WO2020143841A1 - 用于体外规模化生产红细胞的中空纤维管和方法 - Google Patents

用于体外规模化生产红细胞的中空纤维管和方法 Download PDF

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WO2020143841A1
WO2020143841A1 PCT/CN2020/073588 CN2020073588W WO2020143841A1 WO 2020143841 A1 WO2020143841 A1 WO 2020143841A1 CN 2020073588 W CN2020073588 W CN 2020073588W WO 2020143841 A1 WO2020143841 A1 WO 2020143841A1
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hollow fiber
fiber tube
tube
differentiation
scale production
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PCT/CN2020/073588
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English (en)
French (fr)
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赵涌
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赵涌
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Priority claimed from CN201910011450.3A external-priority patent/CN109679834B/zh
Priority claimed from CN201910103065.1A external-priority patent/CN109825434B/zh
Application filed by 赵涌 filed Critical 赵涌
Publication of WO2020143841A1 publication Critical patent/WO2020143841A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/04—Apparatus for enzymology or microbiology with gas introduction means
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/24—Apparatus for enzymology or microbiology tube or bottle type
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the invention relates to the denuclearization of nucleated cells, in particular to a hollow fiber tube and method for large-scale production of red blood cells in vitro.
  • the research on the large-scale production of red blood cells in vitro is generally carried out in a serum-free culture system, which can avoid the influence of some undetermined substances in the serum, and can completely avoid the possible sources of contamination in the serum.
  • red blood cells One of the key technologies to realize the actual production of the required amount of red blood cells is how to efficiently produce completely denucleated red blood cells.
  • hematopoietic support of stromal cells is used to achieve complete denucleation of erythroid progenitor cells, these stromal cells have an adverse effect on late blood transfusion.
  • the present invention provides a solution for improving the efficiency of denucleating red blood cells based on hollow fiber tubes. Specifically, the present invention includes the following.
  • a hollow fiber tube for large-scale production of erythrocytes in vitro.
  • the diameter of the hollow fiber tube is 200-1000 ⁇ m, and its inner cavity is configured to circulate basic culture medium and/or gas. Its end can communicate with the basal medium container and/or oxygen reservoir; the wall thickness of the hollow fiber tube is 20-80 ⁇ m, and is set to allow the components and/or gas of the basal medium to pass from the hollow fiber
  • One side of the lumen of the tube passes through the tube wall to the other side, and at least the outer surface of the tube wall of the hollow fiber tube has a group suitable for cell denucleation, such as a quaternary ammonium group or chitosan.
  • the porous permeable material is selected from the group consisting of polysulfone, polyvinyl chloride, cellulose acetate, and acrylic copolymer.
  • a method for large-scale production of red blood cells in vitro which includes the following steps:
  • the waste generated in the denucleation and differentiation chamber enters the lumen of the hollow fiber tube along with at least part of the culture medium through the wall of the tube, and flows out of the denucleation and differentiation chamber.
  • the steps (1)-(3) are performed simultaneously.
  • the basal medium is SFEM medium
  • the denuclearization and differentiation chamber is further supplemented with at least one selected from the group consisting of BSA, Epo, iron-saturated transferrin, and IGF-1 One.
  • the hollow fiber system of the present invention can provide a very large surface area in a small volume, and the surface area can reach 200 cm 2 /ml, so that a large number of cells can be cultured in a very small volume range.
  • Cells can exchange nutrients and metabolites very efficiently through the wall of the tube, and the filtration performance of the hollow fiber tube can be controlled by known means to retain fiber-specific proteins and cytokines or allow them to enter the circulating matrix through the fiber.
  • the method of producing the cell of the present invention can be obtained in an amount of more than 108 cells / mL, the conventional method of cell culture cells can give up an amount of 10 6 cells / mL as compared with the production method of the present invention may be implemented large-scale production.
  • the hollow fiber tube of the present invention is more conducive to the denucleation and differentiation of erythroid progenitor cells, and more advantageous to the production of large-scale erythrocytes in vitro.
  • FIG. 1 Cell de-nucleation after using different hollow fiber tubes in the same differentiation system for 5 days.
  • the left graph is the result of the hollow fiber tube of the present invention, and the right graph is the result of the control hollow fiber tube.
  • FIG. 2 is a schematic diagram of an exemplary production system of the present invention.
  • FIG. 3 is a structural diagram of an exemplary bioreactor of the present invention.
  • a hollow fiber tube (hereinafter sometimes simply referred to as the "hollow fiber tube of the present invention") used for large-scale production of red blood cells in vitro is provided.
  • the diameter of the hollow fiber tube of the present invention is 200-1000 ⁇ m. It is preferably 300-900 ⁇ m, more preferably 400-800 ⁇ m, for example 550-800 ⁇ m and the like.
  • the “diameter” herein refers to the outer diameter of the hollow fiber tube. The diameter in the above range is very advantageous for the cultivation and denuclearization of erythrocytes, and for providing an appropriate surface area required for cell culture.
  • the diameter is too small, it is not conducive to the penetration of the prescribed amount of basal medium component or gas through the tube wall, or it is necessary to provide greater pressure in the lumen of the hollow fiber tube to promote the basal medium component to penetrate through the tube wall, Too high pressure is not conducive to the growth of cells attached to the hollow fiber tube wall, or although it may not have a substantial effect on cell attachment, it is extremely detrimental to the denucleation of erythroid progenitor cells and affects the efficiency of denucleation.
  • the length of the hollow fiber tube of the present invention is not particularly limited, but considering the uniformity of the pressure distribution in the radial direction of the tube when the basal medium and/or gas (for example, oxygen) permeates, the length of the hollow fiber tube is preferably 10-50 cm, More preferably, it is 20-45 cm.
