KR20070022233A - Continuous culture apparatus with mobile vessel, allowing selection of filter cell variants - Google Patents

Abstract

A method and apparatus for increasing the proliferation rate (by increasing the rate of proliferation and / or increasing the amount of proliferation) through a natural selection process of living cells or any cultivable organism in suspension, the apparatus comprising a) a culture medium Flexible sterile tubes (7), b) separating the tubes (7) into spent culture containing region (downstream region), growth culture containing region (growth chamber) and fresh growth medium containing region (upstream region) Splitting movable gate (clamp) systems (3, 4, 5), c) part of the growth chamber and the culture contained therein can be clamped and separated from the growth chamber, and a portion of the new perfusion containing the unused medium may be And means for moving the gate and perfusion 17 so that they can be mixed with the culture that was in the medium and a portion of the associated medium.

Continuous culture device, mobile container, flexible sterile tube, mobile gate, natural selection

Description

CONTINUOUS CULTURE APPARATUS WITH MOBILE VESSEL, ALLOWING SELECTION OF FILTER CELL VARIANTS}

The present invention relates to methods and apparatus capable of selecting live cells which exhibit increased proliferation and specific metabolic properties in liquid or semisolid media. In the selection process (adaptive evolution), genetically modified organisms (mutants) are produced in one population and compete with other variants of the same origin. The organisms with the fastest growth rates increase in relative proportions over time, leading to populations (and individual organisms) with increased growth rates. This process can improve the performance of organisms used for industrial processes or for academic purposes.

Selection for increased growth rate (adaptability) requires continuous proliferation achieved through regular dilution of the growth culture. In the prior art, this continuous propagation has been carried out in two ways: serial dilution and continuous culture. The two basically differ in dilution.

Curb culture involves the repeated delivery of a small amount of growth culture to a larger volume container containing fresh growth medium. When the cultured organisms are grown to the saturated state in a new vessel, the process is repeated. This method clearly demonstrates a consistent improvement in proliferation rate over decades in Lenski & Travisano: Dynamics of adaptation and diversification: a 10,000-generation experiment with bacterial populations. 1994. Proc Natl Acad Sci USA. 15: 6808-14. Proven experiments have been used to obtain the long-term evidence of sustained culture. This process is usually manual, requiring significant labor costs and being contaminated by exposure to the outside environment. Continuous culture is also inefficient, as described in the next paragraph.

)에 비례하고(Wright: Size of population and breeding structure in relation to evolution. 1938. Science 87: 430-431), 따라서 사이클 중에 최저 집단에 근접하게 될 수 있다.The rate of improvement in selection or growth rate depends on population size (Fisher: The Genetical Theory of Natural Selection. 1930. Oxford University Press, London, UK). In addition, in situations such as continuous delivery where the population size changes rapidly, selection may require a harmonious termination of the group (

(Wright: Size of population and breeding structure in relation to evolution. 1938. Science 87: 430-431) and thus may be close to the lowest population during the cycle.

Population size can be maintained, and selection can therefore be more effective during continuous culture. Unlike continuous dilution, continuous culture involves less relative volume, so that a small amount of growth culture is regularly replaced with the same volume of fresh growth medium. This process increases the minimum size of the population during periodic dilution to maximize the effective population size. Continuous incubators are called "chemostat" if dilution occurs at specific time intervals, and "turbidostat" if dilution occurs automatically when the cultures grow to a certain density.

For simplicity, we will classify both types of devices as "chemistats." Chemostat was invented simultaneously by two groups of researchers in the 1950s (Novick & Szilard: Description of the chemostat. 1950. Science 112: 715-716) and (Monod: La technique de la culture continue-Theorie et applications. 1950 Ann.Inst.Pasteur 79: 390-410). Chemostat was used to demonstrate a rapid increase in proliferation rate over a short period of time (Dykhuizen DE. Chemostats used for studying natural selection and adaptive evolution. 1993. Methods Enzymol. 224: 613-31).

Conventional chemostats cannot sustain long-term selection for increased growth rate due to unintended selection of dilution resistant (static) variants. These variants may adhere to the surface of the chemostat and not dilute, thereby surpassing the less cohesive individuals, including those with higher proliferation rates, thus losing the intended purpose of the device (Chao & Ramsdell: The effects of wall populations on coexistence of bacteria in the liquid phase of chemostat cultures, 1985. J. Gen. Microbiol. 131: 1229-36).

