WO2018058706A1 - 大循环流化床细胞生物反应器及培养动物细胞的方法 - Google Patents

大循环流化床细胞生物反应器及培养动物细胞的方法 Download PDF

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WO2018058706A1
WO2018058706A1 PCT/CN2016/102076 CN2016102076W WO2018058706A1 WO 2018058706 A1 WO2018058706 A1 WO 2018058706A1 CN 2016102076 W CN2016102076 W CN 2016102076W WO 2018058706 A1 WO2018058706 A1 WO 2018058706A1
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truncated cone
culture tank
fluidized bed
cell
cell culture
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PCT/CN2016/102076
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English (en)
French (fr)
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惠倪
惠识瑶
李力
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惠倪
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Application filed by 惠倪 filed Critical 惠倪
Priority to US16/326,826 priority Critical patent/US20190211297A1/en
Publication of WO2018058706A1 publication Critical patent/WO2018058706A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation
    • C12M25/20—Fluidized bed
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J8/00—Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes
    • B01J8/0015—Feeding of the particles in the reactor; Evacuation of the particles out of the reactor
    • B01J8/003—Feeding of the particles in the reactor; Evacuation of the particles out of the reactor in a downward flow
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J8/00—Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes
    • B01J8/02—Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes with stationary particles, e.g. in fixed beds
    • B01J8/0207—Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes with stationary particles, e.g. in fixed beds the fluid flow within the bed being predominantly horizontal
    • B01J8/0228—Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes with stationary particles, e.g. in fixed beds the fluid flow within the bed being predominantly horizontal in a conically shaped bed
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J8/00—Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes
    • B01J8/02—Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes with stationary particles, e.g. in fixed beds
    • B01J8/0285—Heating or cooling the reactor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/02—Percolation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/26—Conditioning fluids entering or exiting the reaction vessel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/32—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of substances in solution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers

Definitions

  • the invention relates to the technical field of cell bioreactors, in particular to a large circulating fluidized bed cell reactor and a method for cultivating animal cells, and more particularly to a large circulating fluidized bed cell reactor for animal cell culture. And methods for large-scale cultivation of animal cells.
  • Fluidized bed cell bioreactors include reaction tanks, temperature control sections, venting sections, inlet and outlet ports, measurement systems, control systems, and other ancillary systems.
  • the existing fluidized bed cell reactors are all constructed by inserting a mechanical agitation device in the tank. Leakage and microbial contamination problems often occur with structures and seals. This leads to a reduction in safety and reliability and a high probability of infection. There is also a low level of dissolved oxygen in the culture solution, which does not meet the requirements for high-density cultured cells. In addition, the working volume of the reactor is small, and although the perfusion culture process is employed, it is difficult to achieve true large-scale cultivation.
  • the object of the present invention is to provide a large circulating fluidized bed cell bioreactor and a method for cultivating animal cells, so as to solve the problem that the fluidized bed cell bioreactor existing in the prior art is prone to microbial contamination, low dissolved oxygen level and work. Defects and deficiencies such as small volume.
  • an aspect of the present invention provides a large circulating fluidized bed cell bioreactor comprising a shaker body, a shaker base, a water inlet silicone hose, a backwater silicone hose, a cell culture tank, a tank base, a return pipe, a water inlet pipe, a shaker, a reactor bottom plate, and a pipe support plate;
  • the rocker body is disposed in the rocker base, and the rocker base and the shaker of the shaker are connected
  • the shaker is mounted on a reactor chassis;
  • the shaker body has a central funnel, and a draft tube is installed at an upper portion of the shaker body, and an inlet of the draft tube connects the shaker
  • the inner wall, the outlet of the draft tube enters the center funnel along an upper tangential direction of the center funnel;
  • the central funnel is located at a center position of the rocker body, and the lower end thereof protrudes from the bottom of the rocker body;
  • the cell culture tank is disposed in the base of the
  • the rocker body has a truncated cone shape with a half cone angle of 25 degrees to 55 degrees (for example, may be 30 degrees, 35 degrees, 40 degrees, 50 degrees, etc.), preferably 30 degrees to 42 degrees;
  • the shaker body has a can lid with various water inlets.
  • the cell culture tank has a bottom truncated cone portion and an upper truncated cone portion, and has a first cylindrical portion between the bottom truncated cone portion and the upper truncated cone portion, and a second cylindrical portion between the upper truncated cone portion and the top portion.
  • the small end of the top of the cell culture tank is connected to the inlet end of the return pipe, and the outlet end of the return pipe is connected to the lower end of the backwater silicone hose; the bottom part of the cell culture tank extends to itself The tangential direction is connected to the lower end of the inlet pipe, and the upper end of the inlet pipe is connected to the lower end of the inlet silicone hose.
  • the cell culture tank has a truncated cone screen, and the truncated cone screen is connected to the inner wall of the second cylindrical portion of the upper portion of the cell culture tank, and the cone top of the truncated cone screen is upward.
  • the bottom truncated cone portion, the upper truncated cone portion, and the truncated cone mesh of the cell culture tank have a half cone angle of 25 degrees to 60 degrees (for example, may be 30 degrees, 35 degrees, 40 degrees, 50 degrees, 55 degrees) Degree, etc.), preferably from 45 degrees to 60 degrees.
  • the top of the cell culture tank has a can lid with various inlet and outlet ports, and the can lid is connected to the top edge flange of the cell culture tank and sealed; the inner wall of the cell culture tank base is heated with heat. rubber Board.
  • the area of the cross section of the upper truncated cone portion is gradually enlarged from bottom to top.
  • Another aspect of the present invention provides a method of culturing animal cells using the large circulating fluidized bed cell bioreactor provided by the present invention and comprising the steps of:
  • the culture medium After adding the culture solution to the static liquid level, the culture medium is heated to 35.5-37 ° C; the shaker is started (rotated within the range of 70-110 rpm), and the microcarriers after disinfection are added to the culture solution in proportion, and then The active animal cells are inoculated into the microcarrier; the culture solution in the upper part of the shaker body is dissolved in the upper space of the shaker body, in the flow tube and in the dissolved oxygen region in the center funnel, and the culture solution after the dissolved oxygen is shaken.
