution, washing solution, and rinsing solution, as well as the release of bubbling gas during the culture, during the filling operation of the culture vessels.

[0151] Advantageously, each of the n culture vessels may further include at least one leveling line. The at least one leveling line allows for control of the volume of the suspension within the culture vessel. Advantageously, a leveling valve controls access to the leveling line. Advantageously, the leveling line is positioned at a height of no more than half the total height of the culture vessel from the bottom surface of the culture vessel.

[0152] More advantageously, each of the n culture vessels may further include: - At least one gas supply device, - At least one discharge device, - At least one leveling line, and - At least one supply line.

[0153] In a particular embodiment, each of the n culture vessels includes: - At least one gas supply device located at the bottom of the culture vessel, - At least one release device located at the top of the culture vessel, - At least one leveling line located at a height of less than half the total height of the culture vessel from the bottom surface of the culture vessel, - At least one supply line located at the bottom of the culture vessel.

[0154] Advantageously, the culture vessel is a closed-system culture vessel. The culture vessel can be disposable or reusable. Particularly advantageous is that the culture vessel is reusable.

[0155] In a particular embodiment, each of the n culture vessels contains living cells in a culture medium. Advantageously, the culture vessels of the apparatus are not empty.

[0156] In certain embodiments, the culture apparatus includes a single sterile fluid supply device to all n culture vessels and at least one mixing vessel.

[0157] In another specific embodiment, the culture apparatus includes a sterile fluid supply device for each culture vessel. Advantageously, each of the n culture vessels is connected individually and independently to the sterile fluid supply device. The sterile fluid supply device is connected to its culture vessel via a supply valve.

[0158] In a particular embodiment of the present invention, at least one mixing vessel is a culture vessel arranged to receive the contents of at least two culture vessels.

[0159] In another specific embodiment of the present invention, at least one mixing vessel is a single vessel, independent of a set of n culture vessels, and is arranged to receive the contents of the set of n culture vessels.

[0160] Advantageously, if the mixing vessel is a single vessel, independent of a set of culture vessels, the mixing vessel includes: - A gas supply device located at the top of the mixing container, - Discharge device located at the top of the mixing container, - A supply line located at the bottom of the mixing container.

[0161] Advantages of the mixing vessel are that it is a closed-system culture vessel and that it is a reusable container.

[0162] In certain embodiments, the mixing vessel includes stirring means, preferably a mechanical stirrer or stirring means by gas injection.

[0163] In certain embodiments, the culture apparatus further includes a control device arranged and configured to operate all different supply means, in particular pumps and valves. It enables the transfer of the contents of at least one culture vessel to at least one mixing vessel, and vice versa.

[0164] Advantageously, the culture apparatus is controlled by the control device.

[0165] In certain embodiments, the culture apparatus further includes a control device arranged and configured to measure physical / chemical indicators, including cell density, in each culture vessel, measure the growth dynamics of the suspension, and control the automatic activation of the suspension mixture in n mixing vessels. [Brief explanation of the drawing]

[0166] [Figure 1a] Figure 1a shows a continuous culture apparatus for living cells according to a specific embodiment of the present invention, the apparatus comprising a sterile fluid supply device and three culture vessels, all of which are arranged to serve as continuous mixing vessels, with one mixing vessel being positioned to receive the contents of at least two culture vessels.

[0167] [Figure 1b] Figure 1b shows the apparatus according to Figure 1a and illustrates step c) of a method according to a specific embodiment of the present invention, in which the entire suspension from the first culture vessel is transferred to a second culture vessel which serves as a mixing vessel.

[0168] [Figure 1c] Figure 1c shows the apparatus according to Figure 1a and illustrates step e) of a method according to a specific embodiment of the present invention, in which at least a fraction of the suspension obtained in step d) in the second culture vessel is transferred to the first culture vessel.

[0169] [Figure 2a] Figure 2a shows a continuous cell culture apparatus according to a second embodiment, which includes a single mixing vessel independent of a set of n culture vessels and is arranged to receive the contents of the set of n culture vessels.

[0170] [Figure 2b] Figure 2b shows the apparatus according to Figure 2a and illustrates step c) of a method according to a particular embodiment of the present invention, in which all suspensions from n culture vessels are transferred to a mixing vessel.

[0171] [Figure 2c] Figure 2c shows the apparatus according to Figure 2a and illustrates step e) of a method according to a particular embodiment of the present invention, in which at least a fraction of the suspension obtained in step d) from the mixing vessel is transferred to each of the n culture vessels.

[0172] [Figure 3] Figure 3 shows a continuous culture apparatus for living cells according to a third embodiment, which includes four culture vessels, each of which is connected individually and independently to a sterile fluid supply device, and the four culture vessels are arranged so that each can serve as a continuous mixing vessel, with one mixing vessel being arranged to receive the contents of at least two culture vessels.

[0173] [Figure 4] Figure 4 shows a continuous cell culture apparatus according to a fourth embodiment, the apparatus comprising four culture vessels, each of which is connected individually and independently to a sterile fluid supply device, and consisting of a single mixing vessel independent of the set of four culture vessels, the mixing vessel being arranged to receive the contents of the set of four culture vessels.

[0174] [Figure 5] Figure 5 illustrates the evolution of bacterial strains in the Pseudomonadaceae family. In this figure, the total number of dilutions occurring each day is plotted against the number of days.

[0175] [Figure 6] Figure 6 plots the temperature changes of culture vessels RC1 and RC2 against the number of culture cycles, illustrating the adaptation of Pseudomonas bacterial strains at 30°C.

[0176] [Figure 7] Figure 7 plots the temperature changes in culture vessels RC1 and RC2 against the number of days in the experiment, illustrating the adaptation of the Pseudomonas bacterial strains at 30°C.

[0177] [Figure 8] Figure 8 shows the dilution ratio adaptation of Pseudomonas bacterial strains cultured at 25°C in a single 15 mL culture vessel (solid rectangle) or a single 80 mL culture vessel (solid circle) based on a method not conforming to the present invention. In this figure, the dilution ratio of time-1 units is a function of days.

[0178] [Figure 9] Figure 9 shows the adaptive evolution of Pseudomonas bacterial strains cultured at a temperature of 25°C and a forced dilution rate of 0.2 hours⁻¹ by sequentially combining and separating suspensions between two 15 mL culture vessels (RC1: solid diamond, RC2: solid triangle) based on the method of the present invention. In this figure, the temperature (°C) is a function of the number of days. [Modes for carrying out the invention]

[0179] The design and function of a continuous culture system for living cells for adaptive evolution are shown in Figures 1a to 4.