One method and chemostat device (the Genetic Engine) was invented to avoid dilution resistance in continuous culture (US Pat. No. 6,686,194-B1, Applicants: PASTEUR INSTITUT (FR) and MUTZEL RUPERT (DE)). This method utilizes valve controlled fluid delivery, which periodically moves proliferation culture between two chemostats, allowing sterilization and cleaning to take place between proliferation periods of the active culture, respectively. Regular sterilization cycles destroy variants that are resistant to dilution and prevent their selection. Although these methods and apparatus have achieved their objectives, they are very corrosive and potentially very reactive in sterile (sealed) environments, which can quickly damage valves and expose containment and waste disposal problems (NaOH). And complicated operations are required.

Summary of the Invention

It is therefore an object of the present invention to provide an improved (completely independent) method and apparatus for continuous cultivation of organisms (eg, bacteria, primitive bacteria, eukaryotes and viruses) without interruption of dilution resistant variants. Like other chemostats, the device provides a means for regularly diluting the growth culture with fresh growth medium, a means for conducting gas exchange between the culture and the external environment, and an automatic actuator such as a chemostat or turbidadostat. do.

The present invention is designed to achieve this object without any fluid delivery, including sterilization or cleaning functions. This is a particular advantage of the present invention over the prior art in that it avoids the risks and difficulties associated with sterilization and cleaning, including complex fluid delivery and containment involving caustic solvents.

Continuous culture is carried out in a flexible sterile tube filled with growth medium. Here, the medium and the chamber surface are stationary with each other, and both interlock the tubing through a "gate" or point that sterilizes the tube by a clamp that blocks the movement of cultured organisms between regions of the tube. By regular and simultaneous substitution. In addition, UV gates can optionally be added upstream and downstream of the culture vessel for additional safety.

The method and apparatus of the present invention is also an improvement over the prior art as the substitution of the deformed adherent surface occurs alongside the dilution process, selecting against the attachment of dilution resistant variants to the chemostat surface continuously rather than periodically. .

The tube is temporarily called a region called a growth chamber, which contains a saturated (fully grown) culture, a region containing fresh medium, and a mixture of grown culture and fresh medium for dilution between the two. Divided. The gates are periodically released at one point of the tube and replenished at another point so that the grown culture is separated from the growth chamber with the growth chamber surface and attached stationary organisms associated therewith and replenished with fresh medium and new chamber surface. By this method, the stationary variant is not specifically selected by being removed in the region (proliferation chamber) in which selection is performed.

One general configuration that is possible without the exclusive and limiting intent is to include several members as described below. The invention will now be described in detail on the basis of the preferred embodiments with reference to the following drawings:

1 is a schematic diagram showing a possible configuration of a device according to the invention, wherein

(1) represents flexible tubing containing several areas of the device, such as upstream fresh medium 7, growth chamber 10, sampling chamber 11, and treated growth culture area 15,

(2) temperature can be adjusted according to the conditions determined by the user, and here

a. The propagation chamber 10,

b. The sampling chamber 11,

c. An upstream gate 3 defining the beginning of the propagation chamber 10,

d. A downstream gate 4 defining the distal end of the propagation chamber 10 and the beginning of the sampling chamber 11,

e. A second downstream gate 5 defining the distal end of the sampling chamber 11,

f. Turbidity that can monitor the optical density of the growing culture by the user or an automatic control system and operate the feedback control system 13 and control move the perfusion 1 based on the culture density (Terbydostat function). Pedometer (6),

g. Shown is a box controlled by a thermostat in which one or several stirrers 9 can be arranged.

It is obvious that the device elements listed in a to g may also exist outside or without the box controlled by the thermostat.

(7) represents fresh medium present in unused flexible perfusion,

(8) represents a barrel loaded with fresh medium filled perfusion for dispensing the fresh medium and perfusion during operation,

12 represents an optional ultraviolet irradiation gate,

(13) may consist of a computer connected by means of signaling with other monitoring or operating interfaces that enable automation and control of operation, such as optical density turbidimeters, temperature measuring and regulating devices, agitators and tilt motors, etc. Control system,

(14) shows an optional treatment barrel that winds up perfusion, including perfusion filled with the treated growth culture,

15 denotes the treated growth culture located downstream of the sampling chamber.