  • the microcarriers carrying the active animal cells are located in the region between the bottom round table portion of the cell culture tank and the upper truncated cone screen; When the culture medium of oxygen and nutrients flows upward, nutrients and dissolved oxygen are delivered to the active animal cells in the microcarrier.
  • the microcarriers are macroporous microcarriers or porous microcarriers having a weight and shape, each of the microcarriers having a relatively downward sedimentation rate in the culture solution; wherein, when the culture solution is upward When the flow rate and the sedimentation rate of the microcarriers are relatively balanced, the microcarriers do not settle down and are not balanced; when the upward flow rate of the culture solution is higher than the equilibrium value, the microcarriers flow upward together with the culture solution; when the culture solution is When the upward flow rate is lower than the equilibrium value, the microcarriers settle downward; through the structural characteristics of the upper truncated cone portion, the cross-sectional area of the portion is gradually enlarged, thereby reducing the upward flow velocity of the culture solution in this interval, which facilitates the downward sedimentation of the microcarriers. Thereby, the rapid and reliable separation of the microcarriers and the culture solution at this site is completed.
  • the diameter of the aperture of the truncated cone screen is smaller than the diameter of the microcarrier.
  • the large circulating fluidized bed cell bioreactor provided by the invention is a brand new structure and principle, and has no stirring device inserted into the tank, the overall sealing property is good, and the safety and reliability are quite high;
  • the problem of dissolved oxygen level in the culture solution solves the problem of dissolved oxygen by stirring the slurry and bubbling, but the dissolved oxygen level is generally present, especially in the process of large-scale, high-density culture of animal cells.
  • the low level of dissolved oxygen can not meet the respiratory requirements of animal cells, and it is difficult to culture animal cells with high density, high quality and high efficiency, and the large circulating fluidized bed cell bioreactor provided by the present invention utilizes the circumferential oscillation of the shaker, A large amount of water droplets and sprays are generated in the shaker tank, which increases the area in which the mixed gas on the surface of the culture liquid and the upper portion of the shaker is in contact with each other, and expands the gas-liquid relative surface area in the oscillating state, thereby achieving a high dissolved oxygen level.
  • the dissolved oxygen level in the oscillating state can be much higher than that in the conventional reactor, which fully meets the requirements of high-density, high-quality cultured cells.
  • the working volume of the cell reactor in general, in the process of large-scale cultivation of animal cells using cell bioreactor, the other conditions are the same, the larger the working volume, the higher the efficiency, the cost is relative
  • the working volume is calculated by multiplying the daily perfusion volume by the perfusion day. The number is the actual working volume; the daily perfusion volume is 0.5-2 times the actual working volume of the culture tank.
  • this coefficient is 1, that is, the daily perfusion volume is the actual volume of the cell culture tank of this product, and the number of days perfusion is generally up to 30 days or so; if the volume of the cell culture tank of the large circulating fluidized bed cell bioreactor provided by the present invention is 500 liters, then the actual working volume is 500 liters/day times 30 days equals 15000 liters; and the present invention provides The large circulating fluidized bed cell bioreactor has been experimentally proved that the ratio of the volume of the cell culture tank driven by the shaker is 1 to 10, while ensuring that the vertical upward flow rate of the culture medium in the cell culture tank is not less than 60 cm/min.
  • the volume of the cell reactor can be driven to 2000 liters.
  • the actual working volume of the cells cultured by the infusion process is 2000 liters/day multiplied by 30 days equals 60,000 liters, which is in biopharmaceuticals.
  • the industry and the biological products industry are all quite large batches, and such a large working volume is rarely used in actual production, which is the large circulating fluidized bed cell bioreactor provided by the present invention. Big advantage, the actual volume of work can do a lot.
  • FIG. 1 is a schematic view showing the structure of a large circulating fluidized bed cell bioreactor according to an embodiment of the present invention
  • FIG. 2 is a schematic top plan view of the large circulating fluidized bed cell bioreactor shown in FIG. 1;
  • Figure 3 is a schematic front view of the rocker body
  • Figure 4 is a side view of the rocker body
  • Fig. 5 is a schematic plan view showing the structure of the rocker body.
  • connection In the description of the present invention, it should be noted that the terms “installation”, “connected”, and “connected” are to be understood broadly, and may be fixed or detachable, for example, unless otherwise explicitly defined and defined. Connected, or integrally connected; can be mechanical or electrical; can be directly connected, or indirectly connected through an intermediate medium, can be the internal communication of the two components.
  • Connected, or integrally connected can be mechanical or electrical; can be directly connected, or indirectly connected through an intermediate medium, can be the internal communication of the two components.
  • the specific meaning of the above terms in the present invention can be understood in a specific case by those skilled in the art.
  • the first embodiment provides a large circulating fluidized bed cell bioreactor comprising a rocker body 01, a rocker base 50, a water inlet silicone hose 06, and a return water silicone hose.
  • 08 the cell culture tank 10, the culture tank base 40, the return water pipe 15, the water inlet pipe 12, the shaker 20, the reactor chassis 30 and the pipe support plate 60;
  • the rocker body 01 is disposed in the rocker base 50, the rocker base 50 is connected to the rocking plate 201 of the shaker 20, and the shaker 20 is mounted on the reactor chassis 30;
  • the shaker body 01 has a central funnel 05, and a draft tube 03 is mounted on the upper portion of the shaker body 01, and the draft tube
  • the inner wall of the rocker body 01 connected to the inlet 031, the outlet pipe 032 enters the center funnel 05 along the upper tangential direction of the center funnel 05;
  • the center funnel 05 is located at the center of the rocker body 01, and the lower end thereof extends out of the bottom of the
  • the rocker body 01 and the attached structure will be described in detail based on the above-described first embodiment.
  • the technical solution in the first embodiment also belongs to the protection range of the second embodiment.
  • the shaker body 01 has a truncated cone shape with a half taper angle of 25 degrees; and, the shaker body 01 has a can lid with a water inlet. That is, the rocker body 01 has a rocker cover 011 with an air inlet, an air outlet, a liquid inlet and a liquid outlet, and the rocker body 01 and the attached portion are mounted in the rocker base 50, the rocker base The bottom flange and the rocker 201 are bolted, and the chassis of the shaker 20 is bolted to the reactor chassis 30 to secure the shaker 20 to the reactor chassis 30.
