CN1260343C

CN1260343C - Cell culture device

Info

Publication number: CN1260343C
Application number: CNB031008534A
Authority: CN
Inventors: 张景明
Original assignee: CESCO Bioengineering Co Ltd
Current assignee: CESCO Bioengineering Co Ltd
Priority date: 2002-01-31
Filing date: 2003-01-23
Publication date: 2006-06-21
Anticipated expiration: 2023-01-23
Also published as: CN1435482A

Abstract

The present invention relates to a novel cell culture device which can be used for efficiently culturing cells with a method to obtain the minimum death rate, and the maximum cells as well as generated cell products. The present invention also provides the method and the device for inoculating cells and enhancing the adhesion of cells to be cultured, and provides a growth substrate to provide the maximum cell adhesion area and act as an oxygen-enriching unit, a filter and a static stirrer. By periodically and intermittently providing nutrients and oxygen for cells, cellular proliferation is promoted to generate the maximum cell products. The economical device can be disposable to reduce sequelae caused by incomplete sterilization. Nutrients and oxygen can be provided for the cells periodically and intermittently by controlling the contact frequency of a culture medium and the grown substrate.

Description

Cell culture apparatus

Technical field

The present invention is a kind of cell culture apparatus, because culturing micro-organisms, cell and/or when organizing, maximum obstruction is that the equipment that uses and/or device volume are huge, expensive, can't uses up promptly and lose, be difficult for sterilization and keep gnotobasis, thereby has increased the chance of pollution; And, cause microorganism, cells injury easily and/or even cause necrocytosis at culturing micro-organisms, cell and/or when organizing.The present invention provides a kind of technology that can be used for any microorganism of vitro culture and cell basically and collect its any by product that generates.More specifically, the present invention is protection and discloses equipment and/or device and method about a kind of novelty, can increase and collect the by product that microorganism and cell are generated effectively, for example: protein.

Background technology

As everyone knows, large-scale cell cultures program is in being developed widely in recent years, and is used in culturing bacterium, yeast and mould etc. and has tough cell walls and/or extracellular matrix (extracellular material) than the rubber-like cell.The whippy structure of microorganism cells is the key factor that this type of high-level efficiency cell cultures program can be fast-developing, for example: bacterial cell can utilize fierce vibrations in a large amount of substratum, rock and ventilating technology makes its abundant ingress of air, and keeps good growth situation.In addition, bacterium also can grow on microbial film, but needs one provide the plane of growth.

On the contrary, the technology of cultivating eukaryotic cell, zooblast, mammalian cell and/or tissue is with regard to difficulty and complicated many, reason is the fragility that this type of cell is next than microorganism cells, and the required nutrient of its growth and oxygen keep also comparatively difficulty and complexity.In addition, different with microorganism cells is: zooblast and/or mammalian cell are impatient at bubbling air or mixed gases, for example: a mixed gases comprises oxygen, nitrogen and carbonic acid gas, violent disturbance that is caused and/or shearing stress (shearing force).And zooblast is ingress of air directly, and the most animals cell only can utilize the oxygen that is dissolved in the substratum.Compared with microorganism cells, zooblast and mammalian cell are easier to sustain an injury by gas or in venting process, therefore increases its mortality ratio.The bio-reactor that is applied to large scale culturing possesses an internal operations device usually, for example: agitating wing (impeller), make cell bear very high liquid shearing stress, and cause the injury or the death of cell, cause the low survival rate of cell and the output of protein and/or byproduct thereof also to decrease.Same, for cell evenly being suspended and/or suitable ingress of air, and the bio-reactor that utilizes him to plant mechanism, fierce gas motion or intensive liquid movement also will cause the injury and the growth that hinders cell or tissue of cell, further cause the byproduct of cell, for example: the minimizing of protein output.

The main function of bio-reactor is to cooperate research to need to cultivate a large amount of cells to extract its deutero-trace activity substance, includes but not limited to protein or the antibody of emiocytosis in the substratum; Another function of bio-reactor is the active protein that large-scale cell cultures is produced in order to the mass production cell or tissue, to cooperate the coml demand.Because eukaryotic cell and/or prokaryotic cell prokaryocyte and/or zooblast and/or mammalian cell be a large amount of needs of cultivating in the laboratory, bio-reactor and culture apparatus have been played the part of important role at research and production cell with the aspect of making active protein and/or antibody and/or any cell byproduct.

Many traditional methods can be used to extensive or the small-scale culturing cell.Aspect the small-scale cultivation, developed the container that many cultivations in recent years, for example: culture dish promptly is an example.Culture dish comprises a chassis that can be used to the splendid attire substratum basically, and the loam cake that can raise.Though the loam cake that can raise is handled easily really, yet frequent flip action often makes cell suffer microbiological contamination also in culturing process.And in fact, how avoiding polluting is the ultimate challenge that the cell or tissue culture technique of a success is faced.

For avoiding using the normal pollution problem that takes place of culture dish, culturing bottle (cultureflasks) so develop.Culturing bottle contains a culturing room basically, a tubular opening is positioned at an end of bottle and the lid of a correspondence.This kind design has been to reduce the chance of cells contacting to dust, bacterium, yeast and other source of pollution.Patent about culturing bottle has, for example: No. the 5th, 398,837, No. the 4th, 851,351, United States Patent (USP) that No. the 4th, 334,028, the United States Patent (USP) that Carver company is had, Akamine company are had and United States Patent (USP) that Degrassi had.

Though culturing bottle comes progressively than culture dish, it is the problem of contaminated solution fully still.In addition, culture dish and culturing bottle all can't provide competent air, and therefore the growth area that culturing bottle provided has limited the scale of cultivating not as the abundance of culture dish.

It is roller culturing bottle (rollerbottles) that another kind is used for the technology of culturing cell and tissue, extensively utilize roller culturing bottle technology in the existing several years of cell cultures, though it has some culture dish or the unexistent advantage of culturing bottle, for example: bigger growth of cell and bond area are provided, yet still can't overcome all shortcomings, especially about enlarged culturing scale aspect.Generally speaking, these shortcomings comprise, but are not restricted to, because the gas of headspace (gas headspace) stirs the huge fluid shearing stress that is caused with a large amount of rotations.Because the high shear stress environment of roller culturing bottle causes the tissue culture of bigger three-dimensional structure almost impossible.Have the injury that can not be subjected to shearing stress only and/or can keep the cell that is attached at roller culturing bottle inwall and just continued to cultivate for some time.Hence one can see that, keeps for a long time the difficulty of cultivating a cell strain in the roller culturing bottle and be: its high shear stress environment and contamination of heavy.Example about the roller culturing bottle comprises: No. the 4th, 962,033, the United States Patent (USP) that No. the 5th, 527,705, United States Patent (USP) that Mussi company is had and Serkes company are had.

In addition, though the entire area of roller culturing bottle greater than culture dish or culturing bottle, yet the area of its each cell attachment that offers might not be greater than the area that culture dish or culturing bottle provided, particularly when large scale culturing.

So, at the example that improves the growth area that the rolling culturing bottle can provide as, the United States Patent (USP) the 5th that Serkes company is had, 010, No. 013 is to describe a kind of roller culturing bottle of promoting the cell attachment surface-area, it is the passage that adds a kind of crape folding shape in the inwall of roller culturing bottle, and the internal surface area that increases the roller culturing bottle is for cell attachment.Yet general roller culturing bottle only provides the area of about 850-1700 square centimeter, so still need a large amount of roller culturing bottles just to be enough to reach mass production.Though and the culture systems of automatization, a large amount of roller culturing bottle can be saved time and manpower, however the suitable costliness and have restricted of its operation.

Except the restriction of the problem of fluid shearing stress and the area of growing, obtaining with keeping sufficient oxygen supply also is the central issue of cell and tissue culture technique.Know as those skilled in the art scholar, prokaryotic cell prokaryocyte, eukaryotic cell, the growth that comprises zooblast, mammalian cell, insect cell, yeast and mould all has a main rate limiting step (rate-limiting step), i.e. oxygen quality conversion (oxygen mass transfer).

Except some carries out the eukaryotic microorganisms of fermentative action, for example: beyond the yeast, prokaryotic cell prokaryocyte and eukaryotic cell for majority, the metabolism of oxygen is considerable for the metabolic function of cell, especially at the culture technique of mammalian cell and zooblast, the supply of oxygen is important especially again for the quick splitted commitment of cell.When cell suspension, to having the call of oxygen, and after cell aggregation or differentiation, the oxygen demand will reduce.Some mammalian cell and zooblast interdependent for adhering to (anchorage-dependent), in addition some mammalian cell and zooblast then can be in liquid environment suspension growth, promptly, non-ly adhere to interdependent (anchorageindependent), yet all cells need grow in all in the sufficient dissolved oxygen environment.In the later stage of cell cultures, no matter be to adhere to the interdependent or non-interdependent cell that adheres to, the cell quantity of per unit volume can roll up, thereby needs a large amount of oxygen quality conversion once again, so that enough oxygen to be provided.

Basically, at least for the cell of apposition growth, can utilize the mode of machine stirring and feeding gas to supply the oxygen of its demand, yet, as previously mentioned, stir and the action of ventilation all can injure cell, thus lower its survival rate and whole cell and/or tissue culture efficient and output.In addition, directly feed gas in cell and tissue culture medium (TCM), can cause bubble to produce, be unfavorable for cell survival equally.

The device that the problem that oxygen provides during for the solution cell cultures is invented, for example: No. the 5th, 153,131, the United States Patent (USP) that Wolf et al. is had is the biological reaction tank (bioreactor vessel) that does not have agitating wing (blades) about a kind of.This biological reaction tank is that air is fed by gas distribution channel (airinlet passageway), (support plate member) passes screen (screen) via a supporting plate element, and by being sandwiched in the flat discous permeable membrane (flat disk permeable membrane) between reactive tank outer container (housing) both sides, utilize the oxygen concentration gradient between the outer container both sides, oxygen can be spread, arrive culturing room (culture chamber) by permeable membrane.

Yet the biological reaction tank of Wolf has many shortcomings:

1, especially, oxygen becomes the key constraints of culturing room's scale via the speed of flat discous permeable membrane diffusion.

2, to be that flat discous permeable membrane is designed to scalable having agitating function for the another one shortcoming, and this point but may cause the death of cell.Utilize agitating function so that even air is distributed in the substratum is considerable, yet, also will increase the shearing stress of culturing room inside in the time of stirring, may cause the injury of pair cell as previously mentioned.Therefore, when design bio-reactor or culture tank, how to provide enough gaseous interchange,, be quite important and be the restriction of a reality to keep the bigger cellularstructure of cultivation.

In order to solve the device of described shortcoming, utilize the reactor of gas permeable material (gas permeable material) manufacturing, for example: the United States Patent (USP) the 5th that Schwarz et al. is had, 702, No. 941, name is called " breathable bio-reactor and using method thereof ", be the container that horizontally rotates about a kind of, and its wall of container is that part adopts gas permeable material, in the hope of reaching the purpose of directly carrying out gaseous interchange by this gas permeable material and substratum.

Yet, the scale of the disclosed device of Schwarz still is restricted, reason is that gaseous interchange needs to decide according to the size of ventilative area, though Schwarz emphasizes to increase when the surface-area of device, the volume of substratum also increases relatively, and the scale of disclosed preferred embodiment is limited to 1 to 6 inch of diameter in its specification sheets, then between 1/4 to 1 inch, such size not proper growth has three-dimensional cell assembly thing and tissue and/or any a large amount of scale production to width.

Similarly, the United States Patent (USP) the 5th that Falkenberg et al. is had, 449, No. 617, title is " culture vessel that is used for cell cultures ", it is the device that horizontally rotates about, this culture vessel is by a dialysis membrane, inner area is divided into cell cultures chamber and nutritive medium storage tanks (nutrient mediumreservoir), gas permeable material is used in this wall of container, make cell culture chamber can carry out gaseous interchange, but the nutritive medium storage tanks is not to be full of nutritive medium fully yet, but a large amount of gas is all kept in the nutrient solution of two chambers top.Yet the Falkenberg container does not design in order to reduce the disturbance of cell cultures chamber interior, and opposite, in order to keep the humidity of dialysis membrane, stirring is a necessary step; In addition, Falkenberg and this container of not mentioned use are assembled the thing or the tissue of any kind in culturing cell.

For the invention of the problem that solves oxygen supply also has, for example: the United States Patent (USP) the 5th, 766 that Liau had, No. 949, title is " method and apparatus of interdependent monolayer cell is adhered in cultivation ", is to describe a kind of substratum that shakes up and down, to increase the culture systems that oxygen provides.Its main drawback is:

1, one of them is the complicacy of its device.The system of Liau comprises two external storage tankss and a separate type culturing room of containing a series of vertical base materials square position (vertical substrate plates), also need most most creeping motion type pump housings (peristaltic pumps) to promote nutrient solutions in addition by the storage tanks culturing room that flows through, flow to another storage tanks again, flow back into first storage tanks at last again.Because the device of Liau is complicated and some is the equipment of culturing room outside, for example: exterior tubing, storage tanks and the pump housing, so very likely the external pollution source is imported inner, in addition, because the equipment of device is huge, sterilizing works is difficult for carrying out, again labor intensive.

2, another shortcoming of the invention of Liau is a nutrient solution when flowing through system, and the fluid shearing stress that is caused may disturb and move the cell that grows on the base material square position, thereby lowers the survival rate of cell.

3, in addition, the design of vertical base material square position also is unfavorable for cell attachment, because some cell that can't adhere at once can drop and be deposited in the bottom of square position, these cells are last mostly can be dead, therefore reduce the survival rate and the protein output of cell, and this system must over and over again restart, and is quite inefficent, and undesirable.

