CN101517063A - Continuous culture apparatus with mobile vessel, allowing selection of fitter cell variants and producing a culture in a continuous manner - Google Patents

Abstract

The invention provides a method and device for growing plant, animal or stem cells in a continuous manner.

Description

Have mobile containers and produce the bactogen of culture in a continuous manner

Invention field

Described invention provides permission selection in liquid or semisolid medium to have the method and apparatus of the viable cell of high reproduction rate and special metabolisming property.For chosen process (adaptive evolution), heritable variation biology (mutant) produces in colony, and competes with other variant of same origin.With the fastest reproduction rate those are pass by relative proportion in time to be increased, and causes producing colony's (and individual organisms) of the reproduction rate with increase.This process can improve the performance of the biology that is used for commercial run or academic purpose.The present invention utilizes bactogen to realize the feasible production of viable cell (for example, prokaryotic organism, archeobacteria (Archaea), virus, eukaryote, fungi, algae, yeast, vegetable cell, zooblast or stem cell).The present invention can be used to produce activeconstituents or the biological products that generated by viable cell.Described activeconstituents or biological products can be used as diagnostic reagent, preventive or therapeutical agent again.

Background of invention

To the growth that the selection of the reproduction rate (fitness) that increases need continue, the latter realizes by the periodic dilution of grown culture.In the prior art, this realizes by three approach: solid culture, and serial dilution and cultured continuously, their main difference is dilute strength.

Solid culture comprise with the grown culture of small volume or cell from the culture dish repetitive displacement to the container that fresh growth medium is housed.Serial dilution comprises grown culture repetitive displacement with small volume to big many containers that fresh growth medium is housed.When cultured cells has grown to when saturated, repeat this process in new container.At document (Lenski﹠amp; Travisano:Dynamics of adaptation and diversification:a 10,000-generation experimentwith bacterial populations.1994.Proc Natl Acad Sci U S is A.15:6808-14) in, this method has been used to realize to continue the longest demonstration of cultivating, and is confirming that clearly reproduction rate is in time period several years in the consistent experiment that improves.This process is manual usually carries out, and quite takes the labor force, and contaminated by being exposed to outside atmosphere.As described in hypomere, it also is inefficient that series is cultivated.

The raising rate dependent of selecting speed or reproduction rate in group size (Fisher:TheGenetical Theory of Natural Selection.1930.Oxford University Press, London, UK).In addition, be similar under the serial transfer case of group size rapid fluctuations, selecting harmonic-mean with colony

Proportional (Wright:Size of population andbreeding structure in relation to evolution.1938.Science 87:430-431), and therefore can estimate by the minimum colony in the working cycle.

Can keep group size by cultured continuously, and therefore make selection more efficient.The cultured continuously that is different from serial dilution comprises littler volume relatively, thereby regularly replaces the small portion grown culture with isopyknic fresh growth medium.Increase by the minimal size that makes it in the circulation dilution, this process makes effective group size maximization.Allow the equipment of cultured continuously to be called " chemostat " (taking place) and " turbidostat " (if when culture grows to specific density, diluting automatically) if be diluted in designated time intervals.

For simply, two kind equipments will be included into term " chemostat " group hereinafter.Chemostat is invented (Novick﹠amp by 2 groups simultaneously in nineteen fifties; Szilard:Description of thechemostat.1950.Science 112:715-716) and (Monod:La technique de laculture continue-Th é orie et applications.1950.Ann.Inst.Pasteur 79:390-410).Chemostat has been used to confirm that the short time period of reproduction rate improves (DykhuizenDE.Chemostats used for studying natural selection and adaptiveevolution.1993.Methods Enzymol.224:613-31) fast.

Because the selection in (immobilized) variant of anti-dilution unplanned, traditional chemostat can not be kept long-time section and select high reproduction rate.These variants can tolerate dilution by adherent to the chemostat surface, and, compete (outcompete) more low-viscosity individuality by doing like this, comprise those with higher reproduction rate, thereby have eliminated the intended purposes (Chao﹠amp of equipment; Ramsdell:The effects of wall populations on coexistence of bacteria in the liquidphase of chemostat cultures .1985.J.Gen.Microbiol.131:1229-36).