  • the rocker body 01 has a truncated cone shape with a half cone angle of 55 degrees; and, the rocker body 01 has a can lid with a water inlet. That is, the rocker body 01 has a rocker cover 011 with an air inlet, an air outlet, a liquid inlet and a liquid outlet, and the rocker body 01 and the attached portion are mounted in the rocker base 50, the rocker base The bottom flange and the rocker 201 are bolted, and the chassis of the shaker 20 is bolted to the reactor chassis 30 to secure the shaker 20 to the reactor chassis 30.
  • the half cone angle of the truncated cone body 01 is generally between 25 and 55 degrees, preferably between 30 and 42 degrees, depending on the circumstances.
  • the inner wall of the shaker base 50 has an electrically heated rubber sheet for holding the shaker body 01.
  • the inlet side of the draft tube 03 is connected to the inner conical surface of the shaker body 01, and the outlet of the draft tube 03 is connected to the upper portion of the center funnel 05.
  • the culture solution is also made.
  • the circumference oscillates.
  • the culture liquid level is changed from the static liquid level 000 to the dynamic liquid surface 666, and a clockwise rotation motion is also performed.
  • the culture liquid of the circumferential oscillating motion flows in from the inlet of the draft tube 03, flows out from the outlet of the draft tube 03, and enters the upper portion of the center funnel 05 from the tangential direction.
  • the vortex flows downward in a clockwise direction, then flows out at the lower port of the center funnel 05, enters the water inlet silicone hose 06, flows into the inlet pipe inlet 121, and then flows down to the inlet pipe outlet 122 to enter the cell culture tank 10. It spirals upward from the bottom truncated portion, flows through the first cylindrical portion of the cell culture tank 10, enters the upper truncated portion 101, and then flows to the second cylindrical portion, thereby reaching the top of the cell culture tank 10. The small end of the top end flows down into the return water pipe inlet 151, and then flows down and back into the return water silicone hose 08, and flows back to the rocker body 01 through the return water outlet 152.
  • the culture liquid returning to the rocker body 01 continues to spiral upward in a clockwise direction, and enters the draft tube inlet 031 again, thus reciprocating to form a mixture between the shaker body 01 and the cell culture tank 10.
  • the culture fluid is circulated.
  • the shaker body 01 there is neither micro-carrier nor cell.
  • the culture liquid in the shake flask After the shaker 20 is started, the culture liquid in the shake flask generates a circular oscillating motion, and the culture liquid in the entire incubator is circulated and flowed.
  • the shake tank is the source of power.
  • the mixed gas entering the tank from the inlet port of the can is in the whole The upper part of the tank body, the guide tube 03, the water droplets in the center funnel 05 and the culture liquid, the surface of the spray is gas-liquid phase mixed, the mixed gas enters the culture liquid, and the undissolved residual gas rises to the liquid.
  • the culture solution in which the mixed gas is dissolved flows into the inlet pipe 12 and then flows into the bottom of the cell culture tank 10.
  • the nutrients and dissolved oxygen carried by the culture solution are supplied to the cells in the macroporous microcarrier 19.
  • the cell culture tank 10 and the auxiliary structure will be described in detail based on the first embodiment and the second embodiment.
  • the technical solution in the first embodiment also belongs to the protection range of the second embodiment.
  • the cell culture vessel 10 has a bottom truncated cone portion and an upper truncated cone portion 101 having a first cylindrical portion between the bottom truncated cone portion and the upper truncated cone portion 101, between the upper truncated cone portion 101 and the top portion The second cylindrical portion.
  • the bottom truncated cone portion of the cell culture tank 10, the upper truncated cone portion 101, and the truncated cone screen 17 have a half cone angle of 25 degrees.
  • the cell culture vessel 10 has a bottom truncated cone portion and an upper truncated cone portion 101 having a first cylindrical portion between the bottom truncated cone portion and the upper truncated cone portion 101, at the upper truncated cone portion 101 and the top portion There is a second cylindrical portion therebetween.
  • the bottom truncated cone portion of the cell culture tank 10, the upper truncated cone portion 101, and the truncated cone screen 17 have a half cone angle of 60 degrees.
  • the half-cone angle of the bottom truncated cone portion, the upper truncated cone portion 101, and the truncated cone-shaped screen 17 of the cell culture tank 10 can be flexibly set in the range of 25-60 degrees according to actual conditions.
  • the semi-cone angle of the conical surface of the truncated cone screen 17 is generally from 40 degrees to 60 degrees, preferably from 45 degrees to 60 degrees, depending on the specific circumstances.
  • the cell culture tank 10 has a truncated cone screen 17 which is connected to the inner wall of the second cylindrical portion of the upper portion of the cell culture tank 10, and which has the cone top of the truncated cone screen 17 upward.
  • Cell culture tank The upper inner cylindrical portion has a truncated cone screen 17, and the lower edge of the truncated cone screen 17 is connected to the inner wall of the cylindrical tank, and the upper edge is higher than the static liquid level 000, specifically 30 mm higher.
  • the diameter of the hole in the truncated cone screen 17 is smaller than the diameter of the microcarrier 19, so that the microcarrier 19 can be reliably filtered out, and only the culture solution can pass.
  • the mixture of the microcarriers 19 is filled between the bottom and the upper truncated cone 17 in the cell culture tank 10, and the cells survive in the microcarrier 19. Due to the action of the truncated cone screen 17, when the microcarriers 19 with cells flow upward with the culture solution, only the culture solution can flow smoothly, and the microcarriers 19 are all blocked under the sieve. Therefore, the survival area of the cells is in the region below the bottom of the cell culture tank 10 to below the truncated cone 17.
  • the small end of the top of the cell culture tank 10 is connected to the return pipe inlet 151, and the return pipe 15 is downward and then upwardly resembling a U-shaped pipe.
  • the outlet end of the return pipe 15 is connected to the lower end of the return water silicone hose 08, and the return water silicone hose
  • the upper end of 08 is connected to the bottom side back water outlet 152 of the shaker body to form a circuit between the cell culture tank 10 and the shaker body 01.
  • the bottom round table portion of the cell culture tank 10 is connected to the lower end of the inlet water pipe 12 in its own tangential direction, and the upper end of the inlet water pipe 12 is connected to the lower end of the water inlet silicone hose 06.