  • the thickness of the wall of the hollow fiber tube of the present invention is not particularly limited, but is generally 20-80 ⁇ m, preferably 25-70 ⁇ m, and more preferably 35-50 ⁇ m.
  • the above tube wall thickness setting allows the components and/or gases of the basal medium to pass through the tube wall from one side (inner side) of the hollow fiber tube to the other side (outer side) under a certain pressure, preferably, further allowing cell culture Or the waste generated during the differentiation process enters the inside from the outside of the hollow fiber tube and is discharged with the flow of the basal medium in the lumen. If the thickness of the tube wall is too small, the hollow fiber tube cannot withstand the pressure required when the basic medium enters, and is easily broken.
  • the end of the hollow fiber tube can communicate with the basal medium container and/or oxygen reservoir.
  • Such a design is beneficial to provide nutrients and/or oxygen to the cells, thereby simulating the growth environment in the cells, and more conducive to the growth of the cells.
  • the tube wall of the hollow fiber tube of the present invention is made of a porous permeable material, and the pore diameter of the pores in the tube wall is less than 0.3 ⁇ m, preferably less than 0.2 ⁇ m.
  • the pore diameter of the pores in the tube wall is sufficient to allow the components of the basal medium to pass through smoothly.
  • the pore diameter is preferably greater than 0.005 ⁇ m, more preferably greater than 0.01 ⁇ m, and even more preferably greater than 0.05 ⁇ m.
  • the porous permeable material of the present invention may be selected from the group consisting of polysulfone, polyvinyl chloride, cellulose acetate, and acrylic copolymer.
  • the present invention may use one or more of the above materials in combination.
  • the water permeability of the hollow fiber tube of the present invention is generally 50-500 mL/m 2 /hr/mmHg, preferably 150-400 mL/m 2 /hr/mmHg.
  • the basic medium components are advantageously permeated and harmful wastes are oozed out, and at the same time, effective macromolecular organic substances (such as albumin, etc.) are formed for cell growth and differentiation, thereby reducing the use of these components, Reduce costs.
  • the hollow fiber tube of the present invention has an asymmetric two-layer structure along the thickness direction of the tube wall: the first layer with the distribution of nano-scale micropores exposed to the outside, and the opposite Loosely facing the second layer of hollow fiber lumen.
  • Such a structure is more conducive to the penetration of the basic medium components into the differentiation chamber, while preventing the differentiation factors in contact with the cells from seeping out of the differentiation chamber.
  • the hollow fiber tube of the present invention has a group suitable for cell denucleation on at least the outer surface of the tube wall.
  • a group suitable for cell denucleation on the outer surface of the tube wall cells that need to be denucleated, such as erythroid progenitor cells, can be efficiently denucleated without relying on stromal cells.
  • the outer surface of the tube wall has a quaternary ammonium group as a group suitable for cell denucleation.
  • the potential ⁇ can be measured based on the principle of electrophoresis. Specifically, when the surface of the tube wall of the fiber tube is in contact with the solution, the ions present in the solution opposite to the surface charge make a directional movement in the external electric field due to dissociation or adsorption. When the surface of the fiber tube has a positive charge, the solvated negative ions near the fiber tube move toward the positive electrode under the action of the electric field. On the contrary, if the surface of the fiber tube has a negative charge, the solvated positive ions will move to the negative electrode, and this movement is related to the amount of charge. Based on this, the zeta potential of the fiber tube surface can be measured.
  • the present invention finds that when the outer surface of the tube wall has the above-mentioned quaternary ammonium groups, it is very advantageous for the differentiation and denucleation of erythroid progenitor cells to form red blood cells and the discharge of wastes generated by the cells. The reason for this is not clear.
  • the inventors speculate that the nucleus component may be negatively charged during the denucleation process.
  • the outer surface of the tube wall has a positive nucleus, due to the interaction of positive and negative charges, nucleus detachment is promoted.
  • the waste generated by cells during metabolism or culture mainly includes small molecule acidic substances such as lactic acid and pyruvic acid.
  • the outer surface of the fiber tube has a positive charge
  • the negative ions solvated by such acidic substances are easily moved to or adsorbed by the wall of the hollow fiber with a positively charged core, and enter the hollow through the flow and free diffusion of the culture solution in the lumen The lumen of the fiber tube is expelled.
  • the hollow fiber tube of the present invention can be prepared by a known method.
  • the preparation method of the hollow fiber tube of the present invention includes the following steps:
  • the activated hollow fiber can be prepared by methods known in the art.
  • activated hollow fiber is prepared by using, for example, a melt extrusion method using chloromethyl ether modified polysulfone as a raw material.
  • a melt extrusion method for producing plastic products is used to extrude the melt of chloromethyl ether modified polysulfone and gradually cool in an atmospheric environment. As the temperature of the melt decreases, the melt fluidity gradually Reduced and cured.
  • an activated hollow fiber having an asymmetric structure is prepared by, for example, a shaped spinneret molding method. Specifically, it includes dissolving chloromethyl ether modified polysulfone in an organic solvent (for example, DMF or DMAc), which contains a pore-forming additive (for example, PEG), which is miscible and defoamed. Extrusion nozzle. After the extruded fiber is exposed to air, it is subjected to a coagulation bath. The solvent in the unformed modified polysulfone solution and the curing agent in the coagulation bath undergo double diffusion, and the modified polysulfone is cured and formed.
  • an organic solvent for example, DMF or DMAc
  • PEG pore-forming additive
  • the activated hollow fiber is prepared by activating the polysulfone hollow fiber.
  • the polysulfone hollow fiber is immersed in an organic solvent containing chloromethyl ether (such as dioxane), and then an inorganic accelerator is added for activation.