2 shows granulation where the thermostat controlled box 2 and other parts of the apparatus associated with the culture chamber may settle to the bottom to avoid dilution for agitation purposes, for gas circulation and removal purposes. Two possible locations of the device are illustrated, illustrating the fact that they can be tilted at various angles, for example, to ensure removal of aggregated (aggregated) cells.

3 to 9 are placed in a box 2 controlled by the thermostat and introduced through gates 3, 4 and 5, through which the perfusion stays during all stages of the process and is self The flexible perfusion 1 is shown in which perfusion moves along a phosphorus peristaltic movement.

FIG. 3 is a representation of state T0 of a device in which all regions of flexible perfusion were filled with fresh medium prior to inoculation of the organism for continuous culture.

4 represents the state T1 of the flexible perfusion immediately after inoculation of an organism strain.

FIG. 5 represents a state T2 of an apparatus which is a multiplier in which culture is growing in a region defined by the growth chamber 10 limited by the gates 3 and 4.

Figure 6 represents the state T3 of the device immediately after the first peristaltic movement of perfusion and associated medium, in which new perfusion and medium are introduced through the movement of gate 3 and at the same time perfusion of equal volume through the movement of gate 4 The start of the second propagation cycle is determined in which the medium and the propagation culture are transferred from the propagation chamber region 10 to the sampling chamber region 11. It is important to understand that perfusion, the medium in this perfusion, and any cultures grown in this medium all move together. Only fluid transfer occurs when fresh medium and grown culture are mixed together through agitation within the growth chamber area.

FIG. 7 is a representation of state T4 of the device in the secondary propagation cycle, wherein the organisms remaining in the propagation chamber after peristaltic movement of perfusion during this cycle now contain the nutrients supplied from the fresh medium mixed with the culture left during this stage. Can be used to multiply.

FIG. 8 represents the state T5 of the device immediately after the secondary peristaltic movement of perfusion and the medium contained therein, in which new perfusion and medium are introduced through the movement of gate 3 and at the same time through the movement of gate 4 The initiation of the third propagation cycle is determined in which volume of perfusion, medium and propagated cultures are transferred from the propagation chamber region 10 to the sampling chamber region 11.

9 represents a state T6 of the device in the third propagation cycle, which step is equivalent to state T4 and shows the tendency for further operations to be repeated. Samples of the selected organisms can be taken at any time in the sampling chamber area 11 using a syringe or other collection device.

FIG. 10 illustrates a possible profile of a tooth that determines a gate in the form of two stacked teeth sandwiching flexible perfusion. The gate may also be determined by squeezing a single tooth against a removable belt, removable clamp or other instrument that can be alternately disposed and removed at various locations along the perfusion while preventing the movement of the organism through the gate.

Detailed description of the invention

The basic operation of the apparatus is shown in FIGS. 3 to 9.

One of the potential configurations of the device is shown in FIG. 1, which shows after a new tube of sterile medium has been loaded (areas A-H divided by gates (3), (4) and (5) above). Loses).

Inoculation of the organisms selected in such a device can be performed by injecting the organisms into the growth chamber (FIG. 3) via injection (FIG. 4, region B). The culture can then be allowed to proliferate to the desired density to begin continuous culture (FIG. 5).

Continuous culture can be progressed by repeatedly moving the gated region of perfusion. At this time, the gate, perfusion, medium and any culture in the perfusion are moved simultaneously. Perfusion always travels in the same direction, where unused perfusion containing fresh medium (hereinafter referred to as "upstream" (7) of the growth chamber) moves into the growth chamber and mixes with the culture remaining there, To provide a substrate for further propagation. Prior to introduction into the growth chamber region, the medium and perfusion containing it will be separated from the growth chamber by the upstream gate 3 and remain sterile. The used perfusion containing the grown culture will move "downstream" and at the same time separate from the growth chamber by the downstream gate 4.