[0180] The continuous culture apparatus for live cells, as shown in Figures 1a to 2c, includes three culture vessels: a first culture vessel RC1, a second culture vessel RC2, and a third culture vessel RC3. The first culture vessel RC1 is adjacent to the second culture vessel RC2, which is adjacent to the third culture vessel RC3. The culture vessels are arranged to contain live cells and culture medium, thereby enabling the culture of these cells.

[0181] Referring to Figure 1a, the culture apparatus includes a sterile fluid supply system 10 consisting of an external gas source GS, a sterilization solution tank AS, a washing solution tank AC, a rinse solution tank AR, and three culture medium tanks M1, M2, and M3. Fluid circulation in the sterile fluid supply system 10, i.e., circulation of gas, sterilization solution, washing solution and rinse solution, and circulation of culture medium, is achieved using pumps and valves, etc. The pumps and valves can be operated, for example, mechanically, and can be automatically controlled electrically and / or electronically, preferably using control means not shown. The sterile fluid supply system 10 includes a sampling means, preferably a syringe 11, for introducing cells into the culture vessel and collecting live cells that have acquired the desired phenotype. For simplicity, the sterile fluid supply system 10 is shown as a block or rectangle in Figures 1b, 1c, 2a, 2b, 2c, 3, and 4.

[0182] Referring to Figures 1a, 1b, 1c, 2a, 2b, and 2c, the culture apparatus includes a main supply line C10 and supply valves 1, 2, 3, Va1, Va2, and Va3 connected to the main supply line C10. The supply line C10 and the valves are located at the bottom of the culture vessel. The sterile fluid supply device 10 is connected to the three culture vessels via the supply line C10 and the valves. These allow gas, sterilization solution, washing solution, rinsing solution, culture medium, and live cells to be supplied to the culture vessels. They also allow, for example, the transfer of contents from the culture vessels when transferring all or part of a suspension from the mixing container to the culture vessels, and allow the emptying of the culture vessels when transferring all or part of a suspension from the culture vessels to the mixing container. Supply valve Va1 allows for filling or emptying of culture vessel RC1. Supply valve Va2 allows for filling or emptying of culture vessel RC2. Supply valve Va3 allows for filling or emptying of culture vessel RC3. Valves 1, 2, 3, Va1, Va2, and Va3 are normally closed when not in operation. When the supply valve Vai is in the open position, the culture vessel RCi can be filled or emptied. When the supply valve Vai is in the closed position, the culture vessel RCi cannot be filled or emptied.

[0183] The culture apparatus includes three gas supply devices G1, G2, and G3. Each culture vessel RC1, RC2, and RC3 is connected to a gas supply device G1, G2, or G3, which allows for the injection of a pressurized gas stream into the culture vessel, the injection of gas into the suspension, the homogenization (bubbling agitation) of the suspension, and pressurization of the culture vessel as needed. Each gas supply device G1, G2, or G3 is connected to the culture vessel from the bottom by a gas supply line CG that opens into the culture vessel at a height of approximately one-quarter of the total height of the culture vessel from the bottom surface of the culture vessel.

[0184] The culture apparatus includes three release devices W1, W2, and W3. Each of the culture vessels RC1, RC2, and RC3 is connected to one of the release devices W1, W2, or W3, enabling the discharge of gases injected into the suspension during culture and the discharge of gases from one or more culture media, sterile solutions, washing solutions, and rinsing solutions during the filling of culture vessels. The release devices are installed on top of the culture vessels. The culture apparatus includes three release valves Vd1, Vd2, and Vd3. Each of the culture vessels RC1, RC2, and RC3 is connected to one of the release valves Vd1, Vd2, or Vd3, respectively, to control the discharge of gases injected during culture and to control the discharge of gases from one or more culture media, sterile solution AS, washing solution AC, and rinsing solution AR during the filling of culture vessels. The release valves Vd1, Vd2, and Vd3 are normally in the open position when not in operation. When the release valves are in the open position, the culture vessels can be filled. When the release valve is in the closed position, the transfer of all or part of the suspension contained in the culture vessel to the mixing vessel can be performed by pressurizing the culture vessel.

[0185] Referring to Figures 1a, 1b, and 1c, the culture apparatus includes three leveling valves Vt1, Vt2, and Vt3. Each culture vessel RC1, RC2, and RC3 is connected to the leveling valves Vt1, Vt2, and Vt3, respectively, via leveling lines CT. Each leveling line CT opens into the culture vessel from the bottom of the vessel at a height of less than half the total height of the vessel. Each leveling valve Vt1, Vt2, and Vt3 is also connected to the main line C10. Each leveling valve controls the volume in each culture vessel, maintaining a constant volume even when culture medium is added. Leveling valves Vt1, Vt2, and Vt3 are normally in the closed position when not in operation. When the leveling valves are in the open position, an amount of suspension exceeding the suspension volume defined by the position of the leveling lines is released from the culture vessel to the supply line C10.

[0186] Figures 1a, 1b, and 1c represent a specific first embodiment in which, during the implementation of this cell culture method, the three culture vessels are arranged to serve as sequential mixing vessels.

[0187] A method for continuous cell culture of living cells, involving the culture apparatus shown in Figures 1a, 1b, and 1c, is described below.

[0188] Based on Figure 1a, each of the three culture vessels RC1, RC2, and RC3 contains live cells in culture medium. Each of the culture vessels RC1, RC2, and RC3 is filled to approximately half its capacity. The live cells are cultured under given selective conditions using defined culture parameters until they reach a given growth stage, in order to obtain a suspension of live cells in each of the three culture vessels. For this purpose, a pressurized gas stream can be injected into each of the three culture vessels via gas supply devices G1, G2, or G3 to perform gas injection and suspension homogenization (bubbling agitation) within each of the three culture vessels. Valves 1, 2, 3, and 4, leveling valves Vt1, Vt2, and Vt3, and supply valves Va1, Va2, and Va3 are in the closed position. Only the release valves Vd1, Vd2, and Vd3 are in the open position.

[0189] Next, Figure 1b shows step c) of the present invention method, which consists of transferring the entire suspension obtained in step b) from at least one culture vessel (RCi) to at least one mixing vessel, and step d), which consists of mixing the suspension from step c) in at least one mixing vessel.