  • the molar ratio of chloromethyl ether to organic solvent is generally 1:5 to 1:15, preferably 1:10.
  • examples of inorganic accelerators include aluminum chloride, tin chloride, zinc chloride, and the like.
  • the activation conditions include reaction at 40-60°C, preferably 45-50°C for 5-24 hours, preferably 8-12 hours.
  • the organic solvent containing chloromethyl ether is preferably stirred. After the activation reaction is completed, it further includes washing the activated hollow fiber tube with deionized water and drying it at 60-85° C., thereby obtaining activated hollow fiber.
  • the surface treatment of the present invention includes immersing the activated hollow fiber in a solution of 20-40% trimethylamine (TMA) for 30-50 hours at room temperature.
  • TMA trimethylamine
  • the group suitable for cell denucleation in the hollow fiber tube of the present invention is chitosan.
  • the present invention found that when the pH of the culture medium is between 6-7, preferably 6.0-6.5, chitosan exhibits a positive charge, erythroid progenitor cells tend to bind to the outer wall of the hollow fiber tube, and are favorable for mature denucleation.
  • the pH of the culture medium increases, for example, to 7.4 or more, the erythroid progenitor cells tend to detach from the outer wall, thereby facilitating the collection of cells.
  • the hollow fiber tube of the present invention has an elongated tubular structure and can be used to form an in vitro cell culture device.
  • the structure of such a device may use a structure known in the art.
  • a method for large-scale production of erythrocytes in vitro (sometimes referred to simply as “the method of the invention") is provided.
  • the method of the present invention includes the process of using the hollow fiber tube described in the first aspect as a differentiation matrix of erythroid progenitor cells. Specifically, it may include the following steps:
  • the basal medium and/or oxygen is continuously entered from the inner cavity of the hollow fiber tube through the tube wall and filled into the denucleation and differentiation chamber;
  • the erythroid progenitor cells are brought into contact with the wall surface of the hollow fiber tube in the denucleation and differentiation chamber, and differentiation factors are added in the denucleation and differentiation chamber;
  • the substances required for cell differentiation and/or growth in the present invention include basal medium and differentiation factors.
  • the basic basal medium is mainly used to provide energy for cell growth and/or differentiation, including a large amount of inorganic substances and small molecular organic substances.
  • a medium known in the art may be used.
  • SFEM medium and IMDM medium a medium known in the art may be used.
  • the SFEM medium is a product produced by Stem Cell, for example, the product number is Cat#09600.
  • IMDM medium is a product known in the art, which can also be referred to as Iskov modified medium. It contains higher concentrations of nutrients and is suitable for high-density cell culture.
  • the components or gas components in the basal medium of the present invention can freely pass through the wall of the hollow fiber tube.
  • wastes such as lactic acid, pyruvic acid, and other small-molecule acidic substances generated during cell growth or differentiation that are unfavorable to cell differentiation can also be discharged outside the denuclear differentiation chamber through hollow fiber tubes.
  • the basal medium of the present invention may also contain other small molecule components.
  • other small molecule components include L-glutamine, 2-mercaptoethanol and iron ions.
  • the concentration of L-glutamine in the basal medium based on the volume of the basal medium is generally 1 mmol/L to 5 mmol/L, preferably 2 mmol/L to 3 mmol/L.
  • the concentration of 2-mercaptoethanol based on the volume of the basal medium is generally 1 ⁇ 10 -4 mol/L to 9 ⁇ 10 -4 mol/L, preferably 1 ⁇ 10 -4 mol/L to 5 ⁇ 10 -4 mol/L .
  • the concentration of iron ions in the basal medium is not limited.
  • the volume of the basal medium Based on the volume of the basal medium, it is generally 200 ⁇ g/ml to 400 ⁇ g/ml, preferably 250 ⁇ g/ml to 300 ⁇ g/ml.
  • the above iron ion concentration can be achieved by adding an appropriate amount such as lron Supplement (product of Sigma, Cat#I3 153).
  • one or more differentiation factors need to be further added in the scale production of erythrocytes in vitro. These differentiation factors ensure the maturation and differentiation of erythroid progenitor cells. Examples of such differentiation factors include, but are not limited to, erythropoietin (Epo), stem cell growth factor (SCF), iron-saturated transferrin, and insulin-like growth factor-1 (IGF-1).
  • Epo erythropoietin
  • SCF stem cell growth factor
  • IGF-1 insulin-like growth factor-1
  • Epo erythropoietin
  • SCF stem cell growth factor
  • IGF-1 insulin-like growth factor-1
  • the amount of Epo added is generally 8-10 U/ml, preferably 8.5-9.5 U/ml.
  • the addition amount of iron-saturated transferrin is generally 400-600 ⁇ g/ml, preferably 450-550 ⁇ g/ml.
  • the amount of IGF-1 added is generally 40-60 ng/ml, preferably 45-55 ng/ml.
  • the amount of SCF added is generally 50-100 ng/ml, preferably 60-80 ng/ml.
  • dexamethasone is further included, and its content is generally 0.5-2 ⁇ M, preferably 1-1.5 ⁇ M.
  • serum albumin needs to be further added to the differentiation chamber. It should be noted that serum albumin cannot enter the differentiation chamber from the hollow cavity of the hollow fiber through infiltration.
  • the amount of serum albumin added is 1-3% based on the weight of the basal medium, preferably 1-2%.
  • the basal medium of the present invention is an IMDM medium containing L-glutamine, 2-mercaptoethanol, and iron ions in the above range, and the above range needs to be further supplemented when cultured in the denucleation and differentiation chamber Amount of BSA, Epo, transferrin and IGF-1.