4 moreover, because the complicacy of this system, it is all pretty troublesome and consuming time to collect any excretory protein or cellular products.At last, when growth media was lower than the matrix square position of cell growth, cell will be exposed in the air,, directly contacts atmosphere surrounding that is, therefore will cause necrocytosis.

Summary of the invention

The present invention provides and discloses the method and apparatus of a kind of cell and tissue culture, because cell and tissue culture technique are for all suitable important of biotechnology research, drug research, patient care and academic research.For defective and the restriction that overcomes conventional art, and supply its inconvenience, the invention provides more reliable, more uncomplicated, more efficient, save trouble, more not expensive, than human-saving and can increase a kind of novel method and the device of the output of cell survival rate and cell by-products, with satisfied needs for a long time.Device of the present invention reduces the life-span of polluting with the increase cell simultaneously.

The purpose of this invention is to provide a kind of method for cultivation of bacteria and device thereof, be used for culturing cell and/or tissue and collect its product reliable, simple and easy and cheap, can use up and promptly lose and efficient method and apparatus.More specifically, the present invention provides and discloses a kind of method and apparatus of novelty, can effectively cultivate the desire cultured cells, for example: prokaryotic cell prokaryocyte, eukaryotic cell, zooblast and mammalian cell, can continue to provide oxygen and nutrient but can not allow any cells contacting air, therefore can reduce the injury even the cell mortality of cell.

In addition, method and apparatus of the present invention is to reduce pollution, avoids cell directly to be subjected to the injury of shearing stress when gas is provided, thereby can prevent the negative impact of bubble and gas pair cell.Device of the present invention can be automatic or manual operation, moreover that device of the present invention provides is one simpler, method production more easily and collecting cell product, for example: protein, and/or antibody, and/or the product of any cell and/or tissue.

The object of the present invention is achieved like this: a kind of cell culture apparatus is characterized in that: which comprises at least:

First chamber has first end, second end, an internal surface and an outside surface, and this internal surface has at least one growth base material, to accept at least one cell and to allow this cell to attach and/or adhere to;

Second chamber is with the handing-over of second end of described first chamber or links to each other that this second chamber has first end, second end, an internal surface and an outside surface, at least one separating between first end that device is positioned over second end of this first chamber and second chamber; This separates device makes cultivation flow based on coming and going between this first chamber and second chamber;

This cell culture apparatus is exposed in air or the substratum this growth base material off and in turn, and the thin layer interface that makes cell pass through air-substratum increases the exchange of oxygen.

Described second chamber is the compressible element with first end and second end, and optionally the outside surface in first end of compressible element provides first lid, and provide second lid in the outside surface of second end of compressible element, this first lid combines with second lid, makes that the compressible element in second chamber is to be compressed between first lid and second lid.

It further comprises: an air ambient is the inside that is positioned at this first chamber; Substratum is the compressible element inside that is arranged in this second chamber, and this substratum separates device and/or film via one, moves freely between this first chamber and second chamber; And optionally use a drive unit to compress and the compressible element that stretches in this second chamber, so that should cultivate, have at least a cell to attach and/or the growth base material that adheres to can be immersed in this substratum when this second chamber compresses based on moving between this first chamber and second chamber; When this second chamber stretches, be exposed to air ambient indirectly by one air-substratum thin layer interface, so that oxygen to be provided.

Described first chamber comprises at least one opening, is to be used for accepting or mobile substratum and cell, and allows cell culture apparatus and extraneous environment exchange of air.The opening of described first chamber optionally is equipped with a stopping device.Described stopping device is optionally to install an air filter screen, to prevent pollution.

Described drive unit is an automatic or manual.Separate device between first end of described second end that places first chamber and second chamber, it is one to have infiltrative film or porousness dividing plate, and this cultivation is flowed based on coming and going between first chamber and second chamber.

Described have infiltrative film for optionally having porous.

Described growth base material is the matrix of a loose assembly.Described matrix has dissolved oxygen hyperplasia device, static mixer or deep-bed filter.

Described growth base material is half permeable sac, or porous particle, static agitation, increase dissolved oxygen, filtration is provided and intercepts cell.

Described growth base material comprises at least one semi-permeable tubular fibre.

Described cell is to be selected from eukaryotic cell or prokaryotic cell prokaryocyte.

The amount of the substratum of described growth base material is to regulate and/or control by a floatoblast and a pneumatic tube.

This second chamber further includes a volume setting device, enters the substratum deal of first chamber with regulation and control; One control device is the described volume adjustment means of control, substratum is flowed between first and second chamber, make when substratum at the volume of second chamber to hour, substratum covers the growth base material fully, and when substratum at the volume of second chamber when being maximum, the interface indirect contact of growth base material by one air-substratum is to the air ambient of first chamber.

Described volume adjustment means is bellows, by compression and stretching, extension, to control the volume of the second chamber interior substratum, make when bellows are compressed to the limit, substratum is minimum at the volume of second chamber, and when bellows were stretched over the limit, substratum was maximum at the volume of second chamber; Or a balloon, by inflation with lose heart, to control the volume of the second chamber interior substratum, make when balloon inflation arrives the limit, substratum is minimum at the volume of second chamber, and when balloon lost heart to the limit, substratum was maximum at the volume of second chamber; Or a piston, wherein push away on the piston or drop-down, to control the volume of the second chamber interior substratum.

Described drive unit is an oil cylinder, a pneumatic cylinder, a screw jack or a gas compression device.

Described growth base material is a porous growth base material, cell is coated within the porous growth base material, and when growth base material emersion during in substratum, the interface indirect contact of the cell on the growth base material by one air-substratum be to air ambient, and obtain enough oxygen.

The material of described porous growth base material is selected from following group: pottery, braiding base material, non-braiding base material, polymeric amide, polyester, urethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol.

The form of described porous growth base material is dish shape, laminar, block, plate-like, sheet, band shape, granular, semipermeability particle or semipolar linkage.

The present invention includes but be not limited to provide a kind of method and apparatus of novelty, in order to preparation, grow and keep cell; The method and apparatus that one novelty is provided is with growth and/or preparation tissue culture;

The method and apparatus that one novelty is provided is with preparation and growth organ cultures;

Provide the method and apparatus of a novelty, culturing cell under the environment of competent oxygen and nutrient supply and minimum pollution;

Provide the method and apparatus of a novelty to go up culturing cell,, but can not make cell directly touch bubble and/or shearing stress because of injecting gas with the oxygen of reinforcement cell and the exchange of nutrient at porous growth base material (porous growthsubstrate);

Provide all one's life long substrate with the sticking power of strengthening cell, increase the contact area of air and nutrient solution, and as the substratum agitator of static state in the device;

The method and apparatus of a novelty is provided, cell can intermittently be immersed or the emersion substratum;

Provide a method and apparatus that cell contamination is minimized, cell mortality is minimized, thereby increase the output of cellular products;

Fluid shearing stress when providing a method and apparatus can make cultivation reduces in a large number even does not have fully, and cell mortality is minimized;

Provide a method and apparatus when cultivating, not need to come supplied with medium oxygen by ventilation;

Provide a method and apparatus that substratum can be moved, allow the sub-distribution and help to promote the adhesive ability of cell again of the cell that drops;

Sustainable supply cell culture medium of one method and apparatus and oxygen are provided, make the output of cellular products reach maximum, provide more area by cell attachment;

Provide the method and apparatus can efficient preparation and protein and other product of collecting cell and/or tissue secretion; And the method and apparatus that a culturing cell is provided, the survival rate of cell can be improved after feasible cultivation for some time.

The present invention provides a cell culture apparatus, and it is to comprise a chamber at least.Described cell culture apparatus can further comprise:

First chamber has first end, second end, an internal surface and an outside surface, and wherein internal surface has at least one structure to accept at least one cell and the attaching of permission cell and/or to adhere to;

Second chamber is to join or link to each other with second end of described first chamber, this second chamber has first end, second end, an internal surface and an outside surface, places comprising at least one film or a communication means between first end of second end of first chamber and second chamber.

Described second chamber is to comprise the compressible element with first end and second end, and optionally the outside surface in first end of compressible element provides first lid (cap), and provide second lid in the outside surface of second end of compressible element, wherein when second chamber does not use, first lid can combine with second lid, makes that the compressible element in second chamber is to be compressed between first lid and second lid.

Described cell culture apparatus still can further include: an air ambient is the inside that is positioned at first chamber; Substratum is the compressible element inside that is arranged in second chamber, and this substratum can move freely between first chamber and second chamber via a communication means and/or film; And optionally use a drive unit to compress and the compressible element that stretches in second chamber, so that cultivate based on moving between first chamber and second chamber, make that the structure that has at least a cell to attach and/or adhere to can be when second chamber compresses, be immersed in the substratum, and when second chamber stretches, by one air-substratum thin layer interface, and be exposed to air ambient indirectly, so that oxygen to be provided.

First chamber of described cell culture apparatus comprises at least one opening, can be used to accept or mobile substratum and cell, and allows cell culture apparatus and extraneous environment exchange of air.The opening of described first chamber can optionally be equipped with a stopping device, and optionally installs an air filter screen, to prevent pollution.By controlling this stopping device, the compression and the stretching, extension of second chamber of arranging in pairs or groups more can reach the ability of the pressure environment of control first chamber, and directly press on the growth base material effect of pressurize.For example: tighten this stopping device, compress second chamber simultaneously, can increase the pressure in first chamber; Tighten this stopping device, stretch second chamber simultaneously, can reduce the pressure in first chamber.By increasing or reduce pressure on the growth base material, can be used as the purposes of cultivating the particular variety cell or tissue, for example: sclerocyte (osteoblast) or artificial bone tissue.

Described cell culture apparatus is the cultivation that can do three-dimensional tissue.

First kind embodiment of the present invention is a cell culture apparatus, it comprises at least: an inside is placed with first chamber that the growth of cell attachment and growth base material (growth substrate means) is provided, the base material of wherein growing is to be positioned in the air ambient of first chamber, the growth base material of novelty of the present invention is can be when inoculating cell (cell plating), as catching and keep here the deep-bed filter (depth filter) of cell, also can increasing the contact area of air and substratum and as the agitator (static mixer) of a static state.This device comprises that also selectivity is connected to second chamber of first chamber, ways of connecting can to have infiltrative and better be the porous film by a kind of, and this first chamber can be positioned at top, below and/or the next door of second chamber, and/or be around mode, can help substratum between first and second chamber, to flow arbitrarily with this.

The inventive system comprises a drive unit, can drive cultivation based on commute between chamber, get back on the growth base material with the cell that the sub-distribution again of gentleness is dropped, thereby can strengthen the cell attachment ability, can pass through the mixing substratum, and help contacting of gas and substratum, and do not injure or the prerequisite of cell killing under, guarantee that nutrient and gas are evenly distributed.

The drive unit that the present invention uses comprises: riser, oil cylinder, pneumatic cylinder, gas compression device, screw jack (screw jack), pulley (pulley) etc.

Another kind of embodiment of the present invention is a cell culture apparatus, it comprises at least: inside comprises and/or supports first chamber that the growth base material of cell attachment and growth is provided, the base material of wherein growing is to be positioned in first chamber with air ambient, device of the present invention also alternative comprises and is connected to first chamber, and be used for preserving second chamber of substratum, wherein second chamber comprises that at least one can regulate and/or regulate and control the device of volume, can be used to regulate the amount of substratum in second chamber, make when the substratum of second chamber is minimum, most substratum can flow in first chamber, yet when the substratum furnishing maximum of second chamber, most substratum all flow back in second chamber.This kind volume adjustment means can include, but are not limited to a compressible chamber, and for example: bellows, piston, balloon, the floatoblast that pneumatic tube is arranged, air pressure and/or any other can move cultivation based on back and forth instrument between chamber.

Device of the present invention also can comprise a drive unit and the volume adjustment means of controlling fluid and/or liquid, can further help substratum intermittently between first chamber and second chamber, to flow, make substratum can cover the growth base material fully so that cell nutrient and dissolved oxygen to be provided.Therefore, when substratum was minimum, the cell on the growth base material just can touch the thin layer interface of one air-substratum, to obtain enough oxygen supplies.And when the substratum of first chamber was maximum, cell then can touch the nutrient in the substratum.By regulating and control the amount of substratum in first chamber, when cells contacting arrives the interface of air-substratum, there is the skim moisture film to separate between cell and the air, so both can provide competent oxygen to cell, but can not allow the cell overdrying, also can not allow cell directly touch shearing stress and/or bubble because of entering of air, pair cell damages or adverse influence.

A class embodiment more of the present invention is the method about a culturing cell, for example: prokaryotic cell prokaryocyte, eukaryotic cell and/or mammalian cell.The method of novelty provided by the present invention may further comprise the steps at least: first chamber is provided; Put into the growth base material and be used for culturing cell and strengthen cell attachment power; Second chamber is provided; Second chamber is linked to first chamber; Provide cultivation based on second chamber; Optionally install a fluid and/or liquid squeezer in second chamber,,, but do not injure or cell killing so that enough nutrients of cell and oxygen to be provided so that substratum intermittently flows between first and second chamber; The cell that drops of sub-distribution again is to increase cell attachment; And the interface that one air-substratum is provided with provide a kind of and can replenish fresh culture, and collect the mechanism of the cellular products in the substratum.Therefore, the present invention not only promotes cell yield by increasing cell attachment, and easy conduit also is provided, and device of the present invention is to sterilize in advance, and use up promptly and abandon, it is minimum that the infection probability of cultivation is reduced to, and also can avoiding repeats to sterilize uses the problem that is caused.In addition, the invention provides the method for a novelty, is can be intermittent, but non-ly directly provides cell oxygen via the thin layer interface of one air-substratum, but can not injure and/or cell killing.