[0190] According to Figure 1b, as indicated by arrow f12, all of the suspension in culture vessel RC1 is transferred to culture vessel RC2. The supply valves Va1, 2, and Va2 are in the operating position (black in Figure 1b) and are open, so the suspension from the first vessel RC1 to the second culture vessel RC2 passes through these valves. The release valve Vd1 of the first vessel is in the operating position (black in Figure 1b) and is closed, thus pressurizing the first culture vessel. The release valve Vd2 of the second vessel is inactive and remains in the open position to allow filling of the second culture vessel RC2. To empty culture vessel RC1, the gas supply device G1 injects a pressurized gas flow called a transfer flow through the gas supply line CG, increasing the pressure inside culture vessel RC1, which allows the suspension to be pushed out like a syringe plunger. Culture vessel RC2 then becomes a mixing vessel, containing both the suspension initially contained in the second culture vessel RC2 and the suspension from the first culture vessel RC1.

[0191] In a manner not shown, a pressurized gas stream is injected into the second culture vessel RC2 via the gas supply device G2, enabling gas injection into the suspension in the culture vessel RC2 and homogenization (bubbling agitation) of the cell suspension.

[0192] With respect to the third culture vessel RC3, the culture process is maintained by injecting a pressurized gas stream into the culture vessel RC3 and by keeping valve Vd3 open.

[0193] For the culture vessel RC1, the sterilization, washing, and rinsing processes are performed by opening valves 1 and Va1. First, the sterilization process begins by adding sterile solution from sterile solution tank AS to culture vessel RC1. Emptying the sterile solution is performed by valve Va1. Next, washing solution is added to culture vessel RC1 from washing solution tank AC. Emptying the washing solution is performed by valve Va1. Then, rinsing solution is added to culture vessel RC1 from rinsing solution tank AR. Emptying the rinsing solution is performed by valve Va1. These processes are not shown.

[0194] As shown in Figure 1c, as indicated by arrow f21, a portion of the suspension in culture vessel RC2 is transferred to culture vessel RC1. The leveling valve Vt2 and supply valves Va1 and Va2 are in the operating position (black in Figure 1c) and are open, so a portion of the suspension from the second vessel RC2 is sent to the first culture vessel RC1 through these valves. The release valve Vd2 of the second vessel is in the operating position (black in Figure 1c) and is closed, so RC2 is pressurized and half of the suspension is transferred to RC1. The release valve Vd1 of the first vessel is inactive and remains in the open position to allow filling of the first culture vessel RC1. To empty culture vessel RC2, the gas supply device G2 injects a pressurized gas flow called a transfer flow through the gas supply line CG, which increases the pressure inside the second culture vessel RC2, allowing the suspension to be pushed out through the transfer line like a syringe plunger.

[0195] A pressurized gas stream is injected into culture vessels RC1 and RC2, respectively, via gas supply devices G1 and G2, in a manner not shown in the diagram, enabling gas injection into the suspensions within culture vessels RC1 and RC2 and homogenization (bubbling agitation) of the suspensions.

[0196] According to Figure 1b, as indicated by arrow f31, all of the suspension in culture vessel RC3 is transferred to culture vessel RC1. The supply valves Va3, 3, 2, and Va1 are in the operating position and open, so the suspension from the third vessel RC3 to the first culture vessel RC1 passes through these valves. The release valve Vd3 of the third vessel is in the operating position and closed, pressurizing the third culture vessel. The release valve Vd1 of the first vessel remains in the non-operating and open position to allow filling of the first culture vessel RC1. To empty culture vessel RC3, the gas supply device G3 injects a pressurized gas flow called a transfer flow through the gas supply line CG, thereby increasing the pressure inside culture vessel RC3 and allowing the suspension to be pushed out like a plunger in a syringe. Culture vessel RC1 then becomes a mixing vessel, containing both the suspension initially contained in the first culture vessel RC1 and the suspension from the third culture vessel RC3.

[0197] In a manner not shown, the pressurized gas flow is injected into the first culture vessel RC1 via the gas supply device G1, enabling gas injection into the suspension and homogenization (bubbling agitation) of the suspension.

[0198] With respect to the second culture vessel RC2, the culture process is maintained by injecting a pressurized gas stream into the culture vessel RC2 and by keeping valve Vd2 open.

[0199] For the culture vessel RC3, the sterilization, washing, and rinsing processes are performed by opening valves 1, 2, 3, and Va3. First, the sterilization process begins by adding sterile solution from sterile solution tank AS to culture vessel RC3. Emptying the sterile solution is performed by valve Va3. Next, washing solution is added to culture vessel RC3 from washing solution tank AC. Emptying the washing solution is performed by valve Va3. Then, rinsing solution is added to culture vessel RC3 from rinsing solution tank AR. Emptying the rinsing solution is performed by valve Va3. These processes are not shown.

[0200] As shown in Figure 1c, as indicated by arrow f13, a portion of the suspension in culture vessel RC1 is transferred to culture vessel RC3. Since the leveling valve Vt1 and supply valves 2, 3, and Va3 are in the operating position and open, a portion of the suspension from the first vessel RC1 is sent to the third culture vessel RC3 through the valves. The release valve Vd1 of the first vessel is in the operating position and closed, so RC1 is pressurized and half of the suspension is transferred to RC3. The release valve Vd3 of the third vessel remains in the non-operating and open position to allow filling of the third culture vessel RC3. To empty culture vessel RC1, the gas supply device G1 injects a pressurized gas flow called a transfer flow through the gas supply line CG, which increases the pressure in the first culture vessel RC1, allowing the suspension to be pushed out through the transfer line like a syringe plunger.

[0201] In a manner not shown, a pressurized gas stream is injected into both culture vessels RC1 and RC3, respectively, via gas supply devices G1 and G3, enabling gas injection into the suspensions within culture vessels RC1 and RC3 and homogenization (bubbling agitation) of the suspensions.

[0202] As shown in Figure 1b, all the suspension in culture vessel RC2 is transferred to culture vessel RC3, as indicated by arrow f23. Since the supply valves Va2, 3, and Va3 are in the operating position and open, the suspension from the second vessel RC2 is sent through these valves to the third culture vessel RC3. The release valve Vd2 of the second vessel is in the operating position and closed, so the second culture vessel is pressurized. The release valve Vd3 of the third vessel is in the non-operating position and remains open to allow filling of the third culture vessel RC3. To empty culture vessel RC2, the gas supply device G2 injects a pressurized gas flow called a transfer flow through the gas supply line CG, which increases the pressure inside culture vessel RC2, making it possible to push out the suspension like a syringe plunger. Culture vessel RC3 then becomes a mixing vessel, containing both the suspension originally contained in the third culture vessel RC3 and the suspension from the second culture vessel RC2.