Invent a kind of method and permanentization equipment (the Genetic Engine) and avoided dilution resistance (the PASTEUR INSTITUT[FR] ﹠amp of continuous culture; MUTZEL RUPERT[DE] the patent US 6,686 that submits to, 194-B1).This method uses the fluid transfer of valve control to come regular mobile grown culture between 2 chemostats, thereby allows each to sterilize between active culture growth time section and wash.Regularly sterilization cycle is by destroying the selection that the variant of anti-the dilution prevents them.This method and apparatus has been realized this purpose, but need carry out independently complex operations to several fluids in aseptic (sealing) environment, comprise a kind of (NaOH), it is extremely corrosive and potential extremely reactive, pollution of destructive valve, and generation rapidly and waste disposal problem.Perseveranceization equipment also is conditional, because do not take measures to provide the upholder of cell growth.Such as the equipment of GeneticEngine and other known technology do not allow the non-suspension growth of cultured continuously and/or as the cell of adherent cell growth.

The a large amount of cultured cells that are difficult to that have some types, this is because cell survival and the required condition of growth.Think that these cells can grow under the condition that the cultured continuously method provides.Be so specifically for eukaryote (for example, algae, yeast, fungi, vegetable cell, zooblast and stem cell) situation.

For example, common following cultivator embryonic stem cell: separate stem cells group also shifts the laboratory plastic culture dish that the nutrient broth that is called substratum into is housed with it.Cell is in division of culture dish surface and diffusion.The internal surface of culture dish is coated with treated associating usually and makes them with nondividing mice embryonic skin cells.This cell coating layer is called feeder layer.The reason that has feeder layer in the culture dish bottom is to provide their viscous surface that can adhere to human embryo stem cell.Equally, feeder cell discharge nutrition in substratum.Recently, scientist has begun to design the mode of the cultivation embryonic stem cell that does not use the mouse feeder cell.This is great scientific progress, because it has avoided viral or other macromole in the mouse cell may pass to the danger of people's cell.

In several days process, the cell proliferation of inner cell mass, and begin to be crowded with culture dish.When this takes place, they are taken out gently, and pave plate and advance in several fresh culture dish.The process that repeats to pave again plate (replate) cell repeatedly with a plurality of months, and this is called the cultivation of going down to posterity.Each cycle of cultured cell line is called goes down to posterity.6 months or more of a specified duration after, the initiating cell of cell mass produces millions of embryonic stem cells.Differentiation ground in cell culture propagation 6 months or more a plurality of months, be multipotency and heredity on the embryonic stem cell of acting normally, be called embryonic stem cell line.

In case established clone, or even before this stage, just can they are freezing in batches, and be transported to other laboratory, be used for other cultivation and test.But, cultured continuously provide suppress maximum to the viable cell pressurization and produce the advantage of the operation of potential pollution source.When beginning to cultivate, cultured continuously conditions permit technician utilizes the continuous production of cell.In case generate stem cell, just can continue to produce stem cell incessantly, to generate the much more stem cell of method of generally using than now.

Summary of the invention

Therefore, the objective of the invention is, be provided for not being subjected to the variant of anti-dilution the (and fully independently) method and apparatus of the improvement of cultured continuously cell (comprising prokaryotic organism, bacterium, archeobacteria, eukaryote and virus) intrusively.As other chemostat, this equipment provides the instrument with fresh growth medium periodic dilution grown culture, is used for carrying out between culture and outside atmosphere gaseous interchange, aseptic and as chemostat or the turbidostat instrument of operation automatically.

In addition, the objective of the invention is, be provided for improvement and unique method and apparatus of cultured continuously cell, described cell is suspension growth and/or grow as adherent cell not, for example eukaryote (for example, vegetable cell, algae, fungi, yeast, zooblast or stem cell) and some prokaryotic organism (for example, streptomycete) and some archeobacteria of possibility and virus.Can utilize stem cell that the present invention cultivates including, but not limited to embryonic stem cell, fetal stem cell, umbilical cord stem cell, placenta deutero-stem cell and adult stem cell.Can utilize adult stem cell that the present invention cultivates (for example including, but not limited to hemopoietic stem cell, bone marrow stem cell, stroma cell, astroglia cell and oligodendrocyte, Hematopoietic Stem Cell Protocols by C.Klug and C.Jordan, Humana Press, Totowa, New Jersey, 2002, be incorporated herein by reference).