Another embodiment of the present invention is the method according to front one embodiment, further comprises by the product of collecting the emiocytosis of being desired in the substratum again.This method comprises the following step at least: collect substratum from cell culture apparatus; Collect substratum inner cell excretory protein; The amount of extremely enough supplying the cell growth with additional fresh substratum to first chamber is provided; And periodicity and the mobile substratum of intermittence mobile coming and going between first and second chamber, make when the substratum of second chamber is transferred to minimum, substratum can flow to first chamber makes the growth base material can be immersed in the substratum, and when the substratum of second chamber is transferred to maximum, substratum can be got back to second chamber again, makes the growth base material touch air ambient by the interface of air-substratum and obtains oxygen.This kind intermittence, but non-ly directly utilize the interface of air-substratum to allow cells contacting on the growth base material, and be immersed in the method in the substratum to rich oxygen containing environment, the supply of one isostatic oxygen and nutrient can be provided, but can not injure and/or cell killing.

An embodiment more of the present invention is a cell culture apparatus, comprises at least: a chamber, have first end and second end, and it can be equipped with substratum and in the second end culturing cell, contain air ambient in first end; All one's life, long substrate was positioned over chamber interior for cell attachment and growth; One substratum storage tanks is that substratum and interconnection with chamber is housed; And a drive unit, be can be intermittent with periodically make cultivation based on flowing between substratum storage tanks and the chamber, make the base material of growing be immersed in the substratum or be exposed in the air ambient indirectly by one layer of air-substratum film; One platform scale combination is to combine with drive unit, and the weight that can respond to chamber is with the unlatching of accessory drive or close.Described cell culture apparatus is further to utilize a rotating shaft and cushion cap combination, and stirring with rotation increases cell inoculation and substratum mixed effect.

Describe in detail below in conjunction with preferred embodiment and accompanying drawing.

Description of drawings

Fig. 1 is embodiment 1 synoptic diagram of cell culture apparatus of the present invention, wherein second chamber is positioned at the below of first chamber, and can optionally have compressibility, make substratum between first and second chamber, to flow, wherein, not compression this moment of compressible second chamber is so the growth base material is to be exposed to the interface of one air-substratum so that oxygen to be provided.

Fig. 2 is that second chamber of Fig. 1 is a compressed state, and the growth base material is to be immersed in the substratum, so that cell obtains nutrient.

Fig. 3 is the synoptic diagram of the embodiment 2 of cell culture apparatus of the present invention, one of them distensible balloon places and/or is arranged in second chamber, this balloon is to be in gas-leaking state, the growth base material of first chamber substratum is detained at second chamber, so just can be exposed in the air ambient by the thin layer interface of one air-substratum.

Fig. 4 is that the balloon of Fig. 3 is to be in inflated condition, thereby promotes substratum and flow to or be transported to first chamber from second chamber, and the cell on the growth base material can be immersed in the substratum.

Fig. 5 is the synoptic diagram of the embodiment 3 of cell culture apparatus of the present invention, one of them piston places second chamber, and fully elongate, make substratum be detained at second chamber, so the growth base material of first chamber just can pass through the thin layer interface of one air-substratum, and be exposed to indirectly in the air ambient, so cell can never directly touch gas or air.

To be Fig. 5 up push away the piston of second chamber Fig. 6, flows to or transport or move to first chamber from second chamber to force substratum, the growth base material can be immersed in the substratum, so that the cell nutrient to be provided.

Fig. 7 is the synoptic diagram of the embodiment 4 of cell culture apparatus of the present invention, and the base material of wherein growing is a tubular fibre.

Fig. 8 is the synoptic diagram of the embodiment 5 of cell culture apparatus of the present invention, and the base material of wherein growing is a semi-transparent membrane vesicle and/or container and/or carrier.

Fig. 9 is the synoptic diagram of the embodiment 6 of cell culture apparatus of the present invention, wherein after the inflation of the inflation mechanism of second chamber, growth base material in first chamber just can float on the substratum, and is exposed in the air ambient indirectly by the interface of one air-substratum.

Figure 10 is that the growth base material in first chamber just can be immersed in the substratum after the inflation mechanism of second chamber of Fig. 9 lost heart.

Figure 11 is the synoptic diagram of the embodiment 7 of cell culture apparatus of the present invention, and its chamber at the middle and upper levels has compressibility, can control the flow direction of the substratum in the chamber of lower floor.

Figure 12 be Figure 11 by compressing the upper strata chamber, the growth base material in lower floor's chamber is immersed in the substratum.

Figure 13 is the synoptic diagram of the embodiment 8 of cell culture apparatus of the present invention, and wherein the substratum content of first chamber is to be controlled by the air pressure that is surrounded on its second chamber on every side.

Figure 14 is a growth base material of the present invention, is by compressing compressible second chamber, the growth base material being immersed in the substratum.

Figure 15 is a growth base material of the present invention, wherein by stretching compressible second chamber, the interface of growth base material by one air-substratum is exposed to indirectly obtains oxygen in the air ambient.

Figure 16 is the synoptic diagram of the embodiment 9 of cell culture apparatus of the present invention, is to combine with embodiments of the invention 1, and wherein the mobile of second chamber is by piston mechanism control, and when second chamber did not compress, substratum can be detained in it.

Figure 17 is that Figure 16 utilizes piston mechanism to control second chamber in compressed state.

Figure 18 is the synoptic diagram of the embodiment 10 of cell culture apparatus of the present invention, is to combine with embodiments of the invention 1 that wherein screw jack combination is used as the instrument that control second chamber compresses.

Figure 19 is that the screw jack combination of Figure 18 is used for compressing second chamber, makes substratum to flow to first chamber by second chamber.

Figure 20 is the synoptic diagram of the embodiment 11 of cell culture apparatus of the present invention, wherein contains a kind of drive unit, is used for compressing the chamber that substratum is housed.

Figure 21 is that Figure 20 contains a kind of drive unit and is used for compressing second chamber that substratum is housed, and makes substratum to flow to first chamber by second chamber.

Figure 22 is the synoptic diagram of the embodiment 12 of cell culture apparatus of the present invention, is to be driven tool with the balloon, as described in Fig. 3,4.

Figure 23 is that the balloon of Figure 22 is in inflated condition.

Figure 24 is the synoptic diagram of the embodiment 13 of cell culture apparatus of the present invention, is with the volume adjustment means of different drive units as this cell culture apparatus, so that substratum is flowed between chamber.

Figure 25 is the synoptic diagram of the embodiment 14 of cell culture apparatus of the present invention, be with another kind of drive unit in conjunction with embodiments of the invention 6, make the pressure of exterior chamber than the internal chamber height, therefore most substratum can flow to internal chamber by exterior chamber.

Figure 26 is the synoptic diagram of the embodiment 15 of cell culture apparatus of the present invention, be in conjunction with embodiments of the invention 1, and comprise two storage tankss that are attached to cell culture apparatus of the present invention in addition, give cell so that replenish fresh substratum, and, collect the cellular products that is dissolved in the substratum via concatenator that is attached to cell culture apparatus and/or pipeline.

Figure 27 is the synoptic diagram of the embodiment 16 of cell culture apparatus of the present invention, be in conjunction with embodiments of the invention 1, and comprise two storage tankss that are attached to cell culture apparatus of the present invention in addition, give cell so that replenish fresh substratum, and collect the cellular products that is dissolved in the substratum via the pump housing that cell culture apparatus includes.

Figure 28 is that Figure 27 optionally uses the electronic apparatus system or the computer control pump housing, so that collect the cellular products that is dissolved in the substratum.

Figure 29 is the synoptic diagram of the embodiment 17 of cell culture apparatus of the present invention, be in conjunction with embodiments of the invention 1, and comprise two storage tankss that are attached to cell culture apparatus of the present invention in addition, give cell so that replenish fresh substratum, and use the pump housing to collect the cellular products that is dissolved in the substratum.

Figure 30 is that Figure 29 optionally uses the electronic system and/or the computer control pump housing, so that collect the cellular products that is dissolved in the substratum.

Figure 31 is the synoptic diagram of the embodiment 18 of cell culture apparatus of the present invention, be to comprise at least one cell growth chamber, the open space that at least one is inner, and further comprise movably grow base material and a drive unit and link, drive unit is a riser, makes the growth base material to be immersed in the substratum.

Figure 32 is that the drive unit of Figure 31 is a riser, makes the growth base material be free to up and down or move left and right, so that the emersion substratum is to growth chamber.

Figure 33 is the synoptic diagram of the embodiment 19 of cell culture apparatus of the present invention, be to comprise at least one cell growth chamber, the open space that at least one is inner, and further comprise movably grow base material and a drive unit and link, drive unit is a screw jack, and the growth base material can be immersed in the substratum.

Figure 34 is that Figure 33 further comprises movably grow a base material and a driven tool binding, driven tool is a screw jack, make the growth base material be free to up and down or move left and right, so that the emersion substratum, and the amount of oxygen of each growth chamber is increased to maximum.

Figure 35 is the synoptic diagram of embodiments of the invention 20, is to comprise a chamber, and filling growth base material attaches growth for cell in it, and other has a storage tanks to take up substratum.Utilize the weight of a platform scale sensing chamber, when chamber weigh less than certain set(ting)value the time, can send signal by controller and start the air pressure pump housing, and unlatching or valve-off, flow between storage tanks and chamber with the control substratum, make the growth base material be soaked in substratum in, or the thin layer interface indirect contact by one air-substratum is to air ambient.In order to promote the stirring efficiency of reactor, utilize the combination of a rotating shaft and cushion cap, stirring with rotation increases cell inoculation and substratum blended effect.

Figure 36 is the synoptic diagram of the embodiment 21 of cell culture apparatus of the present invention, is to comprise the top that first chamber is positioned at compressible second chamber, and first chamber interior contains the growth base material for cell growth, and second chamber then takes up substratum; The activity with movable valve of moving by a lifting gantry can make cell culture apparatus internal liquid flow field unification, that is cause growth base material internal liquid to flow from top to bottom, can help cell retention in carrier.

Figure 37 is the synoptic diagram of the embodiment 22 of cell culture apparatus of the present invention, is the open and close that utilize the pilot-gas valve, with flowing of control chamber room pressure and substratum, so that carry out the action that substratum is changed; This substratum is changed action and can be done process control by computer, do and change volume and the regulation and control of changing number of times.

Figure 38 is the synoptic diagram of the embodiment 23 of cell culture apparatus of the present invention, it is the extension of embodiment 22, utilize a conduit to link the substratum liquid changing device on right side and the high-effect cell culture apparatus in left side, the running of this system is by the compression of inflation chamber and stretching, cause the change of its internal pressure, so mobile substratum by the substratum storage chamber to the cell cultures chamber, the growth base material is immersed in the substratum, or indirect contact is to air ambient by one air-substratum thin layer interface; This device can avoid frequently changing the puzzlement of substratum.

Embodiment

Following embodiment is only for explanation the present invention, and should not be used for limiting to protection scope of the present invention.Detailed description of the present invention can be understood in order to help acquisition in conjunction with the accompanying drawings as a reference more fully.Below partly preferable demonstration example is only narrated in explanation, yet should not therefore add unnecessary restriction to the present invention.Embodiments of the invention can be used for cultivating any cell, for example: eucaryon and prokaryotic cell prokaryocyte, particularly zooblast and/or mammalian cell.The present invention includes, provide a kind of and have at least one chamber, at least one opening and at least one stopping device in order to close opening.Described chamber wherein can comprise, for example: desire cultured cells, substratum and provide cell attachment and the growth base material of growth surface.In a preferred embodiment, the growth base material is a loose assembly thing, can be when inoculating cell, be used as the strainer of catching and keeping cell here, to reach maximum cell attachment degree, the growth base material also can pass through the thin layer interface of one gas-substratum, and increases the contact area of air and liquid, and the growth base material also can be used as the agitator (static mixer) of a static state.The growth base material is preferably the porous base material, and can be any size, shape and can form by any material, porous particularly of the present invention growth base material provides maximum area by cell attachment, growth, mild stirring, gentle mixing and oxygen is provided, and but will not allow cell directly touch air.

Chamber system of the present invention equally can be by periodically and intermittent cells contacting nutrient and the air of making, and promote the growth of cell.

The substratum that system of the present invention also provides an easy method to can be used for collecting to contain cellular products with upgrade substratum.

Cell culture apparatus of the present invention also can protect cell directly not touch any air, bubble or any shearing stress that causes because of ventilation, therefore can avoid any pair cell adverse influence.

Particularly, the invention relates to a kind of reliably, easily, the method and apparatus of not expensive, deserted, aseptic and efficient culturing cell and/or tissue and its product of collection.

More precisely, the invention relates to the method and apparatus of a novelty, can be used to cultivate any cell,, and can but can not hurt cell by required nutrient and the oxygen of ample supply cell growth no matter be eucaryon, protokaryon, Mammals or zooblast.

In addition, method and apparatus of the present invention can prevent or lower any type of pollution in a large number, avoids causing shearing stress and protection cell not directly ingress of air, bubble or gas.

Moreover device of the present invention can be the automatic or manual operation, can save the manpower of labour and/or management, in addition, that the present invention provides is one simpler and easy, method and apparatus easily, can be used to produce and the product of collecting cell or tissue, for example: protein, antibody etc.