[0203] In a manner not shown in the diagram, a pressurized gas stream is injected into the third culture vessel RC3 via the gas supply device G3, enabling gas injection into the suspension in the culture vessel RC3 and homogenization (bubbling agitation) of the suspension.

[0204] With respect to the first culture vessel RC1, the culture process is maintained by injecting a pressurized gas stream into the culture vessel RC1 and by keeping valve Vd1 open.

[0205] For the culture vessel RC2, the sterilization, washing, and rinsing processes are performed by opening valves 1, 2, and Va2. First, the sterilization process begins by adding sterile solution from sterile solution tank AS to culture vessel RC2. Emptying the sterile solution is performed by valve Va2. Then, washing solution is added to culture vessel RC2 from washing solution tank AC. Emptying the washing solution is performed by valve Va2. Then, rinsing solution is added to culture vessel RC2 from rinsing solution tank AR. Emptying the rinsing solution is performed by valve Va2. These processes are not shown.

[0206] According to Figure 1c, as indicated by arrow f32, a portion of the suspension in culture vessel RC3 is transferred to culture vessel RC2. Since the leveling valve Vt3 and supply valves Va2 and Va3 are in the operating position and open, a portion of the suspension from the third vessel RC3 is sent to the second culture vessel RC2 through these valves. The release valve Vd3 of the third vessel is in the operating position and closed, pressurizing RC3 and transferring half of the suspension to RC2. The release valve Vd2 of the second vessel remains in the non-operating and open position to allow filling of the second culture vessel RC2. To empty culture vessel RC3, the gas supply device G3 injects a pressurized gas flow called a transfer flow through the gas supply line CG, which increases the pressure inside the third culture vessel RC3, allowing the suspension to be pushed out through the transfer line like a syringe plunger.

[0207] In a manner not shown, a pressurized gas stream is injected into both culture vessels RC2 and RC3, respectively, via gas supply devices G2 and G3, enabling gas injection into the suspensions within culture vessels RC2 and RC3 and homogenization (bubbling agitation) of the suspensions.

[0208] The pressurized gas flow is reinjected into each of the three culture vessels via gas supply devices G1, G2, or G3, respectively, enabling gas injection into the suspensions within each of the three culture vessels and homogenization (bubbling agitation) of the suspensions. Supply valves 1, 2, and 3, leveling valves Vt1, Vt2, and Vt3, and supply valves Va1, Va2, and Va3 are in the closed position. Only release valves Vd1, Vd2, and Vd3 are in the open position.

[0209] The preceding steps are repeated as many times as necessary until living cells with the desired phenotype are obtained.

[0210] The harvesting step is performed when the cells acquire the desired phenotype. This step is not shown. The harvesting step can also be performed after several mixing cycles. Preferably, the harvesting step is performed using a collection means, particularly a syringe 11.

[0211] Once the recovery process is executed, the release valves Vd1, Vd2, and Vd3 are closed, injecting a pressurized gas stream from the external gas source GS, and the supply valves Va1, Va2, Va3, and 2, 3 are opened, emptying all culture vessels. Subsequently, the sterilization, washing, and rinsing processes are executed by opening valves 1, Va1, Vd1, 2, Va2, Vd2, 3, Va3, and Vd3. First, the sterilization process is initiated by adding sterilizing solution from the sterilizing solution tank AS to each of the culture vessels RC1, RC2, and RC3. Emptying the sterilizing solution is performed by valves Va1, Va2, and Va3, respectively. Then, washing solution is added from the washing solution tank AC to each of the culture vessels RC1, RC2, and RC3. Emptying the washing solution is performed by valves Va1, Va2, and Va3, respectively. Subsequently, the rinse solution is added from the rinse solution tank AR to culture vessels RC1, RC2, and RC3, respectively. Emptying the rinse solution is performed by valves Va1, Va2, and Va3, respectively. These steps are not shown in Figures 1a, 1b, and 1c.

[0212] Figures 2a, 2b, and 2c show a second embodiment in which the culture apparatus includes a single mixing container separate from the culture vessels, and the culture apparatus is arranged to receive the contents of all the culture vessels.

[0213] The culture apparatus in Figure 2a will be described only in terms of its differences from the culture apparatus in Figure 1a. The culture apparatus in Figure 2a further includes a single mixing vessel RM, which is independent of the culture vessels RC1, RC2, and RC3 and is positioned to receive the contents of the culture vessels. The culture apparatus includes a gas supply device GM connected to the top of the mixing vessel RM. The gas supply device GM can pressurize the mixing vessel by injecting a gas stream. The device includes a gas supply valve Vgm that controls the gas supply of the gas supply device GM. The culture apparatus includes a release device Wm connected to the top of the mixing vessel RM. The release device (Wm) can release gas during mixing. This device includes a release valve Vdm that controls the release of gas during mixing. The release valve Vdm is normally in the closed position when not in operation. When the release valve Vdm is in the open position, filling the mixing vessel and / or releasing gas during mixing can be performed. The culture apparatus includes a supply valve Vam that controls the filling and release of the mixing vessel. This valve is located at the bottom of the mixing vessel and is connected to the main supply line C10. It allows for filling the mixing vessel when transferring all or part of the suspension from the culture vessel to the mixing vessel, and allows for emptying the mixing vessel when transferring all or part of the suspension from the mixing vessel to the culture vessel. The supply valve Vam is normally in the closed position when not in operation. When the supply valve Vam is in the open position, the mixing vessel can be filled or emptied. When the supply valve Vam is in the closed position, the mixing vessel cannot be filled or emptied.

[0214] A method for continuous cell culture of living cells, involving the aforementioned culture apparatus, will be disclosed in Figures 2a, 2b, and 2c.

[0215] As shown in Figure 2a, each of the three culture vessels RC1, RC2, and RC3 contains live cells in culture medium. They are filled to approximately 75% of their total volume. The mixing vessel RM is empty, and valves Vdm, Vam, and Vgm are in the closed position.