The present invention is designed to realize these purposes without any fluid transfer (comprising sterilization and washing function).This is representing the concrete advantage of the present invention with respect to prior art in following ranges: it has been avoided and sterilization and wash relevant harm and difficulty, comprises transfer, pollution and because the growth disorder that culture causes to another container transfer from a container of the container (containment) that contains aggressive solvent and complex fluid.

In the flexible sterile tube that has been full of growth medium, realize cultured continuously.Substratum and chamber surface are static each other, and by making tubing system (tubing) wriggling pass " door " or by preventing that cultured cells mobile clip between the territory, area under control from sterilely segmenting the point of pipe, regularly and simultaneously replaces the two.For further safety, also can (randomly) add ultraviolet ray (UV) door at the upstream and downstream of culture vessel.

Present method and equipment also are the improvement that is better than prior art in following ranges: they select continuously rather than termly to overcome the variant of anti-dilution adherent to the chemostat surface, because replace affected surface along with the dilution series connection takes place.Equally for suspension cultured cells with as the cell of adherent cell growth, tubing system provides the continuous upholder of growth, and its advantage is not by cell is shifted and interference cell to another container from a container.

Segment pipe in instantaneous mode, thereby make and have the zone of containing saturated (fully growing) culture, zone and the zone between these two of containing fresh culture, wherein there are one or more chambers that are called growth room or culturing room, to form growth chamber region, grown culture is mixed therein with fresh culture, to realize dilution.A door regularly point from the pipe discharges, and replaces at another point, so that by separating the resting cell of removing grown culture and its bonded growth chamber surface and adhering to from the growth room, and with fresh culture and fresh chamber surface replacement.

The accompanying drawing summary

Exhaustively non-and without limitation, possible general configuration will comprise several components hereinafter described.Hereinafter, on the basis of preferred embodiment, exemplarily explain the present invention, therefore with reference to the accompanying drawings, wherein:

Fig. 1 has shown the panorama of the possible configuration of equipment, wherein:

(1) representative contains the flexible duct system of the different zones of equipment, and described zone is: upstream fresh culture (7), growth room (10), sampling hut (11) and the grown cultures zone (15) of disposing

(2) representative allows the constant temperature controlled box of the condition attemperation determined according to the user, and can place therein:

A. described growth room (10),

B. described sampling hut (11),

C. limit the upstream door (3) of the beginning of described growth room (10),

D. limit the downstream gate (4) of the beginning of the end of described growth room (10) and described sampling hut (11),

E. limit second downstream gate (5) of the end of described sampling hut (11),

F. allow the turbidometer (6) of the optical density(OD) and the operational feedback Controlling System (13) of user or automatic control system monitoring growth culture, thus the motion of control tubing system (1) on culture density (turbidostat function) basis,

G. one or several agitator (9).

Should be pointed out that the equipment component of listing also can be positioned at the outside of constant temperature controlled box in a-g, or do not have the constant temperature controlled box.

(7) fresh culture in the untapped flexible duct of the representative system,

(8) representative is mounted with the bucket (barrel) of the tubing system that fresh culture fills up, with described fresh culture of distribution and tubing system in operating process.

(12) the optional ultra-violet radiation gates of representative,

(13) represent Controlling System, the computer that it can be linked by the instrument with different monitoring or operation interface communication is formed, described instrument such as optical density(OD) turbidometer, temperature survey and conditioning equipment, agitator and incline motor etc., they allow automation of operation and control

(14) the optional disposal barrel of representative, volume is being scratched the tubing system of the tubing system that grown culture that disposal is housed fills up above it,

(15) representative is positioned at the grown culture of the disposal in downstream, described sampling hut.

Fig. 2 has shown two kinds of possible positions of equipment; their illustrations the following fact; being described constant temperature controlled box (2) can tilt to different angles with other parts of the described equipment relevant with described culturing room, and this is may be avoided the purpose of granular (accumulative) cell of diluting by being deposited to the bottom in order to stir purpose, gas circulation and removal purpose and to guarantee to remove.Growth at cell does not need under the condition of stirring, and described equipment also can be positioned at same position (for example, flat position or certain angle is arranged).

The described metal hose (1) that Fig. 3-9 representative is arranged in described constant temperature controlled box (2) appropriate location and imports into by door (3), (4) and (5), all pass through these doors at pipe described in all process steps and stop, and described pipe will pass through these doors according to its vermicular movement.