  • the top of the cell culture tank has a cell culture tank cover 102 with an air inlet, an air outlet, a liquid inlet, a liquid outlet, a sampling port, and various electrode sockets.
  • the cell culture tank 10 and the attached portion are housed in the cell culture tank base 40.
  • the bottom flange of the cell culture tank base 40 and the reactor bottom plate 30 are bolted and fixed to the reactor chassis 30.
  • a heating rubber sheet is adhered to the inner wall of the cell culture tank base 40.
  • a pipe support plate 60 is welded to the reactor chassis 30, and the top of the pipe support plate 60 is connected to the inlet pipe 15 and the inlet pipe 12 to support the two water pipes to be fixed.
  • a method for cultivating animal cells which includes the following steps:
  • the culture liquid is heated to 37 ° C; the shaker 20 is started, specifically rotated at a rotation speed of 110 rpm, and the microcarriers 19 after sterilization are added in proportion.
  • the active animal cells are inoculated into the microcarrier 19; the shaker body 01
  • the inner upper culture solution is dissolved in the flow tube 03 and the dissolved oxygen region in the central funnel 05, and the dissolved oxygen culture solution enters the inflow silicone hose 06 and the inlet pipe 12 under the action of the shaking power;
  • the cell culture tank 10 is introduced along the tangential direction of the truncated cone portion of the bottom of the cell culture vessel 10, and the spiral flow is started until it flows down to the truncated cone 17 to transport nutrients to the active cells in the microcarriers 19 between the regions.
  • the culture solution flows through the truncated cone 17 to the top of the culture tank, flows into the return pipe 15, and then flows through the return water silicone hose 08 and the bottom side return water outlet 152 of the shaker body.
  • the spiral flows upward, re-enters the inlet of the draft tube 03, completes a cycle; after supplementing oxygen and nutrients, the next cycle is started.
  • the culture solution is heated to 35.5 ° C under other conditions; the shaker 20 is activated to rotate at a speed of 70 rpm.
  • the microcarriers 19 carrying the active animal cells are located in the region between the bottom round table portion of the cell culture tank 10 and the upper truncated cone screen 17; When the culture solution of dissolved oxygen and nutrients flows upward, the active animal cells in the microcarrier 19 are transported with nutrients and dissolved oxygen.
  • the microcarrier 19 is a macroporous microcarrier 19 or a porous microcarrier 19 having a weight and a shape, and each microcarrier 19 has a relatively downward sedimentation rate in the culture solution; wherein, when the culture solution is flowed upward and slightly When the downward sedimentation rate of the carrier 19 is relatively balanced, the microcarriers 19 do not settle down and are balanced; when the upward flow rate of the culture solution is higher than the equilibrium value, the microcarriers 19 flow upward together with the culture solution; When the upward flow rate is lower than the equilibrium value, the microcarrier 19 settles downward;