[0216] According to Figure 2b, the suspensions contained in culture vessels RC1, RC2, and RC3 are transferred to the mixing vessel RM, as indicated by arrows f1m, f2m, f3m, and fmp. To this end, the transfer of the suspension from culture vessel RC1 to mixing vessel RM is performed by opening the supply valves Va1, 2, 3, Vam, and the release valve Vdm. The transfer of the suspension from culture vessel RC2 to mixing vessel RM is performed by opening the supply valves Va2, 3, Vam, and the release valve Vdm. The transfer of the suspension from culture vessel RC3 to mixing vessel RM is performed by opening the supply valves Va3, Vam, and the release valve Vdm. Opening the release valve Vdm allows the mixing vessel RM to be filled. The transfer of the suspensions is achieved by the gas supply devices G1, G2, and G3, as described above.

[0217] Once the mixing vessel is filled with all the suspensions from culture vessels RC1, RC2, and RC3, the supply valve Vam is placed in the closed position.

[0218] For culture vessels RC1, RC2, and RC3, the sterilization, washing, and rinsing processes are performed by opening valves 1, Va1, Vd1, 2, Va2, Vd2, 3, Va3, and Vd3. First, the sterilization process begins by adding sterile solution from sterile solution tank AS to each of the culture vessels RC1, RC2, and RC3. Emptying the sterile solution is performed by valves Va1, Va2, and Va3, respectively. Next, washing solution is added from washing solution tank AC to each of the culture vessels RC1, RC2, and RC3. Emptying the washing solution is performed by valves Va1, Va2, and Va3, respectively. Then, rinsing solution is added from rinsing solution tank AR to each of the culture vessels RC1, RC2, and RC3. Emptying the rinsing solution is performed by valves Va1, Va2, and Va3, respectively. These processes are not shown in Figure 2b.

[0219] According to Figure 2c, as indicated by arrows fms and fm1, a portion of the suspension in the mixing vessel RM is transferred to the culture vessel RC1. To this end, the transfer is carried out by opening valves Vgm, Vam, 3, 2, Va1, and Vd1. Valve Vdm is in the closed position, and a pressurized gas stream is injected into the mixing vessel RM through the opening of the gas supply valve Vgm, allowing the mixing vessel RM to be emptied. Opening the release valve Vd1 allows the culture vessel RC1 to be filled. Valves Va2 and Va3 are in the closed position.

[0220] As suggested by Figure 2c, a portion of the suspension in the mixing vessel RM is then transferred to the second culture vessel RC2, as indicated by the dashed arrows fms and fm2. To this end, the transfer is carried out by opening valves Vgm, Vam, 3, Va2, and Vd2. Valve Vdm is in the closed position, and a pressurized gas stream is injected into the mixing vessel RM through the opening of the gas supply valve Vgm, allowing the mixing vessel RM to be emptied. Opening the release valve Vd2 allows the culture vessel RC2 to be filled. Valves Va1 and Va3 are in the closed position.

[0221] As suggested by Figure 2c, a portion of the suspension in the mixing vessel RM is then transferred to the third culture vessel RC3, as indicated by the dashed arrows fms and fm3. To this end, the transfer is carried out by opening valves Vgm, Vam, Va3, and Vd3. Valve Vdm is in the closed position, and a pressurized gas stream is injected into the mixing vessel RM through the opening of the gas supply valve Vgm, allowing the mixing vessel RM to be emptied. Opening the release valve Vd3 allows the culture vessel RC3 to be filled. Valves Va1 and Va2 are in the closed position.

[0222] After the transfer is complete and the mixing container is completely empty, valves 1, 2, 3, Vam, and Vdm are opened to perform the sterilization, washing, and rinsing processes. First, the sterilization process begins by adding sterile solution from sterile solution tank AS to culture container RM. Emptying the sterile solution is performed by valve Vam. Next, washing solution is added from washing solution tank AC to mixing container RM. Emptying the washing solution is performed by valve Vam. Then, rinsing solution is added from rinsing solution tank AR to mixing container RM. Emptying the rinsing solution is performed by valve Vam. These processes are not shown in Figures 2a-2c.

[0223] The pressurized gas flow is again injected into each of the three culture vessels RC1, RC2, and RC3 via gas supply devices G1, G2, or G3, respectively, enabling gas injection into the suspensions within each of the three culture vessels and homogenization (bubbling agitation) of the suspensions. Supply valves 1, 2, and 3, leveling valves Vt1, Vt2, and Vt3, and supply valves Va1, Va2, and Va3 are in the closed position. Only release valves Vd1, Vd2, and Vd3 are in the open position.

[0224] After several culture cycles of viable cells that have acquired the desired phenotype in a culture vessel, a harvesting step is subsequently performed. This step is not shown.

[0225] Once the recovery process is complete, all culture vessels are emptied in the same manner as in the first embodiment. These steps are not shown in Figures 2a to 2c.

[0226] The culture vessel in Figure 3 will only be described from a different perspective than the culture vessel in Figure 1.

[0227] As shown in Figure 3, the apparatus for continuous cell culture of live cells includes four culture vessels: a first culture vessel RC1, a second culture vessel RC2, a third culture vessel RC3, and a fourth culture vessel RC4. In this embodiment, the set of culture vessels is arranged so that each becomes a continuous mixing vessel during the cell culture method, and each mixing vessel is arranged to receive the contents of at least two culture vessels. Each of the culture vessels RC1, RC2, RC3, and RC4 is connected individually and independently to the sterile fluid supply device 10. The sterile fluid supply device (10) is connected to its culture vessels via a supply valve 1.

[0228] As shown in Figure 3, each of the four culture vessels contains live cells in a culture medium. The live cells are cultured to obtain a suspension. For example, a pressurized gas stream is injected into each of the four culture vessels via gas supply devices G1, G2, G3, or G4, respectively, enabling gas injection into each of the four culture vessels RC1, RC2, RC3, and RC4 and homogenization (bubbling agitation) of the suspension. Supply valve 1, leveling valves Vt1, Vt2, Vt3, and Vt4, and supply valves Va1, Va2, Va3, and Va4 are in the closed position. Only release valves Vd1, Vd2, Vd3, and Vd4 are in the open position.

[0229] The culture apparatus includes valves V10, V20, V30, and V40 that enable interconnection of the four culture vessels, and are normally in an inactive closed position.

[0230] The method for continuous cell culture of living cells, associated with the culture apparatus shown in Figure 3, is the same as the culture method associated with Figures 1a, 1b, and 1c.