Fig. 3 represents equipment state T0, and wherein before the injection expection was used for the cell of cultured continuously, the All Ranges of described metal hose was all filled fresh culture.

The state T1 of Fig. 4 representative described metal hose after just injecting cell strain.

Fig. 5 represents equipment state T2, and it is the growth time section, and in the meantime, culture is grown in the zone of the growth room (10) that is defined as described door (3) and (4) qualification.

Fig. 6 representative is managed and the equipment state T3 of wriggling the first time of relevant substratum after just taking place, it has determined the beginning of second growth cycle, motion by door 3 imports fresh tube and substratum, and the motion by door 4 simultaneously migrates out isopyknic pipe, substratum and grown culture growth chamber region (10) and enters zone, sampling hut (11).Key is to recognize, pipe, the substratum in pipe and all motions together of any culture of having grown in described substratum.Fluid transfer only takes place by being stirred in fresh culture and the grown culture mixture scope together in the growth chamber region.

Fig. 7 represents equipment state T4, and it is second growth cycle; In this periodic process, behind pipe wriggling, be retained in cell in the growth room can utilize now with residue culture in this step process mutually the nutrition that provides of blended fresh culture grow.

The equipment state T5 of Fig. 8 representative after the second time of the substratum of managing and comprising, wriggling just took place, it has determined the beginning of the 3rd growth cycle, motion by door 3 imports fresh tube and substratum, and the motion by door 4 simultaneously migrates out isopyknic pipe, substratum and grown culture growth chamber region (10) and enters zone, sampling hut (11).

Fig. 9 represents equipment state T6, and it is the 3rd growth cycle; This step is equivalent to state T4, and indicates the repetitive nature of other operation.Use syringe or other recoverable apparatus, can take out the sample of the cell of selecting at any time from zone, sampling hut (11).

Figure 10 has shown the possible overview of the tooth of the door in the decision configuration, and it piles up tooth by 2 that squeeze metal hose and forms.Door also can be passed other mechanism decision of the motion of door by the single tooth of extruding movable belt, removable clip or prevention cell, and it can alternately place and take off in variable position along pipe.

Detailed Description Of The Invention

The elementary operation of equipment has been described in Fig. 3-9.

A kind of possible configuration that has shown this equipment in Fig. 1 is as its (is shown as by described door (3), (4) and (5) and is divided into regional A-H) that occurs after the fresh tube of loading aseptic culture medium.

By injection (Fig. 4, area B) cell is imported growth room (Fig. 3), can be to the cell of equipment inoculation selection.Allow the density that culture grows to be needed then, and can begin cultured continuously (Fig. 5).

Cultured continuously is carried out in the repeating motion in the gate zone by pipe.Motion when this comprises any culture in door, pipe, substratum and the pipe.Pipe always moves with equidirectional; The untapped pipe " upstream " of (and be called hereinafter in the growth room (7)) that contains fresh culture will move forward into the growth room, and mix mutually with remaining culture wherein, thereby provide matrix for the further growth of the cell that wherein contains.Before importing growth chamber region, by separating with the growth room through upstream door (3), the pipe that this substratum is relevant with it maintains under the aseptic condition.Contain exhausted pipe " downstream " motion simultaneously of grown culture, and separate with the growth room through downstream gate (4).

When having one or more growth room, the growth room can be used for identical or different purpose.For example, viable cell can be grown in first growth room with identical or different condition and second growth room.In one embodiment, first growth room can be used for grown cell, and second growth room then can be used for handling viable cell under different condition.For example, can handle cell, to induce the expression of required product.Before or after cultivating beginning, can add the component or the additive of substratum self.For example, all components or additive can be included in the substratum before cultivating beginning, or can after beginning cultivation component be injected into one or more growth rooms.

The door configuration is not the particular point of present patent application.For example, in given configuration, door can be designed to pass a chain of a plurality of teeth of motion simultaneously, or in another configuration, separates in different synchronous chains as shown in Figure 1.Door can be made up of the system that 2 teeth that squeeze pipe with as shown in figure 10 stack manner are made, thereby accurate by the contact surface between the tooth avoids the G of pipe and the pollution between the H zone.In another configuration, can followingly obtain aseptic door: a tooth is pressed against a side of pipe, and will manage thus and closely press against on the fixed chassis, pipe along this chassis slip, is illustrated as Fig. 3 to 9 mark 3,4 and 5 in its wriggling process.In another configuration, use following mechanism to move pipe, wherein clip is around the stationary shaft rotation about this equipment.