The first embodiment of the present invention is one to comprise the cell culture apparatus of at least one chamber, more specifically, first chamber that cell culture apparatus comprised, its inside is to be placed with the growth of cell attachment and growth base material is provided, and the growth base material can intermittence and periodicity, but non-directly, utilize the thin layer interface of air-substratum and touch air ambient to obtain oxygen, or be immersed in the substratum, grow to help cell, and make cellular products.First chamber comprises at least one film, is preferably two, and film is preferably porous character, and better is to have perviousness, and the growth base material can be placed between double-layer films, so that the oxygen and the nutrient absorbing of control cell.Described film is to make the substratum infiltration, therefore can be used as substratum is leniently entered another chamber by a chamber passage.

Device of the present invention optionally comprises the storage tanks of second chamber as substratum, and is linked to first chamber by film, second chamber partly be device as an adjustable volume, make substratum between chamber, to flow by compression and decompression.First chamber can be positioned at top, below, the next door of second chamber, or around the mode of second chamber.

In addition, the present invention comprises that optionally a drive unit is with the control volume setting device, therefore can further help substratum periodically and intermittently between chamber, to flow, make substratum can cover cell fully so that nutrient to be provided, and the growth base material also can the emersion substratum, and the interface by one air-substratum makes cell obtain sufficient oxygen.This kind is exposed to the mode in the air ambient indirectly, can allow cell obtain sufficient and efficient oxygen supply, but can not injure and/or cell killing.

Another embodiment of the present invention is one to have cell growth and the growth base material that promotes cell attachment are provided, and the base material of wherein growing is to be positioned in the air ambient of first chamber, and first chamber combines with second chamber.Still have a film to be positioned between first chamber and second chamber in addition, described film is preferably has porous, and with as cultivating based on mobile passage between chamber, and film can support the growth base material.Second chamber is to have the device that a volume is regulated and/or liquid is controlled, make substratum between chamber, to flow, therefore, when the gas of second chamber is adjusted to minimum, most substratum all is detained in second chamber, yet when the gas of second chamber was adjusted to maximum, most substratum all flow to first chamber.Volume adjustment means can further optionally regulate, adjust or control by drive unit, and substratum dealing between first chamber and second chamber is flowed.

Therefore, when the culture volume of second chamber for hour, the growth base material of first chamber just can be periodically with intermittently be immersed in the substratum, yet when the culture volume of second chamber is maximum, the growth base material of first chamber just can pass through the interface indirect contact of one air-substratum to air ambient, to obtain sufficient oxygen.This kind is exposed to air ambient indirectly by the interface of air-substratum, so that the method for abundant oxygen to be provided, can't injure and/or cell killing, because cell does not directly touch air-flow.

First chamber of the present invention and second chamber are to be made of any material, include but not limited to polypropylene, plastics or thermoplastics.The volume of each chamber then can change, and can be identical or different volume, the volume of each chamber is preferably between about 10 milliliters to about 5000 milliliters, and better is between about 50 milliliters to about 2500 milliliters, and the best is between about 100 milliliters to about 1000 milliliters.

In another embodiment, cell culture apparatus of the present invention is to comprise that at least one can be separated the film of first chamber and second chamber or separate device, this film or separate device and have perviousness, and be preferably the porous material, and can support and/or support the base material of growing, can also be used as platform (platform).

Described permeable membrane can be made up of any material, for example, but is not limited to semipermeability fiber, semipermeability high molecular polymerization upholder (polymer support), metal or plastics.Permeable membrane can be any form that can support, support or bear the weight of growth base material.In a preferred embodiment, film is that porous and perviousness make substratum to pass in and out between chamber, but is unlikely to the interference cell cultivation and/or moves any cell, can be used as the agitator of a static state simultaneously.The permeable film that especially can add the same or similarity of another and first film is positioned over the opposite side of growth base material, and the position that the growth base material can be maintained fixed in first chamber when immersing substratum is unlikely to move and walks.

Growth base material of the present invention can be any material, include but not limited to, for example: ceramic, biological collapsible substrate, high molecular polymer, braiding base material (woven substrate), non-braiding base material (non-woven substrate), polymeric amide, polyester, urethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol, Trimethylamine 99, glass, silicon and diethylaminoethyl-(DEAE).Porous growth base material can be any form, shape or size, includes but not limited to dish shape, thin slice, bulk, plate-like, sheet, band shape, granular, microcarrier, particulate, semipermeability particle, microparticle, semipolar linkage or semipermeability tubular fibre.Using growth base material of the present invention is to increase the surface-area of cell attachment and the area of air-substratum.

The form of growth base material also can be, but be not limited to, tubular fibre, fibrous bundle, cell growth square (cell growth cube) and the ceramic core (channeled ceramiccore) that passage is arranged, this type of growth advantage that base material had is: roll up cell growth and the surface-area that adheres to.Growth base material of the present invention also can use the microcarrier substrate system that can buy on the market, for example: foregoing tubular fibre, fibrous bundle, cell growth square and the ceramic core of passage is arranged.The microcarrier that the present invention uses is the feature that can wrap up protein and/or other biological or chemical with the outside of its relevant base material, to strengthen cell attachment power or optionally to increase adhering to of specific cell type.

Micro-carrier system can help cell with the required speed growth of commercial mass production, and need not expend a large amount of manpowers, compared to roller culturing bottle and other system, cell culture apparatus of the present invention can be used for small-scale and large-scale cellular replication and cells produce simultaneously, in addition, the microcarrier bioreactor system is well suited for being used for large-scale cultivation of automatization and adheres to interdependent cell.

When tradition utilizes the microcarrier production system to cultivate to adhere to interdependent cell, need a stirring system, so that cell oxygen to be provided, this a system wherein element and another element interaction is suspended in the substratum so that carry the microcarrier particle of cell in a large number.Opposite, the present invention does not then need such stirring system that enough nutrient and oxygen is provided, because substratum periodicity and intermittent mobile between chamber, can help to stir, and can make any cell that drops be attached to the chance of growth base material again, improving the sticking power of cell, thereby can improve the growing state of cell, and increase the production of cellular products.

Material as a microcarrier, its surface chemistry composition must adhere to and growth by sustenticular cell, and can not pair cell or its deutero-product have toxicity, the diameter of ideal microcarrier is about 75-225 μ m, but also there is bigger or less size to be used, for example: (United States Patent (USP) the 5th, 114, No. 855 (in May, 1992); J.Varani, S.Josephs and W.Hillegas, " Human Diploid Fibroblast growth in polystyrenemicrocarriers in aggregates ", Cytotechnology, 22:111-117 (1996)).

The present invention provides a kind of granular growth base material of novelty, and it is various size to exist, and its diameter can be between about 1 millimeter to about 100 millimeters, yet according to individual need, any diameter is suitable diameter.In addition, in the present invention, this particle is to can be any available shape or form, and in preferred embodiment of the present invention, the growth base material is a matrix one irregular, loose assembly.

Particle of the present invention, its ideal density can be between 1.02-1.05g/cc, yet according to the needs on the different application, also can use lighter or heavier material.Except the difference of surface chemistry, particulate hardness, porousness and the degree of absorption of unlike material are all variant, performance characteristic, weather resistance, anti-storage time (shelf life) and the simple and easy degree of disinfectant etc. all can influence the cost of processing procedure, the present invention is can be according to the viewpoint of business potential, and does suitable adjustment.

Preferred embodiment of the present invention provides porous growth base material and/or particle, and this porous substrate can increase the growth and the survival rate of cell at least in three aspects.

First, when the porous particle is immersed in substratum, in the particulate hole, can produce gentle stirring action, for example: use semipermeability particle microcarrier but not whipping appts, when porous growth substrate particles flows in substratum, just created a kind of environment of mild stirring, thereby can be used as the agitator of a static state, the gentle effect that stirs of this kind, can promote cell growth, make the nutrient uniform distribution, and the cell that drops is redistributed, attached and/or adhere again to the possibility of growth on the base material once more to increase it.Redistribute the cell that drops, particularly, can increase the survival rate of cell cultures integral body at adhering to interdependent cell.

Second; when porous growth substrate particles was not immersed in substratum, its internal structure can provide cell enough oxygen, yet can protect cell to avoid its directly contact atmosphere surrounding; therefore, porous growth substrate particles can be used as an efficient dissolved oxygen hyperplasia device (oxygenator).It is unfavorable with pair cell that cell directly touches atmosphere surrounding, and further injure cell or cause death, especially zooblast, when the growth base material by substratum in emersion, some or thin layer substratum will be detained in the surface of porous particle, form the cell that a substratum thin layer covers growth or is soaked in the porous particle surface, so just can form the interface thin layer of one air-substratum, make the efficient diffusion of oxygen, allow the cell can efficient absorption oxygen, but direct ingress of air not.In addition, this porous growth substrate particles can increase the surface-area of cell growth, and allows the interface of cells contacting air-substratum.

The 3rd, this porous growth substrate particles can be used as when square position inoculation (plating) and/or inoculation (inoculate) cell, be used as the strainer of catching cell, to increase the cell attachment number, porous growth substrate particles is the matrix of a loose assembly, can be when inoculating cell, simple and efficient distribution cell, and guarantee the surface of maximum cell attachment in porous growth base material.

Gas used in the present invention can be any gaseous mixture of cultivating any cell that is applicable to, includes, but are not limited to air.First chamber of apparatus of the present invention comprises at least one opening, can be used to accept or mobile substratum and cell, and allows cell culture apparatus and extraneous environment exchange of air.This opening can have a stopping device, and installing one air filter screen between opening and stopping device optionally accepting air, and reduces and pollutes in addition, and this opening can be Any shape or diameter.

This stopping device can be Any shape or form, includes but not limited to screw-cap or spring cup.In addition, this stopping device can be any material, includes but not limited to plastics.In a preferred embodiment, stopping device is to comprise an air filter screen, to avoid microorganism, cell or any pollutent turnover cell culture apparatus.The sterile air filter screen is that to be familiar with this skill person institute known, and can available from, for example: Millipore, MA.

Cell of the present invention can be eukaryotic cell and/or prokaryotic cell prokaryocyte.In a preferred embodiment, its cell is zooblast, mammalian cell, is preferably the human cell.Cell can be any prokaryotic cell prokaryocyte or eukaryotic cell through reorganization or not reorganization, comprising: insect cell, for example: Sf-9; Primate cell, for example: Vero; Mouse cell, for example: BHK or C-127; Grey mouse cell, for example: CHO; Yeast, for example: Saccharomyces or Scizosaccharomyces; Or the human cell, for example: tumour, scleroblast (osteoblast) and mesenchymal stem cell (mesenchymal stemcells).

Prokaryotic cell prokaryocyte can be any aerobic (aerobic) or anaerobism (anaerobic), Gram-positive (Gram positive) or Gram-negative (Gram negative) bacterium or reorganization or non-reorganization, comprise, but be not limited to Escherichia coli and Bacillus subtilis.Any cell all can grow in cell culture apparatus of the present invention, in addition, adheres to and interdependently or non-adheres to interdependent cell and also be applicable to the present invention.Adhere to interdependent cell and need surface attachment growth, yet non-ly adhere to interdependent cell and grow in the aqueous suspension, the cell of all kinds all needs competent oxygen, nutrient and somatomedin to grow for it.

In another embodiment, the present invention provides the tissue that a cell culture apparatus can be cultivated three-dimensional structure, the standard that the tissue that cultivation is used for transplanting need meet FDA (FDA) just can secure permission, the standard of FDA comprises, but be not limited to, have the function of improving disease, the consistence and the reproducibility of tissue culture, and the sterilization process through authenticating.In order to reach the effect of implementing in the body, the tissue of being turned out must have three-dimensional structure, and in order to reach reproducibility, the environment of culturing cell must meet the physiological environment of human body, authentication as for sterilization process, can prove its sterility, and set up specification sheets by the aseptic detecting information of cultured tissue.

Transplantable tissue has three key features:

1) has extracellular matrix (extra cellular matrix), to keep mechanical stability and supportive (scaffolding).

2) has intercellular contact, to keep its survival and normal function.

3) have three-dimensional kenel, can distinguish growth and the daughter cell group (cellsubpopulation) who breeds.

The tissue culture mode of standard (for example: t type culturing bottle, culture dish, rolling culturing bottle and stirring rolling culturing bottle) can't be turned out the transplantable tissue of direct substitution organ dysfunction usually, its reason is: lack multidimensional (multi-dimensional) intercellular contact, and the unwanted daughter cell group of hypertrophy.

The present invention overcomes the restriction of conventional art and defective, for example: the three dimensional growth that overcomes the inhibition cell of the roller culturing bottle of conventional art and stirred reactor; Provide the control of pressure/vacuum environment, with the growing environment of the true tissue of emulation.In addition, about the production of virus, the viral yield of traditional roller culturing bottle is 195pfu/cell, and microcarrier/revolving bottle (spinner flask) is 109pfu/cell, and the present invention then is 313pfu/cell, and being increases by 2 to 3 times output.

Conventional art is because shearing stress (shear) is excessive, so damage for fragile cell, the present invention reduces, reduces and controls the stress that subtracts that disturbance that it applied follows with it, to avoid injuring the cell of some particular types.

In conventional art,, still there is other power operation meeting to influence the cell normal growth, the especially power operation of some feeder cell except the cultivation program of standard roller culturing bottle.When with roller culturing bottle and steel basin bioreactor culture cell, its renewal, the operation of displacement or supplemental medium, i.e. " substratum is new model (medium exchange mode) more ", have several disappearances, especially, to use at the steel basin bio-reactor, for example: " substratum is new models more " such as perfusion (perfusion) or batch feeding (fed-batch) modes is very difficult, because need extra other complicated apparatus that increases, for example, centrifugal filter (spin filters), separate swash plate (inclinedseparation plate) or tangential flow filtration device (tangential flow apparatuses).In addition, when utilizing traditional method and device to implement fill-up mode renewal substratum, the situation that the cell that can adhere to is washed out.