  • human insulin is further supplemented with 5-15 ⁇ g/ml, preferably 6-10 ⁇ g/ml.
  • the basal medium of the present invention is SFEM medium, and further supplemented with BSA, Epo, iron-saturated transferrin, and IGF-1 in the denuclearization and differentiation chamber.
  • the hollow fiber tube is a chitosan modified polysulfone hollow fiber tube, and the method includes:
  • the basal medium with a pH of 6-7 and/or oxygen is continuously entered from the inner cavity of the hollow fiber tube through the tube wall and filled into the denucleation and differentiation chamber;
  • the erythroid progenitor cells are brought into contact with the wall surface of the hollow fiber tube in the denucleation and differentiation chamber, where differentiation factors are added in the denucleation and differentiation chamber, and the pH of the liquid in the denucleation and differentiation chamber is controlled to be maintained to 6-7;
  • Soak 0.2kg polysulfone hollow fiber C2011 (FiberCell Systems, Frederick, MD) in 1000ml of an organic solvent composed of chloromethyl ether and dioxane in a volume ratio of 1:10, and add 8g of chlorination to the organic solvent Zinc.
  • the polysulfone hollow fiber was reacted at 40°C for 12 hours.
  • the organic solvent was stirred intermittently to keep the reaction between the hollow fiber tube and the organic solvent uniform.
  • the activated hollow fiber tube was washed with deionized water for many times. It was then dried at 60°C to obtain activated hollow fibers.
  • the activated hollow fiber was immersed in a 25% TM A solution at room temperature for 45 hours to obtain the hollow fiber tube 1 of the present invention.
  • a hollow fiber membrane spinning machine prepares hollow fibers, and after exposure to air, a coagulation bath (gel bath temperature 25° C., solvent concentration in gel bath 55%, core solution concentration 78%).
  • a coagulation bath gel bath temperature 25° C., solvent concentration in gel bath 55%, core solution concentration 78%.
  • an activated hollow fiber tube having an asymmetric structure is prepared.
  • the activated hollow fiber was immersed in a 25% TM A solution at room temperature for 45 hours to obtain the hollow fiber tube 2 of the present invention.
  • chitosan coating solution dissolve chitosan and N-(2-hydroxyethyl)piperazine in 2wt.% oxalic acid aqueous solution, chitosan and N-(2-hydroxyethyl)piperazine The mass ratio of 20:1-5:1, make up a solution with a mass fraction of 0.1%-2%, stir at room temperature until uniform, then transfer to an autoclave, purge the air with nitrogen and heat to 160°C Range, keep warm for 3 hours, drop to room temperature, and stir at low speed for 2h under vacuum.
  • cross-linking agent Dissolve 50g of cross-linking agent in 1L of methanol or acetone or ether solution with 80% mass fraction; the cross-linking agent is trimesoyl chloride.
  • This example is a test example of the performance of a hollow fiber tube.
  • Membrane potential measurement was performed with reference to the method described in Fudan University Physical Chemistry Experiment Shanghai: Fudan University Press, 1982.
  • This embodiment is an example of a method for producing red blood cells in vitro.
  • the reactor of this embodiment is a modified model of Catalog Nos. C2011 and C2008 of FiberCell Systems, which is a medium-sized reactor of 15 ml and can provide a surface area of 2200 cm 2 .
  • the hollow fiber tubes in the reactor were replaced with the hollow fiber tubes of Examples 1-3 as examples of the production method of the present invention.
  • the same cell culture and differentiation were carried out with this reactor of FiberCell Systems, and this was used as a comparative example.
  • Red blood progenitor cells (from the Institute of Field Blood Transfusion of the Chinese Academy of Military Medical Sciences) generated from cord blood-derived HSCs were used to conduct the following experiments.
  • the basal medium for erythroid progenitor cells uses the serum-free medium StemSpan SFEM.
  • the inventors also added stromal cells derived from fetal liver treated with 10 ⁇ g/ml mitomycin to the reactor for co-culture with erythroid progenitor cells. Differentiation. The specific conditions are the same as above except for the hollow fiber tube. After 10 days, the cultured red blood cells were collected. Stain by Wright-Gimesa, and observe the cell morphology by microscope. See Table 1 for specific observation results.
  • the analysis of conventional indicators through the hematology analyzer showed that the average volume of red blood cells was 10 4 ⁇ 7fl, the average red blood cell hemoglobin concentration was 25 ⁇ 3%, and the average red blood cell hemoglobin content was 30 ⁇ 2pg.
  • the test results were close to normal peripheral red blood cells. Get mature red blood cells.
  • This embodiment is the structure of a cell bionic intelligent production system.
  • the cell bionic intelligent production system of this embodiment includes a bioreactor 100, a culture medium container 200 and an oxygen supplier 300.
  • the medium container 200 is provided with a supply port 210 and a recovery port 220.
  • the supply port 210 and the recovery port 220 are connected to the bioreactor 100 through pipes, respectively.
  • the oxygen supplier 300 is connected to the bioreactor 100 through a hose.
  • the bioreactor 100 includes a sealed chamber 120 formed by a housing 110 and a plurality of hollow fiber tubes 130 disposed in the chamber 120.
  • the housing 110 is a round tube structure, and a first closed end 111 and a second closed end 112 are respectively provided at two ends thereof.
  • a medium inlet 113 is provided at the first closed end, and a medium outlet 114 is provided at the second closed end.
  • the medium inlet 113 is used to connect to the supply port 210, and the medium outlet 114 is used to connect to the recovery port 220.
  • the bioreactor 100 and the culture medium container 200 are connected to form a circuit.
  • the hollow fiber tubes of Embodiments 1 and/or 2 can be used.