Obtain described constant temperature controlled box (2) by known methods, for example with heating and cooling equipment link coupled thermometer.

When the growth of cultured cells or experimental design need ventilation (gaseous interchange), directly realize by using permeability cell, and do not use mechanical assistance.For example and without limitation, can make flexible permeability cell with siloxanes.Ventilation can be by realizing with the ambiance exchange or by exchanging with the artificial atmosphere of determining (liquid or gas) that contacts growth room or whole chemostat.When requirement of experiment anoxybiosis, metal hose can be air-locked.For example and without limitation, flexible airtight pipe can be made by siloxanes coating or that handled.

For anaerobic evolution conditions, also can be in specific and controlled atmosphere zone, with the pilot-gas exchange kinetics with the region limits of pipe.This can followingly realize: by making described constant temperature controlled box airtight, and then to wherein injecting neutral gas, or by entire equipment being placed controlled indoor of atmosphere.

By replacing growth chamber surface, realize the anti-selection of static variants with growth medium.

In addition equipment design is become and that is to say that with a plurality of direction operations tilt as shown in Figure 2, scope is up to 360 ° about gravity.

If thereby aggregating cells can fall into the upstream and avoid removing from the chamber, then the variant of anti-the dilution can be avoided dilution by sticking to each other rather than adhering on the locular wall.Therefore, wish that pipe is downward-sloping usually, thereby make the zone that aggregating cells will fall and will take out from the growth room in pipe moving period process.This configuration comprises makes device inclined, thereby makes downstream gate be lower than the upstream door with regard to gravity.

According to the condition that the experimenter selects, can make growth room's decompression or overvoltage.Can use the mode of different adjusting pressure, for example by it upstream termination and pass the growth room and use vacuum or pressurized air for fresh culture and pipe; By alternately squeezing and be locked in upstream, growth room or inner pipe, can realize that another kind makes the mode of pipe decompression or overvoltage.

When in permeability cell, containing substratum, can in substratum, form bubble.They will rise to the top in seal of tube zone, and be hunted down there, and the motion of (with the door that limits it) is discharged into this zone at the terminal point (Fig. 6 is respectively region D-C, B or A) of growth room, sampling hut or chemostat up to this zone.If equipment is downward-sloping, these bubbles will accumulate in growth room or the sampling hut, and replace culture.Equipment design is paired in pipe the period of motion regularly is inclined upwardly, thereby allow the gas of accumulation to remove from described chamber.

The banking motion of equipment and/or peripheral equipment (9) shake the growth room, can be used to reduce the gathering of growth room's inner cell.Perhaps, can comprise one or several stirring rod filling in the pipe of fresh culture before the sterilization, and in cultivating operating process magnetic agitation.Do not need under the condition of stirring in the cell growth, machine can remain on same position.

The fresh culture base region that the upstream thresholding is fixed and proportional length of culturing room's length will be limited to the dilute strength that reaches in the periodic process.

Can be by regularly determining the frequency (for example, chemostat function) of dilution.For example, can gather in the crops stem cell, thereby begin to collect stem cell before the differentiation at cell in limited time range.Perhaps, can determine the frequency of dilution, measure the density of culture in the growth room thus by turbidometer (Fig. 1-mark 6), and when turbidity reaches threshold value, dilution circulation (turbidostat function) takes place with feedback regulation.Pipe can be transparent or translucent, to measure turbidity.

The sampling hut allows to take out grown culture, the cell that has the growth velocity of raising with analysis experimental result, collection is used for further cultivation, preservation or function execution or other purpose, for example count colony, check the pH of the chemical constitution of substratum or test vector generation for testing IC culture or mass production cell (for example, eukaryote for example stem cell, prokaryotic organism, archeobacteria and virus).In order to realize the lasting monitoring of pH in the growth room, pipe can comprise the pH indicatrix of embedding/crust in tube wall by structure.

The liquid of arbitrary form or semisolid material can be as the growth mediums in this equipment.The ability of utilizing semi-solid growth matrix is the marked improvement that surpasses prior art.Can select and limit growth medium by the user, the latter will limit the metabolic process that improves by chosen process.