  • the cross-sectional area of the portion is gradually enlarged, thereby reducing the upward flow velocity of the culture solution in this interval, which facilitates the downward sedimentation of the microcarrier 19, thereby completing the rapid growth of the microcarrier 19 and the culture solution in this portion.
  • Reliable separation provides reliable basic conditions for the application of the perfusion culture process.
  • the upper truncated portion 101 of the cell culture tank 10 has a function of decelerating the flow rate of the mixed culture liquid flowing upward in the spiral.
  • the upward flow rate of the mixed culture liquid spirally flowing from the bottom to the upper direction changes inversely, that is, the upward flow velocity becomes small.
  • the microcarriers 19 no longer flow upward, but stop or settle vertically downward. For this reason, in this region, the microcarriers 19 are not flowing, and only the culture liquid itself continues to flow upward.
  • the culture medium and the microcarriers 19 can be completely reliably separated here.
  • the microcarriers 19 carry the cells all the way to the bottom of the cell culture vessel 10 to the area under the screen.
  • the culture solution flows to the top of the tank and flows into the return water pipe 15, and returns to the shake tank for the next cycle.

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Abstract

一种大循环流化床细胞生物反应器及培养动物细胞的方法，其中，反应器包括摇罐体(01)、摇罐底座(50)、进水硅胶软管(06)、回水硅胶软管(08)、细胞培养罐(10)、培养罐底座(40)、回水管(15)、进水管(12)、摇床(20)、反应器底盘(30)和管路支撑板(60)；通过上述各个部件的相互配合，有效地解决了现有技术中存在的流化床细胞生物反应器易发生微生物污染，溶氧水平低下和工作容积小等缺陷和不足，利于推广与应用。

Description

大循环流化床细胞生物反应器及培养动物细胞的方法 技术领域
本发明涉及细胞生物反应器技术领域，具体地涉及一种大循环流化床细胞反应器及培养动物细胞的方法，更具体地涉及一种用于动物细胞培养的大循环流化床细胞反应器及大规模培养动物细胞的方法。
背景技术
目前，通过动物细胞体外培养表达和生产各类诊断和治疗用单克隆抗体，疫苗，生长因子等生物活性蛋白质具有十分广泛的市场应用前景。因此大规模高密度培养动物细胞的细胞生物反应器已成为生物技术制药产业中关键设备。它很大程度上决定了生物制药企业的产品成本，质量，生产规模和产品种类。
流化床细胞生物反应器包括反应罐体，控温部分，通气部分，进出料口，测量系统，控制系统及其他附属系统。
现有的流化床细胞反应器均是采用插入罐体内机械搅拌装置的结构。其结构和密封件经常出现泄露和微生物污染问题。导致安全可靠性降低，染菌几率高。还有培养液的溶氧水平低，也满足不了高密度培养细胞的要求。再有反应器工作容积小，尽管是采用灌注培养工艺，但也难以实现真正大规模的培养。
发明内容
本发明的目的在于提供一种大循环流化床细胞生物反应器及培养动物细胞的方法，以解决现有技术中存在的流化床细胞生物反应器易发生微生物污染，溶氧水平低下和工作容积小等缺陷和不足。
为解决上述技术问题，本发明一方面提供了一种大循环流化床细胞生物反应器，其包括摇罐体、摇罐底座、进水硅胶软管、回水硅胶软管、细胞培养罐、培养罐底座、回水管、进水管、摇床、反应器底盘和管路支撑板；所述摇罐体设置于所述摇罐底座中，所述摇罐底座和所述摇床的摇盘连接，所述摇床安装于反应器底盘上；所述摇罐体中具有中心漏斗，且在所述摇罐体的上部装有导流管，所述导流管的进口连接所述的摇罐体内壁，所述导流管的出口沿中心漏斗的上部切线方向进入所述中心漏斗；所述中心漏斗位于所述摇罐体的中心位置，其下端伸出所述摇罐体的底部；所述细胞培养罐设置于培养罐底座中，所述培养罐底座安装于反应器底盘上；所述细胞培养罐的底部、上部分别对应连接有所述进水管、所述回水管；所述进水管、进水硅胶软管、中心漏斗的底部依次连接，所述回水管、回水硅胶软管、摇罐体底部侧位回水出口下端依次连接；所述管路支撑板同时对所述进水管和回水管进行支撑。
可选地，所述摇罐体为圆台形，其半锥角为25度至55度(例如可以为30度、35度、40度、50度等)，优选地为30度至42度；所述摇罐体具有带各种进水口的罐盖。
进一步地，所述细胞培养罐具有底部圆台部分和上部圆台部分，在底部圆台部分与上部圆台部分之间具有第一圆柱部分，在上部圆台部分与顶部之间具有第二圆柱部分。
进一步地，所述细胞培养罐顶部的小头端连接所述回水管的入口端，所述回水管的出口端连接在回水硅胶软管的下端；所述细胞培养罐底部圆台部分延其自身切线方向连接所述进水管的下端，所述进水管的上端连接所述进水硅胶软管的下端。
优选地，所述细胞培养罐内有圆台形滤网，所述圆台形滤网和所述细胞培养罐上部的第二圆柱部分内壁连接，且使所述圆台形滤网的锥顶向上。