The present invention be can be when lowering perfusion shearing stress with reduce the situation that cell drops, make the disappearance of described conventional art up.

The device of adjusting of the present invention and/or adjustment volume can include, but not limited to a telescopic chamber, for example: bellows, floatoblast, piston in combination or balloon.In addition, the device in order to driving inflation chamber of the present invention can be operated for automatic or manual.

Below be further described at the device of regulating and/or adjust volume.Automatic drive of the present invention is can be by electronic installation control, for example: computer.Drive unit of the present invention is to pass through any traditional automated installation of operation, for example: and robot, control its automatization.

Volume adjustment means of the present invention is a compressible chamber, for example: bellows, then can be by compression and these bellows of stretching, extension, regulate the volume of substratum in the bellows, for example: when bellows are compressed to the limit, this moment, the volume of its inner substratum was minimum, and when bellows were stretched over the limit, this moment, the volume of its inner substratum was maximum.Particularly when bellows compress, relative substratum will flow to first chamber from bellows, and the mode that the growth base material in first chamber is placed makes cultured cells can't be subjected to the injury of shearing stress.The chamber of placing substratum can be positioned at top, below, the next door of the chamber that contains the base material of growing, or around form.By compression and stretching, extension bellows, substratum can a rule and/or the erratic timed interval between chamber, flow, to provide cultured cells optimal oxygen concentration, and optimal oxygen concentration by be familiar with this skill person tradition, for example: the personage who generally is familiar with this field all understands too high oxygen concentration and is harmful to particularly well differentiated cell for cell.Similarly, the personage who generally is familiar with this field also understood low oxygen concentration, and growth is harmful to for cell, particularly needs zooblast and the mammalian cell of oxygen to keep metabolism and growth.

The present invention more can be by the stopping device of this first chamber of control, and the compression and the stretching, extension of second chamber of arranging in pairs or groups reach the ability of the pressure environment of control first chamber, and directly presses on the growth base material effect of pressurize.For example: tighten this stopping device, compress second chamber simultaneously, can increase the pressure in first chamber; Tighten this stopping device, stretch second chamber simultaneously, can reduce the pressure in first chamber.By increasing or reducing pressure, can be used as the purposes of cultivating the particular variety cell or tissue, for example: sclerocyte (osteoblast).

Second embodiment of the present invention, its adjusting and/or the device of adjusting volume are inflatable balloons, utilize inflation or lose heart and can regulate substratum and be detained in the volume of second chamber.So when balloon inflation arrived the limit, the volume of the substratum of its second chamber was minimum at this moment, and when balloon lost heart to the limit, the volume of the substratum of second chamber was a maximum at this moment.Balloon of the present invention can be made up of any material, for example: rubber, latex or any telescopic plastics.In addition, because balloon is positioned over the chamber that contains substratum, therefore when balloon inflation, relative substratum will flow to the chamber that device has the growth base material from chamber originally, and can not make cultured cells be subjected to the injury of shearing stress.

In addition, when balloon loses heart, relative substratum will have the chamber of growth base material to flow back to chamber originally from device, the growth base material is exposed in the air ambient, and growth cell thereon just can obtain oxygen by the thin layer interface of one air-substratum.Therefore, no matter cell culture apparatus of the present invention is to use which kind of volume-adjustment and/or volume setting device, all can make cultured cells intermittent with periodically be immersed in the substratum obtaining nutrient, or the non-directly thin layer interface by one deck gas-liquid touches air ambient.The inflation of balloon is with lose heart can a rule and/or the erratic timed interval, and can adjust according to the specific cells kind, so that optimal oxygen supply to be provided.

In the third embodiment of the present invention, be to utilize piston as volume adjustment means, by mobile piston substratum is moved between chamber, for example: shift piston onto the limit, the volume that is positioned at the substratum of second chamber reaches maximum, be withdrawn into the limit and work as piston, the volume that is positioned at the substratum of second chamber just reaches minimum.

In another embodiment, cell culture apparatus of the present invention is to comprise a chamber at least, and this device can optionally comprise two chambers, that is, and and first chamber and second chamber.First chamber can be positioned at top, below, the next door of second chamber, or around mode, vice versa.

The long substrate in all one's life can be housed in first chamber, and to help cell attachment and growth, at least one film is preferably two porous, has infiltrative film, with flowing of promotion substratum, and provides an air ambient, gives the growth base material so that oxygen to be provided.The growth base material is optionally fixing and/or adheres to and/or be suspended in the inwall of first chamber.In second chamber substratum can be housed, and the device that moves based between chamber is cultivated in a control, running gear in second chamber can be automatic and/or manual operation, with a rule and/or interval of erratic any time, to provide optimal oxygen and nutrient to cultured cells.First chamber with combine with second chamber by film, in a preferred embodiment, the growth base material be to be placed between two-layer porous film.

In addition, the present invention provides first chamber that cell growth base material is housed, with second chamber that substratum is housed, wherein first and second chamber all has the difference of first end (or far-end) (distal end) and second end (or near-end) (proximal end).Second chamber is at far-end substratum to be housed, and is an air ambient at near-end.The cell cultures chamber can be with any material manufacturing, for example: plastics.First chamber and second chamber can be fitted, and also optionally are integrated.

The 4th embodiment of the present invention utilizes an airtight floatoblast and/or a container combination as adjusted volume or adjust the device of liquid, it can be fixed in a surface or optionally on the porous film, make the growth base material be positioned at its top, utilize the air of floatoblast inside to make the growth base material be able to move vertically, impel flowing of substratum simultaneously, for example: when floatoblast is inflated, the growth base material of originally being immersed in the substratum can up be pushed away, and makes the growth base material to provide oxygen in non-direct thin layer interface by one air-liquid; And when floatoblast lost heart, the growth base material can down be pushed away and be immersed in and make cell obtain nutrient in the substratum.The floatoblast associated plant can be formed for any suitable material.

Of the present invention optionally possess drive unit, for example: air compressor, with the automatic or manual inflation or the floatoblast associated plant that loses heart, to help to provide oxygen and nutrient to the cell that is attached on the base material of growing.

The 6th embodiment of the present invention provides a cell culture apparatus, comprises first chamber, and it possesses the growth base material and places and be suspended in its inside.First chamber is to contain first end (or near-end) and second end (or far-end), wherein the far-end of first chamber is that an opening towards second chamber is arranged, second chamber also contains first end (or near-end) and second end (or far-end), and wherein near-end is essentially sealing.When gas when the second chamber near-end is squeezed into, the growth base material can be immersed in the substratum; When gas is extracted out from second chamber, the growth base material can be non-directly the thin layer interface by one deck gas-substratum touch air ambient.Gas is in the extraction of second chamber or pour into, and can pass through any drive unit, and for example: air compressor drives, and described drive unit can be operated for automatic or manual.

The present invention provides a kind of method of culturing cell of novelty, can be used for cultivating eukaryotic cell and/or prokaryotic cell prokaryocyte, especially zooblast and mammalian cell, and the operation steps of the inventive method is to comprise at least:

In first chamber of cell culture apparatus of the present invention, add substratum, and make cell suspension on the growth base material;

Provide at least one surface and/or a film to support the growth base material, wherein surface and/or film are preferably porous;

First end in first chamber is provided with an opening at least, is used for adding and/or removing substratum and cell, and at least one stopping device is in order to cut out opening.Described stopping device is that an air filter screen optionally is provided, and enters chamber in order to exchange of air and minimizing pollutent.

This device provides second chamber to receive substratum equally, and a volume setting device makes substratum to move between chamber, and a kind of method of collecting the cellular products in the substratum.

Cell culture apparatus of the present invention still can further comprise at least one layer film or and separate device, film or to separate device be to have perviousness and porousness, and can be used to separate first and second chamber.

In addition, the both sides of growth base material are respectively to have thin film or to separate device in a preferred embodiment, to contain the particulate compartment of cell as restriction.Second chamber can be equipped with substratum, and can be positioned at top, below and/or the next door of first chamber, and/or be around mode, therefore can allow the mobile between first chamber and second chamber of substratum gentleness.Film or separate device material can for, for example: semipermeability fiber, semipermeability high molecular polymerization upholder, metal or plastics.In addition, film or separate device be can support or support and/or limiting growth base material in the fixed position.

The present invention also can further comprise inoculation and/or the step of square position inoculating cell to the base material of growing.

In addition, the present invention also provides the method for a novelty can make growth substrate material or substratum periodicity and/or intermittent mobile in cell culture apparatus, the cell of growth on the base material can be immersed in the substratum to obtain nutrient, or make the cell of growth on the base material can the emersion substratum, non-direct mode allows cell pass through the thin layer interface of one deck gas-substratum, and touch air ambient, to obtain oxygen, still but can not injure and/or cell killing.The present invention also provides the method for a novelty can be by the stopping device of control first chamber, and the compression and the stretching, extension of second chamber of arranging in pairs or groups directly apply pressure on the growth base material, reach the ability of emulation tissue growth environment.For example: tighten this stopping device, compress second chamber simultaneously, can increase the pressure in first chamber; Tighten this stopping device, stretch second chamber simultaneously, can reduce the pressure in first chamber.By increasing or reducing pressure, can be used as the purposes of cultivating the particular variety cell or tissue, for example: sclerocyte (osteoblast).

In further embodiment of this invention, be further to comprise to collect the cellular products of secreting in substratum in the method for described embodiment.The excretory cellular products can include, but not limited to protein, DNA, RNA, plastid, antibody and virus.The step of collecting substratum still comprises except described step in addition:

Utilize to discharge and/or any general method, collect substratum from the opening of first chamber of this cell culture apparatus, and the protein of collecting the emiocytosis that comprises in the substratum.

The method of any traditional collection substratum all can be applicable to the present invention, and for example: the substratum that will cultivate chamber is discharged and/or extracted out.Method of the present invention also provides: after removing the exhausted substratum, refill the method for the fresh culture of appropriate amount to this device, so that the cell nutrient to be provided.The growth base material will be again second periodicity and intermittent being immersed in the substratum, and along with the volume of described second chamber by minimum to maximum, cell is the non-direct thin layer interface of passing through one deck gas-substratum, and touches air ambient.Cell so constantly repeats above-mentioned steps, till can't produce required cellular products again.Because of cell device of the present invention is a simple device, so price is not expensive, and is the jettisonable facility, to avoid deriving the problem of device sterilization aspect.

Further set forth the embodiment of the invention below in conjunction with accompanying drawing,

Embodiment 1

Consult Fig. 1-shown in Figure 2, the embodiment 1 of apparatus of the present invention comprises, first chamber 110 contains growth base material 120, growth base material 120 can be made by any material, for example: ceramic, biological collapsible substrate, high molecular polymer, braiding base material (woven substrate), non-braiding base material (non-wovensubstrate), polymeric amide, polyester, urethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol, Trimethylamine 99 (tri-methyl amine), glass, silicon and diethylaminoethyl-(DEAE).Growth base material 120 optionally has porous, and using growth base material 120 of the present invention is to supply cell attachment, and makes the cell that adheres on it can obtain oxygen, but can directly not be exposed to air ambient.Directly be exposed in the air for cell, especially zooblast and mammalian cell may damage and dead and lose the survival rate of cultivation.

Growth base material 120 can be any form, shape or size, includes but not limited to dish shape, laminar, block, plate-like, sheet, band shape, granular, microcarrier, semipermeability particle, microparticle, semipolar linkage or semipermeability tubular fibre.

First chamber 110 is to link with second chamber 130, and first chamber 110 and second chamber 130 can be fitted, or be integrated with second chamber 130, wherein first chamber 110 and/or second chamber 130 can comprise substratum, and but two chambers all have the opening of intercommunication on one side at least, allow liquid and gas to flow between chamber and mobile.In embodiment 1, compressible element of second chamber, 130 installings is to can be a telescopic bellows form.Second chamber 130 can comprise growth media or substratum.

Shown in Figure 1 is that the compressible element of second chamber 130 is in incompressible state, and so substratum is to riddle second chamber, therefore, growth base material 120 will be non-directly the thin layer interface by one deck gas-substratum touch air ambient with acquisition oxygen.

Shown in Figure 2 is the state that the compressible element of second chamber 130 is in compression, and when two chambers 130 are in the state of compression, substratum is to move to first chamber growth base material 120 is immersed in the substratum.Yet when second chamber 130 was in incompressible state, substratum was to be stranded in second chamber 130, and growth base material 120 just can be immersed in the substratum.First chamber 110 is that at least one opening 140 can be set, to pack into or to discharge cell and substratum to the first chamber 110, opening 140 can be installed a stopping device 150, stopping device 150 comprises at least one air filter screen 160, be used for filtering the air of turnover cell culture apparatus, to reduce the generation of polluting.

Embodiment 2

Consult Fig. 3-shown in Figure 4, the embodiment 2 of apparatus of the present invention comprises, comprise growth base material 220 in first chamber 210, the matrix that growth base material 220 can be loose assembly constitutes, and its material includes but not limited to: ceramic, biological collapsible substrate, high molecular polymer, braiding base material, non-braiding base material, polymeric amide, polyester, urethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol, Trimethylamine 99, glass, silicon and diethylaminoethyl-.

Growth base material 220 in embodiment 2 optionally is a porous, and makes the cell that is attached on the growth base material 220 can obtain oxygen, but can directly not be exposed to air ambient.Directly be exposed in the air for cell, especially zooblast and/or mammalian cell may damage and dead and lose the survival rate of cultivation.

Growth base material 220 can be any particle of any form, shape or size, includes but not limited to dish shape, laminar, block, plate-like, sheet, band shape, granular, microcarrier, semipermeability particle, microparticle, semipolar linkage or semipermeability tubular fibre.