[0231] First, as indicated by arrow f12, all the suspension in the first culture vessel RC1 is transferred to culture vessel RC2. This transfer is carried out by opening valves Va1, V10, V20, Va2, and Vd2. Valve Vd1 is in the closed position, and as described above, culture vessel RC1 can be emptied by the gas supply device. Opening the release valve Vd2 allows culture vessel RC2 to be filled. Subsequently, culture vessel RC2 becomes a mixing vessel, containing both the suspension initially contained in culture vessel RC2 and the suspension from culture vessel RC1. A pressurized gas stream is injected into culture vessel RC2 via gas supply device G2, enabling gas injection into the suspension in culture vessel RC2 and homogenization (bubbling agitation) of the cell suspension. In culture vessels RC3 and RC4, the culture process is maintained by injecting a pressurized gas stream into culture vessels RC3 and RC4 and keeping valves Vd3 and Vd4 open.

[0232] In the culture vessel RC1, the sterilization, washing, and rinsing processes are then performed by opening valves 1, Va1, and Vd1. First, the sterilization process begins by adding sterilization solution from the sterilization solution tank AS to the culture vessel RC1. Emptying the sterilization solution is performed by valve Va1. Next, washing solution is added to the culture vessel RC1 from the washing solution tank AC. Emptying the washing solution is performed by valve Va1. Next, rinsing solution is added to the culture vessel RC1 from the rinsing solution tank AR. Emptying the rinsing solution is performed by valve Va1. These processes are not shown in Figure 3.

[0233] Next, as indicated by arrow f21, a portion of the suspension in culture vessel RC2 is transferred to culture vessel RC1. This transfer is carried out by opening valves Vt2, V20, V10, Va1, and Vd1. With valve Vd2 in the closed position, culture vessel RC2 can be emptied by pressurizing the suspension using gas injected from GC2. Opening release valve Vd1 allows culture vessel RC1 to be filled. Pressurized gas streams are injected into culture vessels RC1 and RC2, respectively, and the gas is injected into the suspension via gas supply devices G1 and G2 to homogenize (bubble agitation) the cell suspension in culture vessels RC1 and RC2. In the third culture vessels RC3 and RC4, the culture process is maintained by injecting pressurized gas streams into culture vessels RC3 and RC4 and keeping valves Vd3 and Vd4 open.

[0234] Subsequently, as indicated by the dotted arrow f13, all the suspension in culture vessel RC3 is transferred to the first culture vessel RC1. This transfer is performed by opening valves Va1, V10, V30, and Va3, and closing valve Vd3. Then, vessel RC3 is sterilized, washed, and rinsed in the same manner as described for vessel RC1. Next, after mixing, some of the suspension in culture vessel RC1 is transferred to culture vessel RC3, as indicated by the arrow f31. This transfer is performed by opening valves Vt1, V30, V10, and Va1, and closing valve Vd1.

[0235] As indicated by the dotted arrow f14, all of the suspension in culture vessel RC1 is transferred to the fourth culture vessel RC4. To this end, the transfer is carried out by opening valves Va1, V10, and V40, and closing valve Vd1. Next, after mixing, as indicated by the arrow f41, some of the suspension in culture vessel RC4 is transferred to culture vessel RC4. To this end, the transfer is carried out by opening valves Vt4, V40, V10, and Va1, and closing valve Vd4.

[0236] Subsequently, as indicated by the dotted arrow f23, all the suspension in culture vessel RC2 is transferred to the third culture vessel RC3. This transfer is performed by opening valves Va2, V20, V30, and Va3, and closing valve Vd2. Then, vessel RC2 is sterilized, washed, and rinsed as described above for vessel RC1. Next, after mixing, some of the suspension in culture vessel RC3 is transferred to culture vessel RC2, as indicated by the arrow f32. This transfer is performed by opening valves Vt3, V30, V20, and Va2, and closing valve Vd3.

[0237] As indicated by the dotted arrow f24, all the suspension in culture vessel RC4 is transferred to the second culture vessel RC2. This transfer is performed by opening valves Va2, V20, V40, and Va4, and closing valve Vd4. Subsequently, vessel RC4 is sterilized, washed, and rinsed as described above for vessel RC1. Next, after mixing, as indicated by the arrow f42, some of the suspension in culture vessel RC2 is transferred to culture vessel RC4. This transfer is performed by opening valves Vt4, V40, V20, and Va2, and closing valve Vd2.

[0238] Finally, as indicated by the dotted arrow f34, all the suspension in culture vessel RC3 is transferred to the fourth culture vessel RC4. To this end, the transfer is carried out by opening valves Va3, V30, V40, and Va4, and closing valve Vd3. Next, after mixing, as indicated by the arrow f43, some of the suspension in culture vessel RC4 is transferred to the third culture vessel RC3. To this end, the transfer is carried out by opening valves Vt4, V40, V30, and Va3, and closing valve Vd4.

[0239] The culture device of FIG. 4 will be described only from a different perspective from the culture device of FIG. 3. FIG. 4 shows a specific fourth embodiment including four culture vessels and a single mixing vessel RM independent of the set of the culture vessels, and the mixing vessel is arranged to receive the contents of the set of culture vessels RC1, RC2, RC3, and RC4. Each of the culture vessels RC1, RC2, RC3, and RC4 is individually and independently connected to the aseptic fluid supply device 10 described above. The aseptic fluid supply device (10) is connected to its culture vessel via a supply valve 1.

[0240] The method for continuously culturing live cells related to the culture device shown in FIG. 4 is similar to the culture methods related to FIGS. 2a, 2b, and 2c.

[0241] Advantageously, the suspensions contained in the culture vessels RC1, RC2, RC3, and RC4 are transferred to the mixing vessel RM.

[0242] Thereafter, the operations of sterilizing, washing, and rinsing the vessels RC1, RC2, RC3, and RC4 are carried out.

[0243] When the mixing step is completed, a part of the suspension in the mixing vessel RM is transferred to the first culture vessel RC1. Thereafter, a part of the suspension in the mixing vessel RM is transferred to the second culture vessel RC2. Thereafter, a part of the suspension in the mixing vessel RM is transferred to the third culture vessel RC3. Thereafter, a part of the suspension in the mixing vessel RM is transferred to the fourth culture vessel RC4.

[0244] Next, the operations of sterilizing, washing, and rinsing the mixing vessel RM are carried out.

Example

[0245] In all the examples described below, the growth condition is a turbidostat, and the dilution rate (unit, hour -1 ) is defined as the ratio of the flow rate of the growth medium to maintain a constant microbial concentration in the culture chamber during evolution to the volume of the culture chamber. In these experiments, a turbidostat was introduced so that the dilution rate was equal to the growth rate of the number of individuals.