  • a plurality of hollow fiber tubes 130 are arranged in parallel along the axial direction of the round tube, and are fixed between the first closed end 111 and the second closed end 112 by a polyurethane bonding agent.
  • An oxygen inlet 115 is also provided on the side of the housing 110 for connecting with the oxygen supplier 300.
  • the housing 110 is further provided with a first chamber port 116 and a second chamber port 117, so that the chamber 120 communicates with the outside world, or add corresponding organisms through the first chamber port 116 and the second chamber port 117 Factor, either seed cells or recover cells after culture.

Abstract

本发明公开用于体外规模化生产红细胞的中空纤维管和方法。本发明的中空纤维管的直径为200-1000μm，且设置为其内腔能够流通基础培养基和/或气体，其末端能够与基础培养基容器和/或氧气储存器连通；中空纤维管的管壁厚度为20-80μm，且设置为允许基础培养基的成分和/或气体从中空纤维管的内腔一侧穿过管壁到达另一侧，且在中空纤维管的至少管壁外表面具有适于细胞脱核的基团。本发明的体外规模化生产红细胞的方法有利于红系祖细胞的脱核分化。

Description

用于体外规模化生产红细胞的中空纤维管和方法 技术领域
本发明涉及有核细胞的无核化，具体涉及用于体外规模化生产红细胞的中空纤维管和方法。
背景技术
来自世界卫生组织(WHO)全球血液安全数据库的数据显示，全球每年收集9200万单位的全血捐献，每天有3.3％的医院因为血液短缺推迟手术，10.3％的医院每年至少有1天紧急手术无血可用。另外，由于艾滋病病毒感染等原因5-10％的血液输入不安全。因此，血液供给的需求在全球范围是非常紧迫的。
随着造血干细胞分化发育的研究深入，体外人工诱导造血干祖细胞扩增生产成熟红细胞逐渐成为解决输血面临困境的潜在可行的方向。目前对于体外大规模产生红细胞的研究一般在无血清培养体系中进行，这可以避免血清中一些尚未确定的物质的影响，而且完全可以避免血清中可能存在的污染源。
实现规模化生产实际所需量的红细胞的关键技术之一在于如何高效地产生完全脱核的红细胞。目前有研究在无血清培养体系中能使造血干细胞在体外持续扩增45天，细胞数目达10 7倍，但是红细胞脱核效率低，不能满足实现所需。虽另有以基质细胞的造血支持实现了红系祖细胞的完全脱核，但这些基质细胞对于后期输血带来不利影响。
发明内容
为解决现有技术中的至少部分技术问题，本发明提供一种基于中空纤维管来提高红细胞脱核效率的方案。具体地，本发明包括以下内容。
本发明的第一方面，提供一种用于体外规模化生产红细胞的中空纤维管，所述中空纤维管的直径为200-1000μm，且设置为其内腔能够流通基础培养基和/或气体，其末端能够与基础培养基容器和/或氧气储存器连通；所述中空纤维管的管壁厚度为20-80μm，且设置为允许所述基础培养基的成分和/或气体从所述中空纤维管的内腔一侧穿过所述管壁到达另一侧，且在所述中空纤维管的至少管壁的外表面具有适于细胞脱核的基团，例如季铵基团或壳聚糖。
在某些实施方案中，所述多孔可渗透材料选自聚砜、聚氯乙烯、醋酸纤维素和丙烯酸共聚物组成的组。
本发明的第二方面，提供一种用于体外规模化生产红细胞的方法，其包括以下步骤：
(1)使至少部分基础培养基和/或氧气连续地从中空纤维管的内腔通过管壁进入并填充至脱核分化室；
(2)使红系祖细胞在所述脱核分化室内与所述中空纤维管的管壁外表面接触，其中在所述脱核分化室内添加有分化因子；
(3)使所述脱核分化室内产生的废物随至少部分培养基一起通过管壁进入至所述中空纤维管的内腔，并流出至所述脱核分化室外。
在某些实施方案中，所述步骤(1)-(3)同时进行。
在某些实施方案中，所述基础培养基为SFEM培养基，且在所述脱核分化室内进一步补充选自BSA、Epo、铁饱和的转铁蛋白和IGF-l组成的组中的至少之一。
本发明的中空纤维系统可以在很小的体积内提供非常大的表面积，其表面积可以达到200cm 2/ml，从而可以在一个非常小的体积范围内规模化培养大量细胞。细胞通过管壁可以十分有效地交换营养和代谢物，并且可通过已知手段控制中空纤维管的过滤性能，使之保留纤维特异蛋白质和细胞因子或者允许它们通过纤维进入循环基质当中。本发明的生产方法可以得到10 8个 细胞/mL以上的细胞量，与传统细胞培养方法最多只能得到10 6个细胞/mL的细胞量相比，本发明的生产方法可实现规模化生产。另外，本发明的中空纤维管更有利于红系祖细胞的脱核分化，更有利于体外规模化红细胞的生产。