First chamber 210 is to combine with second chamber 230, and wherein first and/or second chamber can comprise growth media and/or substratum, and all has opening in the interface of two chambers, to help liquid and exchange gas and to move.In embodiment 2, compressible element 280 of second chamber 230 installing is to can be a balloon 280 in order to as volume adjustment means.Second chamber 230 can optionally comprise substratum.

Fig. 3 is that the balloon 280 of second chamber 230 is in disappointing state, and Fig. 4 is the state that the balloon 280 of second chamber 230 is in inflation.The composition material of balloon 280 can be any material, for example: rubber, latex or any telescopic plastics.

As shown in Figure 4, when balloon 280 is in inflated condition, be minimum in the substratum of second chamber 230, most substratum all is forced to flow to first chamber 210, makes growth base material 220 to be immersed in the substratum.

As shown in Figure 3, when balloon 280 is in gas-leaking state, are maximums in the substratum of second chamber 230, therefore, with the non-directly thin layer interface by one deck gas-substratum, and touch air ambient in the growth base material 220 of first chamber 210, to obtain oxygen.First chamber 210 can be provided with at least one opening 240 with pack into, square position inoculation, inoculation or discharge cell to the base material of growing, make substratum can pass in and out first chamber 210, and the gaseous interchange between first chamber 210 and outside atmosphere be provided.Opening 240 can be installed a stopping device 250, and stopping device 250 comprises at least one air filter screen 260, is used for filtering the air of turnover cell culture apparatus.

Embodiment 3

Consult Fig. 5-shown in Figure 6, the embodiment 3 of apparatus of the present invention comprises, comprise growth base material 320 in first chamber 310, the matrix that growth base material 320 can be loose assembly constitutes, and its material includes but not limited to: ceramic, biological collapsible substrate, high molecular polymer, braiding base material, non-braiding base material, polymeric amide, polyester, urethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol, Trimethylamine 99, glass, silicon and diethylaminoethyl-.Growth base material 320 in embodiment 3 optionally is a porous or can be by the matrix of the loose assembly of granulometric composition, and porous growth base material 320 can make cell attached to it obtain oxygen, but is unlikely to directly to be exposed to air ambient.Because directly be exposed in the air for cell, especially zooblast and/or mammalian cell may damage and dead and lose the survival rate of cultivation.

Growth base material 320 can be any form, shape or size, includes but not limited to dish shape, laminar, block, plate-like, sheet, band shape, granular, microcarrier, semipermeability particle, microparticle, semipolar linkage or semipermeability tubular fibre.

First chamber 310 is to combine with second chamber 330, and wherein first and/or second chamber can comprise substratum, and all has opening in the interface of two chambers.In embodiment 3, second chamber, 330 installings, one piston 380 is as volume adjustment means.Piston 380 can by on push away or drop-down, with control substratum mobile between first chamber 310 and second chamber 330.

Shown in Figure 5 is that piston 380 is in drop-down situation, and wherein the culture volume of second chamber 330 is maximums.

Shown in Figure 6 is that piston 380 is in the situation that pushes away, and wherein the culture volume of second chamber 330 is minimums.The material of piston is for example to can be any material: rubber, plastics, metal, synthetic materials or polypropylene and can any operated in accordance with conventional methods.

When piston 380 is in pull-down state, as shown in Figure 5, substratum is to be detained in second chamber 330, growth base material 320 in first chamber 310 can obtain oxygen supply, when piston 380 is in push mode, as shown in Figure 6, most substratum is pushed toward first chamber 310, makes growth base material 320 to be immersed in the substratum.First chamber 310 can selectivity be provided with at least one opening 340 packing into or to discharge cell and make substratum can pass in and out first chamber 310, and the gaseous interchange of first chamber 310 with outside atmosphere is provided.Opening 340 can be installed a stopping device 350, and stopping device 350 alternative at least one air filter screen 360 of installing are used for filtering the air that passes in and out cell culture apparatus.

Embodiment 4

Consult shown in Figure 7, the embodiment 3 of apparatus of the present invention comprises, in first chamber 410 are the growth base materials 420 that comprise half tubular fibre at least, the growth base material 420 of half tubular fibre can be made of any material, for example: braiding base material, non-braiding base material, biological collapsible substrate, polymeric amide, polyester, urethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol, Trimethylamine 99, glass, silicon and diethylaminoethyl-.The growth base material 420 of half tubular fibre can make cell attached to it obtain oxygen, but is unlikely to directly to be exposed to air ambient.Because directly be exposed in the air for cell, especially zooblast and/or mammalian cell may damage and dead and lose the survival rate of cultivation.

First chamber 410 is to fit or be integrated with second chamber 430, and wherein first and/or second chamber can comprise substratum, and all has opening in the interface of two chambers.In this preferred embodiment 4, second chamber 430 can be the form of bellows, by compressing or decompressing and regulate the volume of the substratum that is positioned at second chamber, 430 inside, as Fig. 1 and shown in Figure 2.

When second chamber 430 is in compressed state (figure do not show), substratum is to flow in first chamber 410, and the growth base material 420 of half tubular fibre is immersed in the substratum, so that cell obtains nutrient.When second chamber 430 is in uncompressed state, substratum be get back to and/or second chamber 430 of being detained in, with provide oxygen to grow in half tubular fibre growth base material 420 go up or interior cell.First chamber 410 can selectivity be provided with at least one opening 441 with cell to the half tubular fibre growth base material 420 of packing into, and can install a stopping device 451 and be repeated to open and close upper shed 441.First chamber 410 also can be provided with second opening 440 packing into or to discharge substratum, and the gaseous interchange with outside atmosphere is provided.Opening 440 also can be installed a stopping device 450, and stopping device 450 is air that at least one air filter screen 460 of installing is used for filtering the turnover cell culture apparatus.

Embodiment 5

Consult shown in Figure 8, the embodiment 5 of apparatus of the present invention comprises, in first chamber 510 are the growth base materials 520 that comprise semi-permeable capsule at least, the growth base material 520 of semi-permeable capsule can be made of any material, for example: braiding base material, non-braiding base material, polymeric amide, polyester, urethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol.The growth base material 520 of semi-permeable capsule can make cell attached to it thin layer interface by one air-substratum, obtains oxygen and but is unlikely to directly to be exposed to air ambient.The growth base material 520 of semi-permeable capsule or container be optionally make gas can by but cell can not pass through.

First chamber 510 is to combine with second chamber 530, and wherein first and/or second chamber can comprise substratum, but and all has the opening intercommunication in the interface of two chambers.In present embodiment 5, second chamber 530 can be a compressible chamber, and for example: bellows make cultivation based on flowing between first chamber 510 and second chamber 530 by compression or stretching, extension.

When second chamber 530 is in compressed state, substratum is to flow to or be forced in first chamber 510, and the growth base material 520 of semi-permeable capsule is immersed in the substratum, so that cell obtains nutrient.When second chamber 530 is in uncompressed state, substratum be get back to and/or second chamber 530 of being detained in, make the growth base material 520 of the semi-permeable capsule in first chamber 510 obtain oxygen.First chamber 510 can further be provided with at least one opening 541, with the extremely growth base material 520 of semi-permeable capsule of cell of packing into, and can install a suitable stopping device 551, is repeated to open and close upper shed 541.First chamber 510 also can further be provided with second opening 540 packing into or to discharge substratum, and the gaseous interchange with outside atmosphere is provided.Second opening 540 also can be installed second stopping device 550, and stopping device 550 is optionally to install the air that at least one suitable air filter screen 560 is used for filtering the turnover cell culture apparatus.

Embodiment 6

Consult Fig. 9-shown in Figure 10, the embodiment 6 of apparatus of the present invention comprises, comprises growth base material 620 in first chamber 610, by controlling floatoblast combination 628 and pneumatic tube 627, makes growth base material 620 float on or is immersed in the substratum.As shown in Figure 9, after floatoblast combination 628 a large amount of inflations, growth base material 620 just can indirect contact arrive the air ambient of cultivating chamber 610, and cell can obtain oxygen by the interface of one air-substratum.As shown in figure 10, when floatoblast combination 628 is a large amount of lose heart after, growth base material 620 just can be immersed in the substratum, makes the cell on it obtain nutrient.

Cultivate chamber 610 and at least one opening 640 can optionally be set packing into or to discharge substratum, and gaseous interchange with outside atmosphere is provided.Opening 640 can be installed a stopping device 650, is repeated to open and close upper shed 640.Stopping device 650 can further be installed at least one air filter screen 660, is used for filtering the air of turnover cell culture apparatus.

Embodiment 7

Consult Figure 11-shown in Figure 12, the embodiment 7 of apparatus of the present invention comprises, comprises substratum in first chamber 710, and second chamber 730 is to combine with first chamber 710, and places the top of first chamber 710.Second chamber 730 is to link to each other with growth base material 720 by cable 729, and growth base material 720 can be any material and for example forms: ceramic, biological collapsible substrate, high molecular polymer, braiding base material, non-braiding base material, polymeric amide, polyester, urethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol, Trimethylamine 99, glass, silicon and diethylaminoethyl-.Growth base material 720 in embodiment 7 optionally is a porous, porous growth base material, can make cell attached to it obtain oxygen, but can directly not be exposed in the air ambient, because the shearing stress that air-flow or bubble produced, directly be exposed in the air for cell, especially zooblast and/or mammalian cell may damage and dead and lose the survival rate of cultivation.

Growth base material 720 can be any form, shape or size, includes but not limited to dish shape, laminar, block, plate-like, sheet, band shape, granular, microcarrier, semipermeability particle, microparticle, semipolar linkage or semipermeability tubular fibre.

First chamber 710 is to combine with second chamber 730, and wherein first and/or second chamber can comprise substratum, and can directly communicate by interface in two chambers.In embodiment 7, second chamber 730 can be a compressible chamber, for example: the compressible or bellows that stretch.Figure 11 is the state that second chamber 730 is in stretching, extension, and Figure 12 is the state that second chamber 730 is in compression.

As shown in figure 12, when 730 compressions of second chamber, growth base material 720 is to reduce, and be immersed in the substratum of first chamber 710, when second chamber 730 does not compress, but in the growth base material 720 emersion substratum of first chamber 710, and the non-air ambient that directly touches second chamber 730 by the thin layer interface of one deck gas-substratum, to obtain oxygen.Second chamber 730 can further be provided with at least one opening 740.Opening 740 can be installed a stopping device 750, and stopping device 750 can further be installed at least one air filter screen 760, is used for filtering the air of turnover cell culture apparatus.

Embodiment 8

Consult shown in Figure 13ly, the embodiment 8 of apparatus of the present invention comprises, cultivates chambers 810 and 810 ' for two, and its middle chamber 810 is installed in the chamber 810 ', and chamber 810 ' is around chamber 810.Chamber 810 is to comprise all one's life long substrate accepting cell, and in its bottom one opening is arranged, and makes that substratum can be freely mobile between chamber 810 and 810 ' by this opening.A surface that raises up or the skeletal lines (contour) 821 ' (for example cone) of a bending are optionally designed in the bottom surface of chamber 810 ', make current be streamlined, and can avoid piling up the cell of failing to be attached to growth base material 820.

Chamber 810 can further be provided with at least one opening 840, to pack into or to discharge cell and/or substratum can be passed in and out and cultivate chamber 810.Opening 840 can be installed a stopping device 850, to repeat to open and close upper shed 840.Stopping device 850 can further be installed at least one air filter screen 860, is used for filtering the air of turnover cell culture apparatus.Chamber 810 ' can further be provided with at least one opening 840 ', and can install a suitable stopping device 850 '.Stopping device 850 ' can further be installed at least one air filter screen 860 ', is used for filtering the air of turnover cell culture apparatus.

The control of the substratum content of chamber 810 is by air is squeezed into chamber 810 ' by air filter screen 860 ', and changes the gas space of chamber 810 ', and this pressure will drive substratum by chamber 810 ' inflow chamber 810.When gas chamber 810 ' diminished, growth base material 820 was to be immersed in the substratum; When gas chamber 810 ' became big, the vacuum of gas chamber 810 ' made substratum flow back to chamber 810 ', thereby made the growth base material 820 of chamber 810 to touch air ambient in non-direct thin layer interface by one deck gas-substratum.

Described growth base material 820 can place chamber 810 separately, place chamber 810 ' or place chamber 810 and chamber 810 ' simultaneously separately.

In addition, consult shown in Figure 14, the growth base material 120 of apparatus of the present invention is to be immersed in the substratum, and can obtain considerable nutrient, when optionally porous growth base material is immersed in substratum, can cause a kind of phenomenon to be called static agitation, be gentle stir culture base and nutrient, and can distribute the cell that drops to get back in the growth base material 120 again equably.

Consult shown in Figure 15; be to be lower than growth base material 120 of the present invention when the substratum height; growth base material 120 can obtain considerable oxygen; Figure 15 provides when from chamber that growth base material 120 is housed when removing substratum; to have the skim substratum is stranded on the porous growth base material; this thin layer can be protected on the growth base material 120 and/or within cell, make it directly not be exposed in the air ambient shearing stress because of air-flow produced.

Embodiment 9

Consult Figure 16-shown in Figure 17, the embodiment 9 of apparatus of the present invention combines with the embodiment 1 of Fig. 2 Fig. 1 with a drive unit 1012, for example: an oil cylinder (oil pressure cylinder) or a pneumatic cylinder (air pres sure cylinder) are done combination, are installed in the bottom of compressible second chamber 1030.When as shown in figure 17, being pressure cylinder 1012 rises; Or as shown in figure 16, be pressure cylinder 1012 when falling, second chamber 1030 is periodically and intermittent compression and stretching, extension.The compression of second chamber 1030 and stretching, extension, growth base material 1020 periodicity and intermittence are moved between chamber 1010 and chamber 1030, thereby growth base material 1020 is immersed in the substratum, obtaining nutrient, or the emersion substratum allows the cell on the growth base material 1020 obtain oxygen.