可选地，所述细胞培养罐的底部圆台部分、上部圆台部分以及圆台形滤网的半锥角均为25度-60度(例如可以为30度、35度、40度、50度、55度等)，优选地为45度-60度。
进一步地，所述细胞培养罐顶部有带各种进出口的罐盖，所述罐盖和所述细胞培养罐顶端边缘法兰相连接并密封；所述细胞培养罐底座的内壁上粘有加热橡胶板。
优选地，所述上部圆台部分的横截面的面积由下至上是逐渐扩大的。
本发明另一方面提供了一种培养动物细胞的方法，其使用了本发明提供的大循环流化床细胞生物反应器，并包括如下步骤：
加培养液至静态液面后，加热培养液到35.5-37℃；启动摇床(以70-110转/分的转速范围内转动)，将消毒后的微载体按比例加入培养液中，再将活性动物细胞接种到微载体中；摇罐体内上部的培养液在摇罐体内上部空间，导流管中和中心漏斗内的溶氧区内进行溶氧，溶氧后的培养液在摇动力的作用下进入进水硅胶软管和进水管；然后沿着细胞培养罐底部圆台部分的切线方向进入细胞培养罐，开始螺旋向上流动，直至流到圆台形滤网下边，给在此区域间的微载体内的活性细胞输送营养物质和溶解氧；完成 这一供给过程后，培养液穿过圆台形滤网流到培养罐顶部，流入回水管，然后通过回水硅胶软管和摇罐体底部侧位回水出口流回到摇罐体内，螺旋向上流动，重新进入导流管进口，完成一个循环；补充氧气和营养后，再开始下一个循环。
进一步地，在细胞培养罐给活性动物细胞输送营养和氧气过程中时，带有活性动物细胞的微载体是位于细胞培养罐底部圆台部分到上部圆台形滤网之间的区域中；当含有溶解氧和营养物质的培养液向上流动时,就给微载体中的活性动物细胞输送了营养物质和溶解氧。
进一步地，所述微载体为有重量和有形状的大孔微载体或多孔微载体，每种所述微载体在培养液中是相对有一个向下的沉降速率的；其中，当培养液向上流速和微载体向下的沉降速率相对平衡时，微载体则不向下沉降而平衡不动；当培养液向上流速高于平衡值时，微载体就随培养液一起向上流动；当培养液的向上流速低于平衡值时,微载体就向下沉降；通过上部圆台部分的结构特性，该部分的横截面积逐步扩大，从而降低培养液在此区间的向上流速，利于微载体向下沉降,从而完成了微载体和培养液在此部位的快速可靠分离。
优选地，所述圆台形滤网的孔的直径小于所述微载体的直径。
根据本发明提供的大循环流化床细胞生物反应器及培养动物细胞的方法，能够带来至少以下有益效果之一：
1、面对动物细胞大规模高密度的培养要求，现行的各种细胞反应器主要存在的问题有:染菌方面的可靠性问题，由于插入罐中的搅拌桨轴承部的密封可靠性差，导致泄露时有发生，降低了可靠性，导致细胞培养失败。 本发明提供的大循环流化床细胞生物反应器是全新的结构和原理，其没有搅拌装置插入罐内，整体密封性好，安全可靠性相当高；
2、动物细胞的剪切力敏感性，现行生物反应器中，因搅拌桨和鼓泡产生的流体剪切力和气泡破裂导致细胞损伤，甚至死亡，造成培养失败，所以本发明提供的大循环流化床细胞生物反应器罐体内就没有搅拌桨，产生大量气泡的溶氧区在摇罐体内，细胞生存区域是在细胞培养罐内,根本不是同一区域，所以细胞受到的损伤程度是非常低的，而且本发明提供的大循环流化床细胞生物反应器是采用大孔微载体培养细胞，细胞在微载体中还受到大孔微载体的硬壳保护；
3、培养液的溶氧水平问题，现行的细胞反应器是靠搅拌浆搅拌和鼓泡解决溶氧问题，但普遍存在溶氧水平底下，特别是在大规模,高密度培养动物细胞过程中,溶氧水平低,总是满足不了动物细胞的呼吸要求，难以高密度、高质量、高效益地培养动物细胞，而本发明提供的大循环流化床细胞生物反应器利用摇床的圆周振荡，摇罐内产生大量的水珠和浪花，增加了培养液表面和摇罐上部的混合气体相互接触溶解的面积，在振荡状态扩大了气-液相比表面积这个参数，所以达到了高溶氧水平，实验中测量发现,在振荡状态下溶氧水平可比传统反应器的溶氧水平高的多，完全满足了高密度、高质量培养细胞的要求。
4、细胞反应器工作容积问题，一般说来,在用细胞生物反应器来大规模培养动物细胞过程中，其他条件不变的情况下,工作容积越大，效率就会越高,成本就相对越低；由于本产品是采用大孔微载体培养动物细胞，采用高效的灌注工艺，其工作容积是这样计算的：每天的灌注量乘以灌注的天 数是实际工作容积；一般每天灌注量是0.5-2倍的培养罐实际工作容积，若我们选此系数为1，即每天灌注量是本产品细胞培养罐的实际容积，灌注的天数一般可达30天左右；若本发明提供的大循环流化床细胞生物反应器的细胞培养罐的容积为500升，那么实际工作容积就是500升/天乘以30天等于15000升；且本发明提供的大循环流化床细胞生物反应器经实验证明，在保证细胞培养罐内培养液垂直向上流速不低于60厘米/分钟的情况下，摇罐带动细胞培养罐的容积之比为1比10，即摇罐容积为200升时，便可带动细胞反应罐容积2000升，在这种条件下，采用灌注工艺培养细胞实际工作容积是2000升/天乘以30天等于60000升，这在生物制药行业，生物制品行业都是相当大的批量，实际生产中很少用的上这样大的工作容积，这是本发明提供的大循环流化床细胞生物反应器的又一大优势,实际工作容积能做的很大。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施方式提供的大循环流化床细胞生物反应器的结构原理示意图；
图2为图1所示的大循环流化床细胞生物反应器的俯视结构示意图；
图3为摇罐体的正视结构示意图；
图4为摇罐体的侧视结构示意图；
图5为摇罐体的俯视结构示意图。
具体实施方式
下面将结合附图对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施方式是本发明一部分实施方式，而不是全部的实施方式。基于本发明中的实施方式，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。
在本发明的描述中，需要说明的是，术语“中心”、“上”、“下”、“左”、“右”、“竖直”、“水平”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。此外，术语“第一”、“第二”、“第三”仅用于描述目的，而不能理解为指示或暗示相对重要性。
在本发明的描述中，需要说明的是，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通。对于本领域的普通技术人员而言，可以具体情况理解上述术语在本发明中的具体含义。
下面通过具体的实施方式并结合附图对本发明做进一步的详细描述。
实施方式1
结合图1-图5所示，本实施方式1提供一种大循环流化床细胞生物反应器，其包括摇罐体01、摇罐底座50、进水硅胶软管06、回水硅胶软管08、细胞培养罐10、培养罐底座40、回水管15、进水管12、摇床20、反应器底盘30和管路支撑板60；摇罐体01设置于摇罐底座50中，摇罐底座50和摇床20的摇盘201连接，摇床20安装于反应器底盘30上；摇罐体01中具有中心漏斗05，且在摇罐体01的上部装有导流管03，导流管进口031连接的摇罐体01内壁，导流管出口032沿中心漏斗05的上部切线方向进入中心漏斗05；中心漏斗05位于摇罐体01的中心位置，其下端伸出摇罐体01的底部；细胞培养罐10设置于培养罐底座40中，培养罐底座40安装于反应器底盘30上；细胞培养罐10的底部、上部分别对应连接有进水管12、回水管15；进水管12、进水硅胶软管06、中心漏斗05的底部依次连接，回水管15、回水硅胶软管08、摇罐体01底部侧位回水出口152依次连接；管路支撑板60同时对进水管12和回水管15进行支撑。