附图说明
图1利用不同中空纤维管在相同分化体系培养5天后的细胞脱核情况。左图为本发明的中空纤维管的结果，右图为对照中空纤维管的结果。
图2为本发明一种示例性生产系统的示意图。
图3为本发明一种示例性生物反应器的结构图。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为具体公开了该范围的上限和下限以及它们之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。除非另有说明，否则“％”为基于重量的百分数。
[用于体外规模化生产红细胞的中空纤维管]
本发明的第一方面，提供一种用于体外规模化生产红细胞的中空纤维管(本发明有时简称为“本发明的中空纤维管”)，本发明的中空纤维管的直径为200-1000μm，优选300-900μm，更优选400-800μm，例如550-800μm等。此处的“直径”是指中空纤维管的外径。上述范围的直径非常有利于红细胞 的培养与脱核分化，并且有利于提供细胞培养所需的适当的表面积。如果直径过小，则不利于规定量的基础培养基成分或气体渗透穿过管壁，或者需要在中空纤维管的内腔中提供更大的压力来促使基础培养基成分渗透穿过管壁，而过高的压力不利于细胞贴附于中空纤维管的管壁生长，或者虽然对细胞贴附可能没实质性影响，但是对于红系祖细胞的脱核极为不利，影响脱核效率。
本发明的中空纤维管的长度不特别限定，但是考虑到基础培养基和/或气体(例如，氧气)渗入时压力在管径向上分布的均匀性，优选中空纤维管的长度为10-50cm，更优选20-45cm。
本发明的中空纤维管的管壁的厚度不特别限定，一般为20-80μm，优选25-70μm，更优选35-50μm。上述管壁厚度设置允许一定压力下基础培养基的成分和/或气体能够从中空纤维管的内腔一侧(内侧)穿过管壁到达另一侧(外侧)，优选地，进一步允许细胞培养或分化过程中产生的废物从中空纤维管的外侧进入内侧，并随内腔的基础培养基的流动而排出。如果管壁的厚度过小，则中空纤维管不能承受基础培养基进入时所需的压力，容易破裂。如果管壁的厚度过大，则不利于物质的渗透，也会影响到细胞的生长或分化。优选地，中空纤维管的末端能够与基础培养基容器和/或氧气储存器连通。这样的设计有利于向细胞提供营养和/或氧气，从而模拟细胞体内的生长环境，更有利于细胞的生长。
在某些实施方案中，本发明的中空纤维管的管壁由多孔可渗透材料制成，且管壁中孔的孔径小于0.3μm，优选小于0.2μm。另一方面，需要管壁中孔的孔径足以使基础培养基的成分顺利通过，为此孔径优选大于0.005μm，更优选大于0.01μm，进一步优选大于0.05μm。本发明的多孔可渗透材料可选自聚砜、聚氯乙烯、醋酸纤维素和丙烯酸共聚物组成的组。本发明可使用上述材料中的一种或多种的组合。本发明的中空纤维管的透水量一般为50-500mL/m 2/hr/mmHg，优选150-400mL/m 2/hr/mmHg。在该透水量范围内有利地 使基础培养基成分透过并使有害废物渗出，同时对于细胞生长、分化有益大分子有机物(例如白蛋白等)形成有效阻挡，从而减少这些成分的使用量，缩减成本。
在某些实施方案中，本发明的中空纤维管在沿管壁的厚度方向上具有不对称的两层结构：暴露在外侧的具有纳米级微孔分布的第一层，和具有较大孔径相对疏松的面向中空纤维内腔的第二层。这样的结构更有利于基础培养基成分渗透进入分化室，而阻止与细胞接触的分化因子等渗出至分化室外。
本发明的中空纤维管的至少管壁外表面上具有适于细胞脱核的基团。通过在管壁外表面上设置适于细胞脱核的基团可以使红系祖细胞等需脱核的细胞在不依赖基质细胞的情况下实现高效脱核。优选地，管壁的外表面具有季铵基团作为适于细胞脱核的基团。管壁外表面具有季铵基团，从而使管壁外表面具有正电荷，由此可使管壁外表面的电位ζ为1.20×10 -3至4.0×10 - 3V，优选1.20×10 -3至3.5×10 -3V。本发明中，电位ζ可基于电泳原理来测定。具体地，当纤维管的管壁表面与溶液接触时，由于离解或吸附作用使溶液中存在的与表面电荷相反的离子在外加电场中作定向运动。当纤维管表面具有正电荷时，纤维管附近溶剂化的负离子在电场作用下向正极运动。相反，如果纤维管表面具有负电荷，则溶剂化的正离子会向负极运动，这种运动与电荷量存在关联。据此可以测定纤维管表面的ζ电位。
本发明发现，当管壁外表面具有上述季铵基团时，对于红系祖细胞分化脱核形成红细胞以及细胞产生的废物的排出非常有利。其原因不清楚，发明人推测可能在于细胞在脱核过程中，细胞核成分显示负电性，而当管壁外表面具有正电核时，由于正负电荷相互作用促进细胞核脱离。另外，细胞在分化或培养时代谢产生的废物主要包括乳酸、丙酮酸等小分子酸性物质。由于纤维管外表面具有正电荷，使此类酸性物质溶剂化的负离子容易向带正电核的中空纤维的管壁移动或被其吸附，并通过内腔中培养液的流动以及自由扩散进入中空纤维管的内腔被排出。
可通过已知的方法来制备本发明的中空纤维管。在示例性方法中，本发明的中空纤维管的制备方法包括以下步骤：
(1’)制备活化中空纤维的步骤
可以本领域已知的方法制备活化中空纤维。在某些实施方案中，以氯甲醚改性的聚砜为原料通过例如熔融挤出方法制备活化中空纤维。