Embodiment 10

Consult Figure 18-shown in Figure 19, the embodiment 10 of cell culture apparatus of the present invention combines with the embodiment 1 of Fig. 2 Fig. 1 with a volume setting device 1117, for example: a screw jack is done combination, is installed in the bottom of second chamber 1130 with a compressing member.When as shown in figure 19, being screw jack 1117 rises; Or as shown in figure 18, be pressure cylinder 1117 when falling, second chamber 1130 is periodically and intermittent compression and stretching, extension.The compression of second chamber 1130 and stretching, extension periodically are immersed in the substratum growth base material 1120 with intermittence, and non-direct thin layer interface by one deck gas-substratum touches air ambient to obtain oxygen.

Embodiment 11

Consult Figure 20-shown in Figure 21, the embodiment 11 of apparatus of the present invention combines volume adjustment means 1219 and 1220 with Fig. 1 with the embodiment 1 of Fig. 2, for example: drive shaft rod (shaft driver) and drive unit 1218, for example: motor or step motor (step motor) are installed in the bottom of compressible second chamber 1230.When as shown in figure 21, being the drive shaft rod rise; Or as shown in figure 20, be drive shaft rod when falling, second chamber 1230 is periodically and intermittent compression and stretching, extension.The compression of second chamber 1230 and stretching, extension, the growth base material 1220 of first chamber 1210 can periodically be immersed with intermittence or emersion in substratum.

Embodiment 12

Consult Figure 22-shown in Figure 23, the embodiment 12 of apparatus of the present invention is that the embodiment 2 of Fig. 3 and Fig. 4 combines with a gas compression drive unit.Porthole hole (valve port) the 1322nd is in opened condition, and when open in porthole hole 1324, and porthole hole 1323 is when closing, and air is just squeezed into balloon 1380 by the air pump housing 1325 and made its inflation.Therefore, as shown in figure 23, can reduce in the volume of second chamber 1330, substratum will be pushed by second chamber 1330 and be extruded onto first chamber 1310, and growth base material 1320 is immersed in the substratum.Helical porthole hole 1323 and 1324 conversion can be by following controls, for example: a timer 1326 is controlled, and timer 1326 can change the On/Off state of helical porthole hole 1323 and 1324.When porthole hole 1324 is closed, and porthole hole 1323 is when opening, and the gas in the balloon can be extruded because of the pressure that substratum weight caused, and substratum thereby flow to second chamber 1330 by first chamber 1310 makes growth base material 1320 be exposed in the air.The inflation and the gas-leaking state of conversion balloon 1380, make growth base material 1320 periodically and intermittence immerse with emersion in substratum, and the non-directly thin layer interface by one deck gas-substratum touches air ambient.

Embodiment 13

Consult shown in Figure 24ly, the embodiment 13 of apparatus of the present invention is that the embodiment 6 of Fig. 9-Figure 10 combines with a gas compression drive unit.By regulation and control gas at floatoblast 1428 content with pneumatic tube 1427, can make growth substrate material 1420 immerse with emersion in substratum.Floatoblast 1428 is the bottoms that are attached at growth base material 1420, utilizes the drive unit be connected at least one screwed pipe porthole hole, and for example: the air pump housing 1430 can allow floatoblast 1428 inflations or lose heart.During operation, always open in porthole hole 1433 and 1436, and when open in porthole hole 1432 and 1437, porthole hole 1434 and 1435 close, air is just squeezed into floatoblast 1428 by the air pump housing 1430 and is made floatoblast 1428 inflations, so growth base material 1420 is up pushed away, and makes the indirect thin layer interface of passing through one deck gas-substratum of cell, and touches the air ambient of first chamber 1410.The switch in screwed pipe porthole hole can utilize following control, and for example: a timer 1431 is controlled.Timer 1431 can change the On/Off state in helical porthole hole, when porthole hole 1432 and 1437 is closed, and porthole hole 1434 and 1435 is when opening, and the gases in the floatoblast 1428 can be made growth base material 1420 be immersed in the substratum by 1430 emptyings of the air pump housing.

Embodiment 14

Consult shown in Figure 25ly, the embodiment 14 of apparatus of the present invention is that the embodiment 8 with Figure 13 combines with a gas compression drive unit.Porthole hole 1533 and 1536 is to open, and when open in screwed pipe porthole hole 1534 and 1535, other porthole hole 1532 and 1537 is for closing, gas compression device 1530 can push air the inner face of first chamber 1510, and therefore, the volume of substratum reduces because of air pressure in first chamber, because chamber 1510 is to communicate with the bottom of chamber 1510 ', so when culture volume in the chamber 1510 reduced, the culture volume of chamber 1510 ' just can rise, anti-is as the same.Secondly, porthole hole 1532 and 1537 is for opening, and when porthole hole 1534 and 1535 is closed, porthole hole 1533 and 1536 is still can not change for opening, afterwards, air can be pushed into outside cultivation chamber 1510 ', so that the minimizing of the culture volume of chamber 1510 ', and the culture volume of chamber 1510 increases simultaneously.By repeating this operation, substratum in chamber 1510 and chamber 1510 ' is moving between two chambers of periodicity and indirect, growth base material 1520 is immersed in the substratum obtaining nutrient, or the thin layer interface of emersion substratum by one deck gas-substratum obtain oxygen.

Embodiment 15

Consult shown in Figure 25ly, the embodiment 15 of apparatus of the present invention is that the embodiment 1 of Fig. 1 and Fig. 2 combines with a substratum exchange system.The substratum exchange system can be operated by two kinds of different modes: substratum switch mode and growth pattern.In growth pattern, valve 1643 and 1644 is to close, so as to shutting colligator 1645 and 1646, similarly, valve and gas inlet 1647 are to open, to allow as the embodiment 1 of Fig. 1 and Fig. 2 can to carry out gaseous interchange, under this state, substratum can't pass in and out cell culture apparatus via air filter screen 160 (scheme not be).In case substratum is exhausted and/or the protein of cells produce has reached a q.s, then can carry out the collection of substratum, obtaining protein, and change fresh substratum.

In the substratum switch mode, valve and gas inlet 1647 are to close, and in addition, valve 1643 is to close.(for example: bellows), valve 1644 can be opened, and allows the exhausted substratum flow out, and arrives storage tankss 1642 via colligator 1646 to utilize the residual gas pressure of first chamber 1610 to add the compression of second chamber 1630.After old substratum flowed out, valve 1644 can be closed, and then valve 1643 can be opened, and the fresh culture that is positioned at storage tanks 1641 will be inhaled into, and arrived second chamber 1630 via colligator 1645.The stretching, extension of second chamber 1630 has attracted fresh culture by storage tanks 1641 process colligators 1645, and arrives cell culture apparatus.In case substratum reaches enough amounts, valve 1643 can be closed.For getting back to normal growth pattern once again, valve and gas inlet 1647 are opened conditions, and the control of all valves can be passed through an electronic installation, and for example: computer 1648, operating method can be the automatic or manual operation.

Embodiment 16

Consult Figure 27-shown in Figure 28, the embodiment 16 of apparatus of the present invention is that the embodiment 1 of Fig. 1 and Fig. 2 combines with a substratum exchange system.Wherein Figure 27 is manual operation, and Figure 28 is automatic operation.

This cell culture apparatus can be operated by two kinds of different modes: substratum switch mode and growth pattern.

In growth pattern, the

pump housing

1743 and 1744 is to be in closing condition, similarly, gas inlet 1760 is to open, can be via air filter screen 160 (figure is not) to carry out gaseous interchange with the embodiment 1 that allows Fig. 1 and Fig. 2, under this state, fresh substratum can't pass in and out first and/or second chamber of cell culture apparatus, in case substratum is exhausted and/or the protein of cells produce has reached a q.s, then can carry out the collection of substratum, obtaining protein, and change fresh substratum.

In the substratum switch mode, valve and gas inlet 1760 are to close, and start the pump housing 1744 exhausted substratum in second chamber 1730 is released, and via colligator 1750, are transported to storage tanks 1742 then.After old substratum flowed out, the pump housing 1744 promptly cut out.For getting back to normal growth pattern once again, gas inlet 1760 is opened conditions, and the control of all pump housings can drive by a computer and/or electronic installation 1748, and operating method can be the automatic or manual operation.

Embodiment 17

Consult Figure 29-shown in Figure 30, the embodiment 17 of apparatus of the present invention is that the cell culture apparatus as Fig. 1 and Fig. 2 combines with a substratum exchange system.Figure 29 is manual operation, and Figure 30 is automatic operation.

The basal culture medium switch can be operated by two kinds of different modes: substratum switch mode and growth pattern.

In growth pattern, the pump housing 1843 and 1844 is to be in closing condition, similarly, gas inlet 1860 is to open, to allow as the cell culture apparatus of Fig. 1 and Fig. 2 can be via air filter screen 160 (scheme not be) to carry out gaseous interchange, under this state, fresh substratum can't pass in and out first and/or second chamber of cell culture apparatus, in case substratum is exhausted and/or the protein of cells produce has reached a q.s, the collection that then can carry out substratum to be obtaining protein, and changes fresh substratum.

In the substratum switch mode, gas inlet 1860 is to close, and the pump housing 1844 is a starting state, the exhausted substratum being released cell culture apparatus, and is transported to storage tanks 1842.After old substratum flowed out, the pump housing 1844 promptly cut out.The pump housing 1843 is to start, so that the fresh culture in the storage tanks 1841 is added to cell culture apparatus.When reaching enough fresh culture, just close the pump housing 1843.For getting back to normal growth pattern once again, gas inlet 1860 is opened conditions, and the control of all pump housings can drive by a computer and/or electronic installation 1848, and operating method can be the automatic or manual operation.

Embodiment 18

Consult Figure 31-shown in Figure 32, the embodiment 18 of apparatus of the present invention comprises, at least one cell growth chamber, and it contains at least one chamber inner wall 1992, and wherein each chamber all has first end, second end, an internal surface and an outside surface to be used for defining an opening.Cell culture apparatus of the present invention can have second chamber, and it is top, below and/or the next door that can be positioned at first chamber, and/or be inside/outside around mode.Having a growth chamber at least is with wheel or support 1990, makes the cell culture apparatus of present embodiment can be by a planar support, for example: floor, desktop or laboratory operation table top.Each growth chamber has at least one growth base material 1991, to grow at least one cell and allow cell to attach and/or adhere to.

At least one growth chamber is to include substratum and atmosphere surrounding, make substratum place second end of cultivating chamber, and air ambient is positioned at and/or be contained in first end of cultivating chamber.Embodiments of the invention comprise a drive unit 1995 too, be used for up and down or move left and right growth base material is travelled to and fro between between first end and second end of growth chamber, make the growth base material periodically to immerse and/or the emersion substratum, so that suitable nutrient of cell and oxygen to be provided with intermittence.Drive unit 1995 alternatives comprise first connected unit 1994, be used for linking the drive unit 1995 and second connected unit 1993, drive unit 1995 is to link to each other with growth base material 1991, its role is to make the growth base material repeatedly, periodically with intermittently to move to second end by first end of growth chamber, promptly, growth base material in the mobile growth chamber can immerse and the emersion substratum it, so that suitable nutrient and oxygen to be provided repeatedly.Drive unit of the present invention can be a riser, and elevator system can help to grow base material up and down or move left and right.

Growth base material 1991 in the present embodiment 18 optionally has porous, and porous growth base material can make cell attached to it obtain oxygen, but can directly not be exposed in the air ambient shearing stress because of air-flow or bubble produced.Because directly be exposed in the air for cell, especially zooblast and/or mammalian cell may damage and dead, and lose the survival rate of cultivation.

Growth base material 1991 can be any form, shape or size; include but not limited to; dish shape, laminar, block, plate-like, sheet, band shape, granular, microcarrier, semipermeability particle, microparticle, semipolar linkage or semipermeability tubular fibre; also optionally utilize a porous Membrane cover to hold the growth base material; for example: porous particle or microcarrier can flow substratum.In addition, also can utilize a fixed support structure,, make it can move into or shift out substratum to support the growth base material.

Embodiment 19

Consult Figure 33-shown in Figure 34, the embodiment 19 of apparatus of the present invention comprises that first growth chamber contains at least one chamber inner wall 2092, and wherein each chamber all has first end, second end, an internal surface and an outside surface to be used for defining an opening.Cell culture apparatus of the present invention can have second chamber, and it is top, below and/or the next door that can be positioned at first chamber, and/or be inside/outside around mode.Having a growth chamber at least is with wheel or support 2090, makes the cell culture apparatus of present embodiment 19 can be by a planar support, for example: floor, desktop or laboratory operation table top.Each growth chamber has at least one growth base material 2091, with at least one the cell of growing, and allows cell to attach and/or adheres to.

At least one growth chamber is to include substratum and atmosphere surrounding, make substratum place second end of cultivating chamber, and air ambient is disposed at and/or be contained in first end of cultivating chamber.Embodiments of the invention 19 comprise a drive unit 2095 too, be used for up and down or move left and right growth base material is travelled to and fro between between first end and second end of growth chamber, make the growth base material can be repeatedly, periodically with intermittently immerse and/or the emersion substratum, so that suitable nutrient of cell and oxygen to be provided.Drive unit 2095 alternatives comprise first connected unit 2094, be used for linking the drive unit 2095 and second connected unit 2093, drive unit 2095 is to link to each other with growth base material 2091, its role is to make the growth base material repeatedly, periodically with intermittently to move to second end by first end of growth chamber, growth base material in the promptly mobile growth chamber, it can be immersed and the emersion substratum repeatedly, so that suitable nutrient and oxygen to be provided.Drive unit of the present invention can be a screw jack, and the screw jack system can help to grow base material up and down or move left and right.