实施方式2
结合图1-图5所示，本实施方式2在上述实施方式1的基础上，对摇罐体01以及附属结构进行详细的说明。当然，实施方式1中的技术方案也属于本实施方式2的保护范围。
在一个实施例中，具体地，摇罐体01为圆台形，其半锥角为25度；而且，摇罐体01具有带进水口的罐盖。也就是说，摇罐体01上具有带进气口、出气口、进液口和出液口的摇罐盖011，该摇罐体01及附带部分被装在摇罐底座50中，摇罐底座50底部法兰盘和摇盘201用螺栓连接，而摇床20的底盘与反应器底盘30用螺栓连接，把摇床20固定在反应器底盘30上。
可选地，在另一实施例中，摇罐体01为圆台形，其半锥角为55度；而且，摇罐体01具有带进水口的罐盖。也就是说，摇罐体01上具有带进气口、出气口、进液口和出液口的摇罐盖011，该摇罐体01及附带部分被装在摇罐底座50中，摇罐底座50底部法兰盘和摇盘201用螺栓连接，而摇床20的底盘与反应器底盘30用螺栓连接，把摇床20固定在反应器底盘30上。
根据本申请，圆台形的摇罐体01的半锥角一般在25度至55度，优选在30度到42度之间较好，可视具体情况而定。
优选地，摇罐底座50内壁有电加热橡胶板，为摇罐体01保温。
更加具体而言，导流管03进口一侧连接在摇罐体01内侧圆锥表面上，导流管03出口连接中心漏斗05上部，摇罐沿顺时针摇动方向555摇动时,使培养液也做圆周振荡流动。在此时培养液面由静态液面000变成动态液面666，同时还做顺时针旋转运动。圆周振荡运动的培养液从导流管03进口流进,从导流管03出口流出，从切线方向进入中心漏斗05的上部。按顺时针方向成旋涡向下流动，然后在中心漏斗05的下端口流出，进入到进水硅胶软管06后流入进水管入口121，再向下流到进水管出口122，进入细胞培养罐10内,从底部圆台部分处螺旋向上，流过细胞培养罐10第一圆柱部分，进入到上部圆台部分101，然后再流到第二圆柱部分，进而到达细胞培养罐10顶部。在顶部的小头端向下流进回水管入口151，再向下然后反上回流进回水硅胶软管08，通过回水出口152流回到摇罐体01中。由于摇罐体01的圆周振荡运动，回到摇罐体01内的培养液继续顺时针螺旋向上流动，再次进入导流管进口031，如此循环往复，形成摇罐体01和细胞培养罐10间的培养液循环流。
其中，在摇罐体01内，既无微载体也无细胞，摇床20启动后，使摇罐内培养液产生圆周振荡运动，带动整个培养器内的培养液循环流动。摇罐就是源动力。在摇罐体01内，由罐盖上进气口进入罐内的混合气体在整 个罐体的上部，导流管03内,中心漏斗05内和培养液的水珠，浪花的表面进行气-液相混合，溶混合气进入培养液中，未溶入的剩余气体上升到液面后逸出，与此同时,溶入混合气体的培养液流进进水管12，再流进细胞培养罐10底部。把培养液所携的营养物质和溶解氧供给在大孔微载体19内的细胞。
实施方式3
结合图1-图5所示，本实施方式3在上述实施方式1、实施方式2的基础上，对细胞培养罐10以及附属结构进行详细的说明。当然，实施方式1中的技术方案也属于本实施方式2的保护范围。
在一个实施例中，具体地，细胞培养罐10具有底部圆台部分和上部圆台部分101，在底部圆台部分与上部圆台部分101之间具有第一圆柱部分，在上部圆台部分101与顶部之间具有第二圆柱部分。优选地，细胞培养罐10的底部圆台部分、上部圆台部分101以及圆台形滤网17的半锥角均为25度。
在另一实施例中，可选地，细胞培养罐10具有底部圆台部分和上部圆台部分101，在底部圆台部分与上部圆台部分101之间具有第一圆柱部分，在上部圆台部分101与顶部之间具有第二圆柱部分。优选地，细胞培养罐10的底部圆台部分、上部圆台部分101以及圆台形滤网17的半锥角均为60度。
当然，根据本申请，细胞培养罐10的底部圆台部分、上部圆台部分101以及圆台形滤网17的半锥角具体可根据实际情况在25-60度范围内灵活设置。其中，圆台形滤网17的圆锥表面的半锥角一般为40度-60度，优选为45度-60度之间较好，可视具体情况而定。
细胞培养罐10内有圆台形滤网17，圆台形滤网17和细胞培养罐10上部的第二圆柱部分内壁连接，且使圆台形滤网17的锥顶向上。细胞培养罐 10内上部圆柱形部分有圆台形滤网17，圆台形滤网17的下边缘与圆柱形罐内壁相连接，上边缘高于静态液面000，具体要高出30毫米。
圆台形滤网17上孔的直径要小于微载体19的直径，这样才能可靠的滤去微载体19，只让培养液通过。细胞培养罐10内从底部到上部圆台形滤网17之间充满带有微载体19的混合液，细胞在微载体19中生存。由于圆台形滤网17的作用，带有细胞的微载体19随培养液螺旋向上流动时，只有培养液能顺利流过，而微载体19全部被挡在滤网下。所以细胞的生存区域是在细胞培养罐10底部到圆台形滤网17下面的这个区域内。
细胞培养罐10顶部的小头端连接回水管入口151，回水管15向下然后又向上类似U形管，回水管15的出口端连接在回水硅胶软管08的下端，回水硅胶软管08的上端连接摇罐体底部侧位回水出口152，在细胞培养罐10和摇罐体01之间形成回路。对应地，细胞培养罐10底部圆台部分延其自身切线方向连接进水管12的下端，进水管12的上端连接进水硅胶软管06的下端。
而且，细胞培养罐顶部具有带进气口，出气口，进液口，出液口，取样口和各种电极插口的细胞培养罐盖102。细胞培养罐10及附带部分被装在细胞培养罐底座40中。细胞培养罐底座40底部法兰盘和反应器底盘30用螺栓连接，固定在反应器底盘30上。而且，在细胞培养罐底座40内壁上还粘有加热橡胶板。此外，反应器底盘30上焊有管路支撑板60，管路支撑板60顶部于回水管15和进水管12相连接，支撑两水管,使其固定。
实施方式4
结合图1-图5所示，本实施方式4在上述实施方式1、实施方式2、实施方式3的基础上，还提供一种培养动物细胞的方法，其包括如下步骤：
在一个实施例中，加培养液至静态液面000后，加热培养液到37℃；启动摇床20，具体以110转/分的转速范围内转动，将消毒后的微载体19按比例加入培养液中，再将活性动物细胞接种到微载体19中；摇罐体01 内上部的培养液在导流管03中和中心漏斗05内的溶氧区内进行溶氧，溶氧后的培养液在摇动力的作用下进入进水硅胶软管06和进水管12；然后沿着细胞培养罐10底部圆台部分的切线方向进入细胞培养罐10，开始螺旋向上流动，直至流到圆台形滤网17下边，给在此区域间的微载体19内的活性细胞输送营养物质和溶解氧；完成这一供给过程后，培养液穿过圆台形滤网17流到培养罐顶部，流入回水管15，然后通过回水硅胶软管08和摇罐体底部侧位回水出口152流回到摇罐体01内，螺旋向上流动，重新进入导流管03进口，完成一个循环；补充氧气和营养后，再开始下一个循环。
在另一实施例中，其他条件不变的情况下，加热培养液到35.5℃；启动摇床20，以70转/分的转速范围内转动。