If desired, this equipment can contain a plurality of growth rooms, thereby makes the downstream gate of a growth room become another upstream door.For example, this will allow the growth separately in first Room of a kind of cell, and be used as the nutrition source of second kind of cell (or virus) in second Room then.

The present invention can be used to produce preparation, for example is used for medicine, vaccine or antitoxic biological products, and it is synthetic from cultured cells of the present invention or their product.Described biological products can be used as diagnosis, prevention or therapeutical agent.For example, the present invention can be used for manufacture of therapeutic protein, for example Regular Insulin.

In preferred embodiments, described equipment and/or method can circulate in a certain way, are in the stem cell of their undifferentiated state with continuous collection.In addition, can modify culture condition, to suppress the differentiation of stem cell.For example, can in substratum, add differentiation of stem cells inhibitor (for example, the inhibitor of the inhibitor of aldehyde dehydrogenase, phosphoinositide 3-kinase, TGF receptor kinase inhibitor, TGF-B receptor kinase inhibitor etc.).Perhaps, can raise or reduce process condition, for example be delivered to the amount of the oxygen of substratum, with the growth that improves some stem cell and/or slow down or improve the differentiation of stem cell.

Because the growth needs matrix of some cell can add physics upholder or structure in vessel culture chamber.In preferred embodiments, can add continuous upholder in pipe, the continuous fibre bed as being made of thin continuous fibre sample support structure can add in the vessel culture chamber, and it allows the growth of cell three-dimensional ground.For example, described upholder can be a fibre bed, it provides the upholder of cell growth, described cell is the cell of this support structure of preference of stem cell, vegetable cell and other type for example, or cannot suspension growth, or as the adherent cell growth, and in some certain conditions or condition variation, to carry out the natural selection of target mutation.

In preferred embodiments, the people such as Huang that are incorporated herein by reference at this paper, Continuous Production of butanol by Clostridium acetobutylicumimmobilized in a fibrous bed reactor, Appl Biochem Biotechnol.2004Spring; Fibrous material described in the 113-116:887-98.Also can change the structure and the size of pipe, need not that support structure is mixed movably vessel culture chamber.In preferred embodiments, use to have the more pipe of minor diameter, thereby cell can be adherent in more natural mode, or, need not the adherent of some adherent cell the effective natural support of doing.

This equipment and method allow researchist and product developer by continuing to grow (cultured continuously) but any strain of the viable cell of the suspension culture of evolving out or on the tube wall or on upholder, grow can not suspension culture any strain of viable cell, described upholder can be the fibre bed in the pipe; The improved cell that obtains can constitute new strain or species.By the sudden change that obtains in the culturing process, can differentiate these new cells, and these sudden changes can allow new cell and their ancestral gene type characteristic area to separate.This equipment and method allow the new strain of researchist by any viable cell of following selection: by natural chosen process, separate the individuality of the reproduction rate with raising.The present invention also provides improved and diverse method and apparatus, and it is used for the cultured continuously cell, eukaryote for example, (for example, yeast, fungi, vegetable cell, algae, zooblast or stem cell), prokaryotic organism, archeobacteria and virus.

In another embodiment, can make cell suffer at least one radiation wave, light wave, x ray, sound wave, electromagnetic field, radioactivity field, radiating medium or their combination enduringly or temporarily with radiator.Following publication is incorporated herein by reference: Biofizika.2005Jul-Aug, 50 (4): 689-92; Bioelectromagnetics.2005Sep, 26 (6): 431-9; Chem Commun (Camb).2005Jan 14, (2): 174-6; BiophysJ.2005Feb, 88 (2): 1496-9; Bioelectromagnetics.1981,2 (3): 285-9; Sb Lek.1998,99 (4): 455-64; Antimicrob Agents Chemother.2004Dec, 48 (12): 4662-4; J Food Prot.2003Sep, 66 (9): 1712-5; Astrobiology.2006Apr, 6 (2): 332-47; Life Sci Space Res1970,8:33-8; Adv Space Res.1995Mar, 15 (3): 211-4; Radiat Res.2006May, 165 (5): 532-7; Mutagenesis.2004Sep, 19 (5): 349-54; Cancer Sci.2006Jun, 97 (6): 535-9; Appl Environ Microbiol.2006May, 72 (5): 3608-14; With Pol J Microbiol.2005,54Suppl:7-11.