Growth base material 2091 in the present embodiment 19 optionally is a porous, and porous growth base material can make cell attached to it obtain oxygen, but can directly not be exposed in the air ambient shearing stress because of air-flow or bubble produced.Because directly be exposed in the air, for cell, especially zooblast and/or mammalian cell may damage and dead and lose the survival rate of cultivation.

Growth base material 2091 can be any form, shape or size; include but not limited to; dish shape, laminar, block, plate-like, sheet, band shape, granular, microcarrier, semipermeability particle, microparticle, semipolar linkage or semipermeability tubular fibre; also optionally utilize a porous Membrane cover to hold the growth base material; for example: porous particle or microcarrier can flow substratum.In addition, also can utilize a fixed support structure,, make it can move into or shift out substratum to support the growth base material.

Embodiment 20

Consult shown in Figure 35ly, the embodiment 20 of apparatus of the present invention comprises, the interior filling growth base material 2120 of a chamber 2110 attaches growth for cells, and other has a storage tanks 2130 to take up substratum.Utilize the weight of a platform scale 2180 sensing chambers 2110, when chamber 2110 weigh less than certain set(ting)value the time, can send signal by controller 2181, start the air pressure pump housing 2125 and valve-off 2144, and open valve 2143 and close the air pressure pump housing 2126, so, will be by the gas that the air pressure pump housing 2125 is released through air filter screen 2160, make substratum be pushed to chamber 2110 by storage tanks 2130, this moment grow base material 2120 be soaked in substratum in.When the weight of platform scale 2180 sensing chambers 2110 surpasses certain set(ting)value, can send signal by controller 2181, start the air pressure pump housing 2126 and valve-off 2143, and open valve 2144 and close the air pressure pump housing 2125, so, substratum flow to storage tanks 2130 by chamber 2110, and the base material 2120 of growing this moment will arrive air ambient by the thin layer interface indirect contact of one air-substratum.

In order to promote the stirring efficiency of reactor, utilize a rotating shaft and cushion cap combination 2170, stirring with rotation increases cell inoculation, and substratum blended effect.

Embodiment 21

Consult shown in Figure 36, the embodiment 21 of apparatus of the present invention, at least comprise one first chamber 2210 and compressible second chamber 2230, wherein first chamber 2210 is the tops that are positioned at second chamber 2230, and growth base material 2220 is contained for the cell growth in first chamber, 2210 inside, and 2230 of second chambers take up substratum.In order to make cell culture apparatus internal liquid flow field unification, that is cause growth base material 2220 internal liquids to flow from top to bottom, can help cell retention within carrier.When a lifting gantry 2250 moved up, the movable valve 2240 that is positioned at second chamber, 2230 inside moved up, and cap holes, so substratum then upwards flows via middle hollow circuit cylinder 2270 and overflow, and flowing downward covers growth base material 2220 again.And when lifting gantry 2250 moves down, movable 2240 of valves move down, and the unlatching hole, therefore first chamber, 2210 interior substratum then flow into second chamber 2230 downwards via the outer shroud hole, make growth base material 2220 arrive air ambient by the thin layer interface indirect contact of one air-substratum.

Embodiment 22

Consult shown in Figure 37, the embodiment 22 of apparatus of the present invention, when desiring to carry out substratum replacing action, gas valve 2345 is closed, and leaks outside to prevent the chamber internal gas pressure, simultaneously, valve-off 2343 is drawn into to avoid fresh culture, opens valve 2344 and compression below, the air pressure in compressible devices 2330 thereby the increase chamber, so that substratum in the chamber is extruded, enter first storage tanks 2341 through connector 2350.After this action is finished, gas valve 2345 still is a closing condition, to prevent the leakage of chamber internal gas pressure, simultaneously, valve-off 2344 is to avoid air from connector 2350 suction, and Open valve 2343, the negative pressure of utilizing compressible devices 2330 to descend and produced by second storage tanks 2342, sucks fresh culture by connector 2351.This substratum is changed action can do process control by computer, with the control valve folding condition, and the compression number of times of compressible devices 2330, do the regulation and control of changing volume.

When usual cultivation, valve 2343 and 2344 is a closing condition, and therefore, the connector 2351 and 2350 that is connected in the chamber both sides is closing stage, and gas valve 2345 is an opened condition, to carry out gaseous interchange.

Embodiment 23

Consult shown in Figure 38, the embodiment 23 of apparatus of the present invention is extensions of embodiment 22, as shown in figure 38, the right side is the substratum liquid changing device, be to comprise top substratum storage chamber 2410 and below inflation chamber 2430 at least, the left side is high-effect cell culture apparatus, comprises top cell cultures chamber 2411 and below inflation chamber 2431 at least, utilizes a conduit 2460 to link these two devices.The running of this system is by the compression of inflation chamber 2430 and stretching, cause the change of its internal pressure, so it is substratum is interior by connector 2450 by substratum storage chamber 2410, with the cell cultures chamber 2411 that is squeezed to cell culture apparatus through conduit 2460 and connector 2451, no matter this moment, whether its below inflation chamber 2431 compressed, growth base material 2421 is immersed in the substratum, and when inflation chamber 2430 stretches, substratum will suck back by following same passage in the cell culture apparatus.This device is by promoting the volume of total substratum, to avoid frequently changing the puzzlement of substratum.

The present invention illustrates by preferred embodiment; need be appreciated that: the present invention should not be subject to the enforcement aspect that is provided; and should comprise various enforcement aspects of the present invention (as being familiar with present technique field person institute tradition); therefore; protection scope of the present invention should be made generalized and explain, to comprise all modifications and similar combination.

The present invention utilizes a non-restrictive example to further specify, and present embodiment is in order to describing content of the present invention, and should not be used for tangible, the possible difference of shrinkage limit the present invention and many present invention of disengaging spirit and category.

Embodiment 24

Cell cultures of the present invention and when cell is grown the proteinic Application Example of collecting cell excretory.

Obtain a strain through the Chinese grey mouse gonad cell strain (CHO cell line) of genetic engineering modified, as to contain a stable insertion and performance DNA, this cell strain is inoculated on the semipermeability microcarrier growth base material of cell culture apparatus of the present invention with the extraneous protein of coding institute desire performance.Cell culture apparatus of the present invention contains two chambers: first chamber and second chamber, and wherein first chamber is positioned at the top of second chamber, and contains growth base material of the present invention, and second chamber is to contain compressible bellows and contain substratum.

In case after adding substratum and cell, cell culture apparatus of the present invention can be loaded on the drive unit, intermittent compression and stretching, extension bellows with periodicity, when compressing bellows automatically by drive unit, substratum can be pushed to first chamber by second chamber, microcarrier growth base material is immersed in the substratum, and when stretching bellows by drive unit, substratum can flow to second chamber by first chamber, make microcarrier growth base material and on cell be maintained at the interface of the one air-substratum of growth substrate surface by one deck, and being exposed to the air ambient of first chamber indirectly, this air ambient is to contain the ideal oxygen concentration.

In addition, after substratum is got back to second chamber, will keep an a small amount of substratum,, avoid coming to harm because of ingress of air with the protection cell in the microcarrier substrate surface.The compression of bellows with stretch substratum periodically contact with intermittent moving grow base material and on cell so that provide maximum nutrient to cell.Moreover cell cycle property and intermittently move between chamber can help any cell that drops to redistribute and adhere to again, and then can increase whole survival rate and productivity of cultivating

Utilize described cell culture apparatus, cultivate a strain and contain the stable performance of inserting, and the Chinese grey mouse gonad cell strain (CHO cell line) of the DNA of the extraneous protein desired of codified, by periodicity and intermittence substratum is moved between chamber, and the providing alternately of oxygen and nutrient, do not make cell directly be exposed to air ambient, to reach ideal cell growth situation and extraneous protein production and secretion, the extraneous protein of being desired will be secreted to substratum, and its concentration will be along with accumulated time.

In case the cell growth reaches perfect condition, for example: all nutrients in the substratum use up, and just must remove substratum old in the cell culture apparatus, and replenish fresh substratum, contain the recombinant protein of desiring to some extent in the old substratum, can be purified out according to general traditional method.

Above narration is to propose preferred embodiment of the present invention, and the embodiment of this narration must not be used for limiting the present invention.

Claims

1, a kind of cell culture apparatus is characterized in that: which comprises at least:

2, cell culture apparatus according to claim 1, it is characterized in that: described second chamber is the compressible element with first end and second end, and optionally the outside surface in first end of compressible element provides first lid, and provide second lid in the outside surface of second end of compressible element, this first lid combines with second lid, makes that the compressible element in second chamber is to be compressed between first lid and second lid.

3, cell culture apparatus according to claim 1, it is characterized in that: it has further comprised:

One air ambient is the inside that is positioned at this first chamber; Substratum is the compressible element inside that is arranged in this second chamber, and this substratum separates device and/or film via one, moves freely between this first chamber and second chamber; And optionally use a drive unit to compress and the compressible element that stretches in this second chamber, so that should cultivate, have at least a cell to attach and/or the growth base material that adheres to can be immersed in this substratum when this second chamber compresses based on moving between this first chamber and second chamber; When this second chamber stretches, be exposed to air ambient indirectly by one air-substratum thin layer interface, so that oxygen to be provided.

4, cell culture apparatus according to claim 1 is characterized in that: described first chamber comprises at least one opening, is to be used for accepting or mobile substratum and cell, and allows cell culture apparatus and extraneous environment exchange of air.

5, cell culture apparatus according to claim 4 is characterized in that: the opening of described first chamber optionally is equipped with a stopping device.

6, cell culture apparatus according to claim 5 is characterized in that: described stopping device is optionally to install an air filter screen, to prevent pollution.

7, cell culture apparatus according to claim 3 is characterized in that: described drive unit is an automatic or manual.

8, cell culture apparatus according to claim 1, it is characterized in that: separate device between first end of described second end that places first chamber and second chamber, be one to have infiltrative film or porousness dividing plate, this cultivation is flowed based on coming and going between first chamber and second chamber.

9, cell culture apparatus according to claim 8 is characterized in that: described have infiltrative film for optionally having porous.

10, cell culture apparatus according to claim 1 is characterized in that: described growth base material is the matrix of a loose assembly.

11, cell culture apparatus according to claim 10 is characterized in that: described matrix also includes dissolved oxygen hyperplasia device, static mixer or deep-bed filter.

12, cell culture apparatus according to claim 1 is characterized in that: described growth base material is half permeable sac.

13, cell culture apparatus according to claim 1 is characterized in that: described growth base material is a porous particle, static agitation, increase dissolved oxygen, filtration is provided and intercepts cell.

14, cell culture apparatus according to claim 1 is characterized in that: described growth base material comprises at least one semi-permeable tubular fibre.

15, cell culture apparatus according to claim 1 is characterized in that: described cell is to be selected from eukaryotic cell or prokaryotic cell prokaryocyte.

16, cell culture apparatus according to claim 1 is characterized in that: the amount of the substratum of described growth base material is to regulate and/or control by a floatoblast and a pneumatic tube.

17, cell culture apparatus according to claim 1 is characterized in that: this second chamber further includes a volume setting device, enters the substratum deal of first chamber with regulation and control;

One control device is the described volume adjustment means of control, substratum is flowed between first and second chamber, make when substratum at the volume of second chamber to hour, substratum covers the growth base material fully, and when substratum at the volume of second chamber when being maximum, the interface indirect contact of growth base material by one air-substratum is to the air ambient of first chamber.

18, cell culture apparatus according to claim 17, it is characterized in that: described volume adjustment means is bellows, by compression and stretching, extension, to control the volume of the second chamber interior substratum, make when bellows are compressed to the limit, substratum is minimum at the volume of second chamber, and when bellows were stretched over the limit, substratum was maximum at the volume of second chamber.

19, cell culture apparatus according to claim 17, it is characterized in that: described volume adjustment means is a balloon, by inflation and disappointing, to control the volume of the second chamber interior substratum, make when balloon inflation arrives the limit, substratum is minimum at the volume of second chamber, and when balloon lost heart to the limit, substratum was maximum at the volume of second chamber.

20, cell culture apparatus according to claim 17 is characterized in that: described volume adjustment means is a piston, wherein pushes away on the piston or drop-down, to control the volume of the second chamber interior substratum.

21, cell culture apparatus according to claim 3 is characterized in that: described drive unit is an oil cylinder.

22, cell culture apparatus according to claim 3 is characterized in that: described drive unit is a pneumatic cylinder.

23, cell culture apparatus according to claim 3 is characterized in that: described drive unit is a screw jack.

24, cell culture apparatus according to claim 3 is characterized in that: described drive unit is a gas compression device.

25, cell culture apparatus according to claim 1, it is characterized in that: described growth base material is a porous growth base material, cell is coated within the porous growth base material, and when growth base material emersion during in substratum, cell on the growth base material arrives air ambient by the interface indirect contact of one air-substratum, and obtains enough oxygen.

26, cell culture apparatus according to claim 25 is characterized in that: the material of described porous growth base material is selected from following group: pottery, braiding base material, non-braiding base material, polymeric amide, polyester, urethane, fluorocarbon polymer, polyethylene, polypropylene or polyvinyl alcohol.

27, cell culture apparatus according to claim 25 is characterized in that: the form of described porous growth base material is dish shape, laminar, block, plate-like, sheet, band shape, granular, semipermeability particle or semipolar linkage.