在细胞培养罐10给活性动物细胞输送营养和氧气过程中时，带有活性动物细胞的微载体19是位于细胞培养罐10底部圆台部分到上部圆台形滤网17之间的区域中；当含有溶解氧和营养物质的培养液向上流动时,就给微载体19中的活性动物细胞输送了营养物质和溶解氧。
微载体19为有重量和有形状的大孔微载体19或多孔微载体19，每种微载体19在培养液中是相对有一个向下的沉降速率的；其中，当培养液向上流速和微载体19向下的沉降速率相对平衡时，微载体19就不向下沉降而平衡不动；当培养液向上流速高于平衡值时，微载体19就随培养液一起向上流动；当培养液的向上流速低于平衡值时,微载体19就向下沉降；
通过上部圆台部分101的结构特性，该部分横截面积逐步扩大，从而降低培养液在此区间的向上流速，利于微载体19向下沉降,从而完成了微载体19和培养液在此部位的快速可靠分离，为灌注培养工艺的应用，提供了可靠的基本条件。细胞培养罐10的上部圆台部分101，有一个对螺旋向上流动的混合培养液流速减速的功能。在这部分，因为培养罐的圆台体部分的截面积的变大，从下向上螺旋流动的混合培养液向上流速就产生相反变化，即向上流速变小。当向上速度值小于和微载体19的沉降速率平衡值 时,微载体19便不再向上流动，而是停止或垂直向下沉降。正因如此，在这区域中,微载体19不在随流而上，只培养液本身继续向上流动。加之滤网作用，使的培养液和微载体19在此能完全可靠的分离。微载体19带着细胞一直保留在细胞培养罐10的底部到滤网下边的区域中。培养液流到罐顶部流入回水管15，回到摇罐中，进入下一循环。
最后应说明的是：以上各实施方式仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施方式对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施方式所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施方式技术方案的范围。

Claims (16)

  1. 一种大循环流化床细胞生物反应器，其特征在于，包括摇罐体、摇罐底座、进水硅胶软管、回水硅胶软管、细胞培养罐、培养罐底座、回水管、进水管、摇床、反应器底盘和管路支撑板；
    所述摇罐体设置于所述摇罐底座中，所述摇罐底座和所述摇床的摇盘连接，所述摇床安装于反应器底盘上；所述摇罐体中具有中心漏斗，且在所述摇罐体的上部装有导流管，所述导流管的进口连接所述的摇罐体内壁，所述导流管的出口沿中心漏斗的上部切线方向进入所述中心漏斗；所述中心漏斗位于所述摇罐体的中心位置，其下端伸出所述摇罐体的底部；
    所述细胞培养罐设置于培养罐底座中，所述培养罐底座安装于反应器底盘上；所述细胞培养罐的底部、上部分别对应连接有所述进水管、所述回水管；所述进水管、进水硅胶软管、中心漏斗的底部依次连接，所述回水管、回水硅胶软管、摇罐体底部侧位回水出口依次连接；所述管路支撑板同时对所述进水管和回水管进行支撑。
  2. 根据权利要求1所述的大循环流化床细胞生物反应器，其特征在于，所述摇罐体为圆台形，其半锥角为25度至55度。
  3. 根据权利要求1或2所述的大循环流化床细胞生物反应器，其特征在于，所述摇罐体具有带各种进水口的罐盖。
  4. 根据权利要求1所述的大循环流化床细胞生物反应器，其特征在于，所述细胞培养罐具有底部圆台部分和上部圆台部分，在底部圆台部分与上部圆台部分之间具有第一圆柱部分，在上部圆台部分与细胞培养罐顶部之间具有第二圆柱部分。
  5. 根据权利要求4所述的大循环流化床细胞生物反应器，其特征在于，所述细胞培养罐顶部的小头端连接所述回水管的入口端，所述回水管的出口端连接在回水硅胶软管的下端；所述细胞培养罐底部圆台部分延其自身切线方向连接所述进水管的下端，所述进水管的上端连接所述进水硅胶软管的下端。
  6. 根据权利要求5所述的大循环流化床细胞生物反应器，其特征在于，所述细胞培养罐内有圆台形滤网，所述圆台形滤网和所述细胞培养罐上部的第二圆柱部分内壁连接，且使所述圆台形滤网的锥顶向上。
  7. 根据权利要求6所述的大循环流化床细胞生物反应器，其特征在于，所述细胞培养罐的底部圆台部分、上部圆台部分以及圆台形滤网的半锥角均为25度-60度。
  8. 根据权利要求1所述的大循环流化床细胞生物反应器，其特征在于，所述细胞培养罐顶部有带各种进出口的罐盖，所述罐盖和所述细胞培养罐顶端边缘法兰相连接并密封。
  9. 根据求权利要求8所述的大循环流化床细胞生物反应器，其特征在于，所述培养罐底座的内壁上粘有加热橡胶板。
  10. 根据权利要求1所述的大循环流化床细胞生物反应器，其特征在于，所述摇罐体为圆台形，其半锥角为30度至42度。
  11. 根据权利要求4所述的大循环流化床细胞生物反应器，其特征在于，所述上部圆台部分的横截面的面积由下至上是逐渐扩大的。
  12. 根据权利要求6所述的大循环流化床细胞生物反应器，其特征在于，所述细胞培养罐的底部圆台部分、上部圆台部分以及圆台形滤网的半锥角均为45度-60度。
  13. 一种培养动物细胞的方法，其特征在于，使用了如权利要求1-12中任一项所述的大循环流化床细胞生物反应器，并包括如下步骤：
    加培养液至静态液面后，加热培养液到35.5-37℃；启动摇床，将消毒后的微载体按比例加入培养液中，再将活性动物细胞接种到微载体中；摇罐体内上部的培养液在摇罐体内上部空间，导流管中和中心漏斗内的溶氧区内进行溶氧，溶氧后的培养液在摇动力的作用下进入进水硅胶软管和进水管；然后沿着细胞培养罐底部圆台部分的切线方向进入细胞培养罐，开始螺旋向上流动，直至流到圆台形滤网下边，给在此区域间的微载体内的活性细胞输送营养物质和溶解氧；完成这一供给过程后，培养液穿过圆台形滤网流到培养罐顶部，流入回水管，然后通过回水硅胶软管流回到摇罐体内，螺旋向上流动，重新进入导流管进口，完成一个循环；补充氧气和营养后，再开始下一个循环。
  14. 根据权利要求13所述的培养动物细胞的方法，其特征在于，
    在细胞培养罐给活性动物细胞输送营养和氧气过程中时，带有活性动物细胞的微载体是位于细胞培养罐底部圆台部分到上部圆台形滤网之间的区域中；当含有溶解氧和营养物质的培养液向上流动时，就给微载体中的活性动物细胞输送了营养物质和溶解氧。
  15. 根据权利要求13所述的培养动物细胞的方法，其特征在于，
    所述微载体为有重量和有形状的大孔微载体或多孔微载体，每种所述微载体在培养液中是相对有一个向下的沉降速率的；其中，当培养液向上 流速和微载体向下的沉降速率相对平衡时，微载体则不向下沉降而平衡不动；当培养液向上流速高于平衡值时，微载体就随培养液一起向上流动；当培养液的向上流速低于平衡值时，微载体就向下沉降；通过上部圆台部分的结构特性，该部分的横截面积逐步扩大，从而降低培养液在此区间的向上流速，利于微载体向下沉降，从而完成了微载体和培养液在此部位的快速可靠分离。
  16. 根据权利要求13所述的培养动物细胞的方法，其特征在于，所述圆台形滤网的孔的直径小于所述微载体的直径。
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