[0246] Example 1: Evolutionary adaptation of bacterial strains to 30°C At sub-optimal temperatures lower than the optimal temperature, the growth rate of an organism is lower than at the optimal temperature. If an organism is intended to be used at a temperature lower than the optimal temperature, adapting this organism to said temperature is relevant.

[0247] In the following Experiments 1 and 2, the strain used was a soil bacterium of the Pseudomonadaceae family. The specific growth rate of this bacterium in a synthetic growth medium containing 20 g / L of sucrose as a carbon source was 0.315 h -1 at the optimal temperature of 35°C.

[0248] For each of the two experiments, this strain was aimed to be adapted to a temperature of 30°C, aiming for the growth rate at 30°C to be comparable to that of the starting strain at 35°C.

[0249] To achieve this goal, two adaptive evolution experiments were carried out using the same strain as above, with the same settings as above and using the same reference medium.

[0250] 1 / Experiment 1: Comparative method not included in the present invention: Turbidostat with a target temperature of 30°C Experiment 1 evolves the aforementioned bacterium adaptively based on a simple evolution protocol not according to the present invention, and only implements the turbidostat selection situation in a single culture vessel.

[0251] At the start of the experiment, the above strain is inoculated into the culture vessel.

[0252] During the experiment, the temperature is fixed and maintained at 30°C.

[0253] The selection situation is carried out discontinuously as follows. Every 10 minutes, the transparency measured by optical measurement is compared with an arbitrarily set threshold value of 80. · If the measured value exceeds the threshold value, no action occurs. If the measured value is below the threshold, dilution of the suspension is performed by adding 4 mL of the above growth medium to the culture vessel, maintaining a constant volume of suspension in the culture vessel at 13.5 mL, and then withdrawing the same volume V from the culture vessel.

[0254] The results obtained are shown in Figure 5.

[0255] Results obtained After 20 days of adaptive evolution in a turbid stat at 30°C, the theoretical dilution rate or growth rate was 0.358 hours. -1 Reaching 29 daily dilution cycles, which corresponds to this number of cycles.

[0256] 2 / Experiment 2: The present invention method using two culture vessels Experiment 2 involved evolutionarily adapting the same bacteria as described above based on the method of the present invention, and conducting the experiment in two culture vessels, RC1 and RC2, at different temperatures. This protocol was carried out using the same apparatus as that used in Experiment 1.

[0257] At the start of the experiment, the same bacterial strains described above, which were planted at the start of Experiment 1, were inoculated into each culture vessel RC1 and RC2, and the initial temperature for both vessels RC1 and RC2 was 35°C.

[0258] The two culture vessels, RC1 and RC2, were each subjected to a turbidstat selective environment, using the same parameters as those used in Experiment 1, and were performed discontinuously as described in Experiment 1: • Growth medium as defined above Transparency threshold = 80 • Add 4 mL of growth medium to the culture vessel, maintain a constant volume of suspension at 13.5 mL, and dilute the suspension by withdrawing the same volume V from the culture vessel.

[0259] The growth stage is arbitrarily defined as 12 hours, at which point the entire contents of culture vessel RC1 are transferred to culture vessel RC2, which will serve as a mixing vessel, and step c) is performed. Therefore, one cycle is 12 hours, and there are two cycles per day.

[0260] For each new cycle, the temperature of each of these two culture vessels RC1 and RC2 is automatically adjusted based on the calculation of the average dilution rate in vessel RC1, and the dilution rate of vessel RC1, which is the average of the immediately preceding cycle, is 0.358 hours obtained in Experiment 1 after being placed in a turbidostat at 30 °C for 20 days -1 If it exceeds an arbitrarily set threshold value, it is immediately instructed to lower the culture temperature.

[0261] Table 1 below shows the temperatures applied in consecutive cycles in each of the two culture vessels RC1 and RC2, taking into account that only one cycle was carried out on the 7th day of the experiment.

Table 1

[0262] Results obtained The results obtained are shown in Figures 6 and 7.

[0263] During the 18th culture cycle, that is, 10 days after adaptive evolution, the temperature in culture vessel RC1 is 30 °C. During this cycle, the average dilution rate is 0.387 hours -1 which is higher than the average dilution rate obtained after 20 days of adaptive evolution in a 30 °C turbidostat with the same apparatus.

[0264] To compare the growth rates, it is legitimate to compare the dilution rate in one cycle of Experiment 2 with that measured in Experiment 1. This is because in each cycle, the suspension is exposed to a turbidostat selective situation where it is carried out with the same parameters and in the same apparatus (the culture vessel RC1 in Experiment 2 is exactly the same culture vessel as that used in Experiment 1) based on the same batch protocol.

[0265] Therefore, the suspension grown on the 10th and 18th cycles of Experiment 2 shows a slightly higher growth rate than the growth rate at 30 °C obtained on the 20th day of Turbidostat Experiment 1. <000098​ conclusion Therefore, in the protocol of Experiment 2, which implements the parallel evolutionary adaptation of two subpopulations that are periodically mixed and redistributed into two culture vessels based on the method of the present invention, it was possible to double the rate at which bacteria are adapted to suboptimal temperatures compared to a conventional turbidstat situation in which a single population grows in a single vessel.

[0267] Example 2: Evolutionary adaptation of bacterial strains to 25°C. In all of these experiments, the goal is to increase the growth rate of this strain while adapting it to a temperature of 25°C.

[0268] To achieve this objective, the same strain as in Example 1 was used, under the same conditions, and with the same reference medium, at 35°C for a dilution of 0.315 hours. -1 We conducted two adaptive evolution experiments, in which the initial evolution was carried out using this method.

[0269] 1 / Experiment 3: Comparative method not included in the present invention: Turbidostat at a target temperature of 25°C This experiment was conducted under the same conditions as Experiment 1 / shown in Example 1 above, but the temperature was set to 25°C.

[0270] The experiments were conducted using single culture vessels with a capacity of 15 mL or 80 mL.

[0271] Results obtained The results are shown in the attached Figure 8. In this figure, the dilution ratio (time) -1 The curve shown by the solid circle corresponds to the experiment conducted with a volume of 80 mL, and the curve shown by the solid rectangle corresponds to the experiment conducted with a volume of 15 mL.

[0272] Whether using a 15 mL or 80 mL culture vessel, the dilution ratio or growth rate at 25°C over 18 days was 0.2 hours. -1 This was confirmed to be possible, demonstrating that the volume of the culture vessel is not an important parameter for microbial evolution.