The gate shape does not form a feature of the present invention. For example, certain types of gates can be designed in one chain of multiple teeth moving simultaneously, or in other forms, each separated from a synchronous chain, as shown in FIG. The gate consists of a two-teeth system that cuts through the perfusion in a stacked manner, as shown in FIG. 10, to avoid contamination between the areas G and H of the perfusion through the precision of the interface between them. have. In another form, the sterile gate squeezes one tooth against one side of the perfusion and is secured when perfusion is slipped during peristaltic movement, as indicated by reference numbers 3, 4 and 5 in FIGS. It can be obtained by strongly pressing the perfusion against the chassis.

The box 2 controlled by the thermostat is obtained by known means such as a thermometer combined with a heating and cooling device.

Exhalation (gas exchange) is achieved directly with no mechanical aids using gas permeable perfusion when necessary for the propagation of cultured organisms or as necessary for experimental design. For example, flexible gas permeable perfusion can be made of silicon, but is not limited to such. Exhalation can be carried out through exchange with an artificially defined atmosphere (liquid or gas) in contact with the growth chamber or the entire chemostat, or through exchange with the surrounding atmosphere. When the experiment requires anaerobic life, flexible perfusion can be gas impermeable. For example, flexible gas impermeable perfusion can be made of, but not limited to, coated or treated silicone.

In anaerobic evolution, the areas of perfusion may be confined within a specific controlled atmosphere to control gas exchange kinetics. This can be done by making the box controlled by the thermostat airtight and then injecting neutral gas or by placing the entire apparatus in an atmospheric control chamber.

Inhibition of selection of the stationary variant is achieved by replacing the growth chamber surface with the growth medium.

The device is also designed to be able to operate in a variety of orientations with respect to gravity, i.e. can be tilted along a range of up to 360 ° as shown in FIG.

Dilution resistant variants may not be diluted by attaching to one another rather than to the chamber wall when aggregated cells are upstream and not removed from the chamber. Thus, the perfusion is generally inclined downward so that the aggregated cells fall towards the area where they can be removed from the growth chamber during the tube migration cycle. This configuration includes tilting the device so that the downstream gate is below the upstream gate in terms of gravity.

The growth chamber may be depressurized or over pressurized depending on the conditions chosen by the experimenter. There are many ways to adjust the pressure, such as applying vacuum or pressurized air to the fresh medium and perfusion through its upstream end along the growth chamber, or by alternately clamping the perfusion upstream of the growth chamber and Locking may also reduce or overpressurize the perfusion.

When the medium is contained in gas permeable perfusion, air bubbles may form in the medium. It rises up to the top of the sealed region of perfusion so that the movement of this region (and the gate defining it) will release this region to the end point of the growth chamber, sampling chamber or chemistat (region DC, B or A in FIG. 6, respectively) Until it is gathered there. If this device is tilted downwards, such bubbles will accumulate in the growth chamber or sampling chamber and be present in place of the culture. Thus, the device can be designed to be periodically inclined upwards every tube movement cycle to remove accumulated gas from the chamber.

In order to reduce the aggregation of cells in the growth chamber, oblique movement of the device and / or shaking of the growth chamber by the external device 9 may be used. Alternatively, one or several stir bars may be placed in a perfusion filled with fresh medium prior to sterilization and magnetically agitated during the culture operation.

Compared to the length of the incubation chamber, the ratio of the length of the fresh medium region defined by the upstream gate will determine the degree of dilution achieved during the cycle.

Dilution frequency is measured by the timing (chemistat function) or the density of the culture in the growth chamber with a turbidity meter (FIG. 1-sign 6), whereby the feedback control (Terbydostat is started when the turbidity reaches the threshold value) Function).

Sampling chambers can be used to analyze experimental results, to collect organisms with increased growth rates for additional culture, storage, or functioning, or for other purposes such as population counting, chemical composition testing of media, or pH testing of grown cultures. This is where the cultures grown for the purpose can be recovered. Perfusion can be included with a buried / enveloped configuration of pH indicator lines on the walls of the perfusion to permanently monitor the pH in the propagation chamber.

Any form of liquid or semisolid material can be used as the growth medium of the device. The ability to use semisolid growth substrates is a significant advance over the prior art. The proliferation medium, which determines the metabolic process enhanced by the selection process, can be selected and determined by the user.

If desired, such an apparatus may include multiple propagation chambers, such that the downstream gate of one propagation chamber becomes the upstream gate of another propagation chamber. The device, for example, allows one organism to only multiply in the first chamber and then act as a nutrient source for the other organism (or virus) in the second chamber.