In another embodiment, can make the growth chamber region of equipment make cell be in different gravity enduringly or temporarily.For example, can be in microgravity environment culturing cell.Following publication is incorporated herein by reference: J Gravit Physiol.2004Mar; 11 (1): 75-80; Immunol Rev.2005Dec; 208:267-80; With J Gravit Physiol.2004Jul; 11 (2): P181-3.

Those skilled in the art are from the detailed description of front of the present invention, apparent of the present invention modification and the variation relevant with apparatus and method.These modifications and variation are intended to fall in the scope of appended claims.

Claims (45)

1. be used for cultivating in a continuous manner the equipment of viable cell, it comprises:

The metal hose and the surface of substratum are housed, and viable cell therein can suspension growth or suspension growth not, adheres to or does not adhere to, and wherein said pipe is transparent or translucent, to measure turbidity; With

Clip systems, each self energy are in and open and close the position, thereby clip is placed feasible pipe can being divided into like this:

I) upstream region of untapped substratum is housed;

The downstream area of exhausted substratum ii) is housed; With

Iii) place the growth chamber region that is used to cultivate described cell between the described upstream and downstream zone;

Wherein said clip systems is configured and arranges to open and close, so that open at the clip of the growth chamber region of the pipe between the upstream and downstream zone of pipe and be limited to the growth chamber region of the pipe between the upstream and downstream zone of pipe, and periodically limit the growth chamber region of pipe again, thereby the first part of the growth chamber region that limits before making becomes the part of pipe downstream area, and the part of the upstream region of the pipe that limited in the past becomes the part of the growth chamber region of pipe.

2. according to the equipment of claim 1, wherein said clip systems is configured and arranges, thereby when described clip was in the closed position, each clip was not with respect to the pipe motion.

3. according to the equipment of claim 1 or 2, wherein said surface is the internal surface of pipe.

4. according to the equipment of claim 3, wherein said surface is the continuous upholder in the tubular stinger.

5. according to the equipment of claim 4, wherein said surface is a continuous fibre.

6. according to the equipment of one of claim 1-5, wherein said pipe is gas-pervious.

7. according to the equipment of one of claim 1-5, wherein said pipe is air-locked.

8. according to the equipment of one of claim 1-7, wherein said pipe comprises siloxanes or any flexible material.

9. according to the equipment of one of claim 1-8, wherein said equipment comprises pressure-regulator in addition, and described setter is fabricated the pressure with the corresponding environmental stress of growth room's part that changes pipe.

10. according to the equipment of one of claim 1-9, wherein said pipe comprises the pH indicator.

11. according to the equipment of one of claim 1-10, comprise thermoswitch in addition, described setter is fabricated the temperature with the growth chamber region that allows control tube.

12. according to the equipment of one of claim 1-11, wherein said equipment comprises agitator in addition, described agitator is fabricated to allow growth room's part of stirring pipe.

13. according to the equipment of claim 12, wherein said agitator comprises at least one stirring rod.

14. according to the equipment of one of claim 1-13, comprise radiator in addition, described radiator is fabricated so that the grown cultures chamber region suffers following at least one: radiation wave, light wave, x ray, sound wave, electromagnetic field and radioactivity field.

15., comprise in addition and make growth chamber region suffer the instrument of different gravity according to the equipment of one of claim 1-14.

16. according to the equipment of one of claim 1-15, wherein said growth chamber region comprises one or more growth rooms that substratum is housed.

17., comprise the turbidometer that is used for the turbidity in the measuring tube in addition according to the equipment of one of claim 1-16.

18. according to the equipment of claim 1-17, wherein each clip can move around the stationary shaft rotation about described equipment, thereby can move pipe.

19. according to the equipment of one of claim 1-18, be additionally contained in the pH indicator in pipe composition or the lining, to allow to measure the pH of substratum.

20. according to the equipment of one of claim 1-19, comprise inclination device in addition,, or be inclined upwardly to remove air so that growth room's pipe is downward-sloping with the removal aggregating cells.

21. according to the equipment of one of claim 1-20, wherein said equipment allows the cultured continuously cell, wherein said cell is eukaryote, prokaryotic organism, archeobacteria and virus, and wherein said cell is suspension growth in substratum not, or suspension growth in pipe not.