[0273] 2 / Experiment 4: Specified dilution ratio, 0.2 hours -1 The present invention uses two culture vessels. Experiment 4 consisted of evolving and adapting the same bacteria according to the present invention method, based on the protocol described in Experiment 2 of Example 1, using two culture vessels RC1 and RC2, each with a capacity of 15 mL. However, the temperature was changed from 35°C to 25°C.

[0274] At the start of the experiment, the same bacterial strain as in Example 1 was planted in each culture vessel RC1 and RC2, and the initial temperature for both vessels RC1 and RC2 was 35°C.

[0275] For each new cycle, the temperatures of the two culture vessels, RC1 and RC2, are automatically adjusted based on the calculation of the average dilution ratio within the vessel, which is the average of the previously completed cycles. -1 If the set threshold value, which was obtained in Experiment 3 after placing the culture in a turbid stat at 25°C for 18 days, is exceeded, instructions are given to immediately lower the culture temperature.

[0276] Table 2 below shows the temperatures applied in a continuous cycle for each of the two culture vessels, RC1 and RC2. [Table 2]

[0277] The results obtained are shown in the attached Figure 9.

[0278] In this figure, temperature (°C) is a function of the number of days. The solid diamond-shaped curves correspond to the experiments conducted in RC1, and the solid square-shaped curves correspond to the experiments conducted in RC2.

[0279] These results indicate that, according to the present invention's method of repeatedly mixing the contents of two culture vessels, the dilution ratio or growth rate at 25°C is 0.2 hours. -1 This shows that adaptation of the bacteria can be achieved in 7 days, which is 2.6 times faster than the method in Experiment 3, which does not conform to the present invention.

[0280] The advantages observed for evolutionary adaptation methods are indeed brought about by the present invention, which consists of periodically combining and separating suspensions coming from at least two culture vessels.

Claims

1. A method for adaptively evolving living cells, excluding human germline cells, through continuous culture of living cells, wherein n culture vessels (RCi) are used, i is in the range of 1 to n, n ≥ 2, and the method is a) A step of introducing at least one liquid culture medium and living cells into each of the n culture vessels, b) In each of the n culture vessels, using predetermined culture parameters based on a given selective condition, the viable cells are cultured in at least one of the n culture vessels until a determined growth stage is reached, and a suspension of the viable cells in the liquid medium is obtained in each of the n culture vessels. c) A step of mixing at least a portion of the suspension of living cells from at least two culture vessels (RCi) obtained in step b) to obtain a mixed suspension of living cells. d) A step of homogenizing the mixed suspension of living cells obtained in step c) to obtain a homogenized suspension of mixed living cells. e) Distributing at least a portion of the homogenized suspension of mixed living cells obtained in step d) into at least two culture vessels (RCi), f) A process that repeats steps b) to e), g) A step of culturing viable cells that have acquired the desired phenotype in at least one of the n culture vessels for several cycles and then harvesting them. A method for adaptively evolving living cells, characterized by including the following:

2. The method according to claim 1, characterized in that the living cells are selected from human, animal, or plant eukaryotic or prokaryotic cells.

3. The method according to claim 1 or 2, characterized in that the selective condition in step b) is selected from a chemostat, a turbidostat, a medium swap, and a repeat batch.

4. The method according to any one of claims 1 to 3, characterized in that the predefined culture parameters in step b) are selected from temperature, pH, cell density, culture medium composition, gas composition, exposure to electromagnetic waves of a specific wavelength, exposure to mutagenic substances, or a combination thereof.

5. The method according to any one of claims 1 to 4, characterized in that step c), which consists of mixing at least a portion of the suspension of living cells from at least two culture vessels (Ri) obtained in step b), is carried out by using one of the at least two culture vessels as a mixing vessel, or by enabling a mixing vessel independent of the at least two culture vessels to contain all or part of the contents of the at least two culture vessels.

6. The method according to any one of claims 1 to 5, characterized in that step c) is performed using a mixing vessel, and at least a portion of the suspension obtained in step b) is transferred from at least two culture vessels to at least one mixing vessel.

7. The method according to any one of claims 1 to 6, characterized in that the homogenization step d) is carried out in whole or in part by a stirring means selected from mechanical stirring and injection of a gas stream.

8. The method according to any one of claims 1 to 7, characterized in that step e) comprises transferring at least a portion of the homogenized suspension of mixed living cells obtained in step d) to at least two culture vessels (RCi).

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

10. The method according to any one of claims 1 to 9, characterized in that when step b) is repeated, the selective conditions and / or culture parameters used during the culture cycle may be the same as or different from those used in the preceding culture cycle.

11. n culture vessels (RCi) are used, where i is in the range of 1 to n and n is at least equal to 2, and at least n-1 mixing vessels (RMj) are used, where j is in the range of 1 to n-1, and each of the at least n-1 mixing vessels is a culture vessel (RCi) arranged to receive the contents of at least two culture vessels, and steps c) to e) are, i) Transferring all or part of the suspension obtained in step b) from a culture vessel (RCi) called a culture start vessel to a mixing vessel (RMj) called a transfer destination vessel, thereby carrying out the transfer to the destination. ii) In the destination container (RMj), homogenize the suspension in the culture initiation container (RCi) with the suspension in the destination container (RMj) to obtain a homogenized suspension of mixed living cells. iii) Transfer at least a portion of the suspension obtained in step ii) from the destination container (RMj) to the culture start container (RCi), and perform a return transfer. iv) Repeat steps i) to iii) above, while varying RCi and RMj, so that all suspensions are mixed together in pairs at least once. The method according to any one of claims 1 to 10, further characterized by being performed in [a specific location].

12. The method according to claim 5, 6, or 11, characterized in that the mixing container is a single container independent of the set of culture containers and is arranged to receive the contents of the n culture containers.

13. n culture vessels (RCi) are used, where i is in the range of 1 to n and n is equal to at least 2, and at least one mixing vessel (RM) is used, where the at least one mixing vessel is a single vessel independent of the set of culture vessels and is arranged to receive the contents of the n culture vessels, and steps c) to e) are, c) Transfer all or part of the suspension of living cells obtained in step b) from at least two culture vessels (RCi) to at least one mixing vessel (RM) to obtain a suspension of living cells that has been mixed. d) Homogenize the mixed suspension of living cells obtained in step c) in at least one mixing container (RM) to obtain a homogenized suspension of mixed living cells. e) Transfer at least a portion of the suspension obtained in step d) from the at least one mixing vessel (RM) to each of the at least two culture vessels (RCi), The method according to any one of claims 1 to 10, characterized in that it is performed by [a specific method].

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