在示例性方法中，用生产塑料制品的熔融挤出方法将氯甲醚改性的聚砜的熔体挤出，在大气环境中逐渐冷却，随着熔体温度的降低,熔体流动性逐渐减小而固化成型。
在另外的示例性方法中，通过例如多采用异型喷丝板成型法制备具有不对称性结构的活化中空纤维。具体地包括将氯甲醚改性的聚砜溶解于有机溶剂(例如，DMF或DMAc)中，其中含有致孔作用的添加剂(例如PEG)混溶脱泡，由计量泵通过中心通液式插入管式喷头挤出。挤出纤维在经空气暴露后，进行凝固浴，未成型的改性聚砜溶液中的溶剂与凝固浴中的固化剂进行双扩散，改性聚砜固化成型。由于改性聚砜溶液中的部分溶剂在空气间的蒸发，溶剂与固化剂在纤维径向的交换的扩散作用以及聚砜溶液中添加剂对相分离速度的调整和水溶性添加剂洗脱后留下的空位的影响，沿径向形成所需的孔道。
在某些实施方案中，对聚砜中空纤维进行活化处理制备活化中空纤维。首先，将聚砜中空纤维浸泡于含氯甲醚的有机溶剂(例如二氧乙烷)中，然后加入无机促进剂进行活化。其中氯甲醚与有机溶剂的摩尔比一般为1:5至1:15，优选1:10。无机促进剂的实例包括氯化铝、氯化锡、氯化锌等。活化条件包括在40-60℃，优选45-50℃下反应5-24小时，优选8-12小时。在活化时优选搅拌含氯甲醚的有机溶剂。活化反应结束后，进一步包括用去离子水清洗活化后的中空纤维管，并将其在60-85℃烘干，从而得到活化中空纤维。
(2’)活化中空纤维的表面处理步骤
本发明的表面处理包括将活化中空纤维于室温下，在20-40％三甲胺(TMA)的溶液中浸泡30-50h得到。
在某些实施方案中，本发明的中空纤维管中适于细胞脱核的基团为壳聚糖。本发明发现当培养基的pH在6-7，优选6.0-6.5之间时，壳聚糖呈现正电荷，红系祖细胞倾向于结合至中空纤维管的外壁，并且有利于成熟脱核。而当培养基的pH升高，例如，升至7.4以上时，红系祖细胞倾向于从外壁脱离，从而有利于细胞的收集。
本发明的中空纤维管为细长管状结构，可用于形成体外细胞培养的装置。此类装置的结构可使用本领域内已知的结构。
[用于体外规模化生产红细胞的方法]
本发明的第二方面，提供用于体外规模化生产红细胞的方法(有时简称为“本发明的方法”)。本发明的方法包括以第一方面所述的中空纤维管作为红系祖细胞分化基质的过程，具体地，可包括以下步骤：
(1)使基础培养基和/或氧气连续地从中空纤维管的内腔通过管壁进入并填充至脱核分化室；
(2)使红系祖细胞在脱核分化室内与中空纤维管的管壁表面接触，其中在脱核分化室内添加有分化因子；
(3)使脱核分化室内产生的废物随至少部分培养基成分一起通过管壁进入至中空纤维管的内腔，并流出至脱核分化室外。
本发明中细胞分化和/或生长所需的物质包括基础培养基和分化因子。基基础培养基主要用于为细胞生长和/或分化提供能量，包括大量无机物质和小分子有机物。基础培养基可使用本领域已知的培养基。例如，SFEM培养基和IMDM培养基。其中SFEM培养基是由Stem Cell公司生产的产品，例如产品号为Cat#09600的产品。IMDM培养基为本领域内已知的产品，其又可称为伊思柯夫改良培养液。它含有更高浓度的营养成分，适合于高密度细胞 培养。
本发明的基础培养基中的成分或气体成分可自由地通过中空纤维管的管壁。另外，细胞生长或分化过程中产生的对细胞分化不利的废物(例如乳酸、丙酮酸等小分子酸性物质)也可通过中空纤维管排出至脱核分化室外。
在某些实施方案中，本发明的基础培养基还可包含其他小分子成分。此类其他小分子成分的实例包括L-谷氨酰胺、2-巯基乙醇和铁离子。基础培养基中L-谷氨酰胺的浓度基于基础培养基的体积一般为1mmol/L至5mmol/L，优选2mmol/L至3mmol/L。2-巯基乙醇的浓度基于基础培养基的体积一般为1×10 -4mol/L至9×10 -4mol/L，优选1×10 -4mol/L至5×10 -4mol/L。基础培养基中铁离子的浓度不限定。基于基础培养基的体积，一般为200μg/ml至400μg/ml，优选250μg/ml至300μg/ml。上述铁离子浓度可通过添加适量例如lron Supplement(Sigma公司产品，Cat#I3 153)来实现。
除了上述基础培养基外，在体外规模化生产红细胞时需进一步添加一种或多种分化因子。这些分化因子确保红系祖细胞的成熟分化。此类分化因子的实例包括但不限于促红细胞生成素(Epo)、干细胞生长因子(SCF)、铁饱和的转铁蛋白和胰岛素样生长因子-1(IGF-l)。基于基础培养基的体积，Epo的添加量一般为8-10U/ml，优选8.5-9.5U/ml。铁饱和的转铁蛋白的添加量一般为400-600μg/ml，优选450-550μg/ml。IGF-l的添加量一般为40-60ng/ml，优选45-55ng/ml。SCF的添加量一般为50-100ng/ml，优选60-80ng/ml。优选地，进一步包括地塞米松，其含量一般为0.5-2μM，优选1-1.5μM。
优选地，在体外规模化生产红细胞时需要向分化室内进一步添加血清白蛋白，需要说明的是血清白蛋白不能从中空纤维的内腔通过渗透进入分化室。血清白蛋白的添加量基于基础培养基的重量为1-3％，优选1-2％。
在某些实施方案中，本发明的基础培养基为含有上述范围量的L-谷氨酰胺、2-巯基乙醇和铁离子的IMDM培养基，并且在脱核分化室内培养时需进一步补充上述范围量的BSA、Epo、转铁蛋白和IGF-l。优选进一步补充5- 15μg/ml，优选6-10μg/ml的人胰岛素。