22. according to the equipment of one of claim 1-21, wherein said equipment allows the cultured continuously cell, wherein said cell is eukaryote, prokaryotic organism, archeobacteria and virus, and wherein said cell is grown as adherent cell.

23. the method for culturing cell in a continuous manner, it comprises:

A) provide flexible, transparent or translucent pipe and the surface that substratum is housed, wherein viable cell can be grown in described pipe, and clip systems, and each clip can be in and open and close the position, thereby clip is placed feasible pipe can being divided into like this:

I) upstream region of untapped substratum is housed;

The downstream area of exhausted substratum ii) is housed; With

Iii) place the growth chamber region that is used to cultivate described cell between the described upstream and downstream zone;

B) with the turbidity in other measurement device pipe of turbidometer or measurement turbidity;

C) close clip on the pipe of selection, introduce growth chamber region with the pipe growth chamber region between the upstream and downstream zone that is limited to pipe with viable cell;

D) turbidity in the monitoring pipe is up to the growth of the relevant needs that obtain culture;

E) periodically close and open selected clip, to limit the growth chamber region of pipe again, thereby the first part of the growth chamber region that limits before making becomes the part of pipe downstream area, and the part of the upstream region of the pipe that limited in the past becomes the part of the growth chamber region of pipe; With

F) repeating step e), up to the cell of cultivating q.s.

24. according to the method for claim 23, it comprises the additional step that takes out some viable cell from the described substratum of downstream area.

25., comprise in addition from downstream area and separate described viable cell according to the method for claim 23 or 24.

26. according to the method for one of claim 23-25, wherein said viable cell is selected from: algae, fungi, yeast cell, zooblast, vegetable cell and stem cell.

27. according to the method for claim 26, wherein said viable cell is a stem cell.

28. according to the method for claim 27, wherein said viable cell is selected from: hemopoietic stem cell, bone marrow stem cell, stroma cell, astroglia cell, oligodendrocyte, embryonic stem cell, fetal stem cell, umbilical cord stem cell, placenta deutero-stem cell and adult stem cell.

29. according to the method for one of claim 23-28, wherein the surface of cell growth is the internal surface of pipe.

30. according to the method for one of claim 23-29, wherein the surface of cell growth is a continuous fibre.

31., be additionally contained in culturing cell in the one or more growth rooms that exist in the growth chamber region according to the method for one of claim 23-30.

32., be additionally contained in the cell that growth chamber region is cultivated one or more types according to the method for one of claim 23-31.

33. according to the method for one of claim 23-32, wherein said pipe is gas-pervious.

34. according to the method for one of claim 23-32, wherein said pipe is air-locked.

35., comprise the pressure of the corresponding environmental stress of growth room's part of adjustable pipe in addition according to the method for one of claim 23-34.

36., comprise the pH that measures substratum in the growth chamber region in addition according to the method for one of claim 23-35.

37. according to the method for one of claim 23-36, comprise the temperature of regulating growth chamber region with thermoswitch in addition, described thermoswitch is fabricated the temperature with the growth chamber region of control tube.

38., comprise in addition with the substratum in the agitator stirring growth chamber region according to the method for one of claim 23-37.

39. according to the method for one of claim 23-38, wherein said agitator comprises at least one stirring rod.

40. according to the method for one of claim 23-39, comprise in addition and make the grown cultures chamber region suffer following at least one: radiation wave, light wave, x ray, sound wave, electromagnetic field and radioactivity field.

41., comprise in addition and make growth chamber region suffer different gravity according to the method for one of claim 23-40.

42. method according to claim 23, wherein step e) allows following dilution: provide the fresh culture from upstream region of specified quantitative to enter the growth room, another end by described growth room simultaneously, separate from the growth room and the culture that takes out equivalent to downstream area.

43. method according to claim 23 or 42, wherein step e) is additionally contained in and moves pipe between each growth cycle, thereby make growth room's pipe upgrade with the importing of untapped substratum, thereby prevent anti-possible propagation of diluting colony, avoid the possible pollution of isolating growth room simultaneously.

44. according to the method for one of claim 23-43, wherein said cell is suspension growth not.

45. according to the method for one of claim 23-44, wherein said cell is grown as adherent cell.

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