According to these devices and methods, researchers and product developers can allow any strain of cultivable live cells to evolve through continuous proliferation (continuous culture) in suspension, and the improved organisms obtained constitute new strains or species. Can be. Such new organisms can be identified through mutations acquired during the culturing process, and these mutations can distinguish their progenitor genotypic features from the new organisms. According to these devices and methods, researchers can select new strains of all living organisms by separating individuals with improved proliferation rates through natural selection.

Claims (17)

A device for increasing the proliferation rate (by increasing the rate of proliferation and / or increasing the amount of proliferation) through a natural selection process of living cells or any cultivable organism in suspension, a) flexible containing culture medium ) A sterile tube, b) a movable gate (clamp) that divides this tube into spent culture containing region (downstream region), growth culture containing region (growth chamber) and fresh growth medium containing region (upstream region) C) a culture in which a portion of the growth chamber and the culture contained therein can be clamped and disconnected and separated from the growth chamber, and a portion of the fresh tubing containing the unused medium is present in the existing growth chamber and Means for moving the gate and perfusion so that it can be mixed with a portion of the associated medium. The device of claim 1, wherein the perfusion is flexible so that the perfusion can be separated into a separate chamber. The device according to claim 1, wherein the perfusion is gas permeable, mainly composed of silicon or the like such that gas exchange occurs between the cultured organism and the external environment according to the type of experiment. The device of claim 1, wherein the perfusion is gas impermeable to block gas exchange between perfusion and the external environment if the experiment requires anaerobic life. The device of claim 1, wherein the perfusion is transparent or translucent to enable turbidity measurements. The device of claim 1, wherein the propagation chamber perfusion and the medium and culture contained therein can be depressurized or over-pressurized above atmospheric pressure as needed according to experimental requirements. The device of claim 1, wherein the perfusion comprises a pH indicator in the perfusion composition or on the inner surface of the tube to allow pH measurement of the medium. The device of claim 1, wherein the growth chamber perfusion and the medium and culture contained therein can be heated or cooled if suitable for the experimental conditions. The device of claim 1, wherein the growth chamber perfusion and the medium and culture contained therein can remain stationary or agitated according to any known method. 10. The device of claim 9, wherein the perfusion can comprise one or several stir bars for agitation. The device of claim 1, wherein the areas of perfusion can be placed in a specifically controlled atmospheric area to control gas exchange kinetics. The method of claim 1, wherein the growth chamber perfusion and the medium and culture contained therein can be inclined downward for removal of aggregated cells or upwards for air removal through the function described in c) of claim 1. Characterized in that the device. Suspending live cells or any cultivable organism through natural selection processes to increase the proliferation rate (by increasing the rate of growth and / or increasing the amount of proliferation), a) in a sterile tube containing sterile growth medium Sterile injection of the starter culture to effect initial culture of the described growth chamber; b) maintaining growth conditions in accordance with experimental requirements; c) after a certain time and propagation of the associated culture, adjust the position of the gate so that the equivalent portion of fresh medium and the propagated culture (each) is moved into and out of the area defined by the propagation chamber, so that the remaining portion of the propagated culture Mixing with a portion of the fresh medium introduced to continue the growth; d) reproducing steps b) and c) until the end of the experiment in which continuous cultures are achieved and selection of variants with increased proliferation is achieved; e) recovering a sample of the culture grown from the sampling chamber when necessary. The method of claim 13, wherein the simultaneous peristaltic movement of the gates is applied such that the perfusion and the medium and culture contained in the perfusion provide an equal amount of culture to the growth chamber while providing a specific amount of fresh medium to the growth chamber. Separating and removing through the other terminus to terminate the growth cycle and initiate another new growth cycle. The method of claim 13, wherein the experiment can include as many proliferation cycles as the experimenter requires without the possibility of contamination of a separate growth chamber, and without the possibility of proliferation of a dilution resistant population. The method of claim 13, wherein during operation the experimenter can maintain growth conditions in accordance with experimental requirements, which may include temperature, pressure, optical density, chemical acidity, agitation, and expiration with various gases. The method of claim 13, wherein the combination of tilt control of the device and the operable stirrer induces proper agitation to mix the growing culture to prevent or inhibit aggregation of living organisms.

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