在某些实施方案中，本发明的基础培养基为SFEM培养基，且在脱核分化室内进一步补充上述规定范围量的BSA、Epo、铁饱和的转铁蛋白和IGF-l。
在示例性实施方案中，用于体外规模化生产红细胞的方法中，中空纤维管为壳聚糖改性聚砜中空纤维管，所述方法包括：
(1)使pH为6-7的基础培养基和/或氧气连续地从中空纤维管的内腔通过管壁进入并填充至脱核分化室；
(2)使红系祖细胞在脱核分化室内与中空纤维管的管壁表面接触，其中在脱核分化室内添加有分化因子，且控制脱核分化室内液体的pH维持至6-7；
(3)使脱核分化室内产生的废物随至少部分培养基成分一起通过管壁进入至中空纤维管的内腔，并流出至脱核分化室外；
(4)使脱核分化室内液体的pH升至7.4以上，并从脱核分化室的液体收集红细胞。
实施例1
将0.2kg聚砜中空纤维C2011(FiberCell Systems,Frederick,MD)浸泡于1000ml由体积比为1:10的氯甲醚和二氧乙烷组成的有机溶剂中，在该有机溶剂中加入8g氯化锌。在40℃下使聚砜中空纤维反应12小时。在反应期间间断性搅拌有机溶剂，使中空纤维管与有机溶剂之间的反应保持均一性。之后用去离子水清洗多次活化后的中空纤维管。然后将其在60℃烘干，从而得到活化的中空纤维。将活化的中空纤维于室温下在25％TM A的溶液中浸泡45小时得到本发明的中空纤维管1。
实施例2
将氯甲醚改性的聚砜溶解于DMF有机溶剂中，以PEG6000为添加剂，经混溶脱泡，使用包括计量泵(规格为1.2ml/r)、插入管式喷丝头、氮气钢瓶等的中空纤维膜纺丝机制备中空纤维，经空气暴露后，进行凝固浴(凝胶浴温度25℃，凝胶浴中溶剂浓度55％，芯液中溶剂浓度78％)。由此制备具有不对称性结构的活化中空纤维管。将活化的中空纤维于室温下在25％TM A的溶液中浸泡45小时得到本发明的中空纤维管2。
实施例3
1.参考Lan等人(Lan,WJ；Li,SW；Lu,YC；Xu,JH；Luo,GS.Controllable preparation of microscale tubes with multiphase co-laminar flow in a double co-axial microdevice Lab Chip,2009,9(22):3282-3288)的方法制备聚砜(PSF)中空纤维微管。
2.中空纤维管的改性
2.1壳聚糖涂覆液的制备：将壳聚糖和N-(2-羟乙基)哌嗪溶解于2wt.％草酸水溶液中，壳聚糖与N-(2-羟乙基)哌嗪的质量比20:1-5:1，配成质量分数为0.1％-2％的溶液，在常温下搅拌至均匀，然后转移至高压釜内，用氮气吹扫空气后加热至温度为160℃范围，保温3小时，降至室温，抽真空状态下低速搅拌2h。
2.2交联剂的制备：将50g的交联剂溶解于1L质量分数为80％的甲醇或丙酮或乙醚水溶液中；交联剂为均苯三甲酰氯。
2.3聚砜中空纤维微管的处理：将中空纤维微管用质量分数为1.0wt.％-5.0wt.％的十二烷基苯磺酸钠水溶液在30℃下浸泡2h，然后蒸馏水冲洗后室温晾干备用。
2.4壳聚糖/聚砜复合中空纤维管的制备：将聚砜中空纤维管浸入壳聚糖涂覆液中维持1-15min后取出，在50℃的烘箱中干燥2h。
2.5将涂覆了壳聚糖的中空管浸泡在交联剂中，在50℃温度下反应1h， 取出后在氮气气氛下50℃烘干15min，得到中空纤维管3。
实施例4
本实施例为中空纤维管性能的测试例。
1.中空纤维管的孔径观察
截取一小段膜，在光学显微镜下观察内、外表面和断面的孔结构。
2.管壁表面电位测量
将多根中空纤维管平行排列于平面基质上形成膜状。参考复旦大学《物理化学实验》上海:复旦大学出版社,1982中所述的方法进行膜电位测量。
结果如表1所示。
实施例5
本实施例为体外生产红细胞的方法例。
本实施例的反应器为FiberCell Systems公司catalog nos.C2011 and C2008的改造型号，其为15ml的中号反应器，能提供2200cm 2的表面积。将该反应器中的中空纤维管分别替换为实施例1-3的中空纤维管作为本发明的生产方法的实施例。同时，以FiberCell Systems公司的该反应器进行同样的细胞培养和分化，并将其作为比较例。
采用体外诱导脐带血来源的HSC生成的红系祖细胞(来自中国人民解放军军事医学科学院野战输血研究所)进行下述实验。红系祖细胞的基础培养基采用无血清培养基StemSpan SFEM。
在改造的反应器中再次加入所需体积的StemSpan SFEM，向其中加入Epo 5U/mL、SCF 100ng/mL、IGF-l 50ng/mL、Dex l/-lM等细胞因子组成培养体系，以5×10 5/mL的浓度加入所需的红系祖细胞，开启反应器，使新鲜的StemSpan SFEM不断地经中空纤维管管壁向反应器加入反应器，并通过管壁不断排出细胞培养所产生的废物，由此构成循环体系，同时不断向反应器中供给所需的氧等。在该条件下培养红系祖细胞8天，当观察到细胞体积 变大，细胞形态趋于均一时，之后收集细胞。
在改造的反应器中更换新的StemSpan SFEM，并向其中加入Epo 10U/mL，铁饱和的转铁蛋白500μg/mL，IGF-l 50ng/mL，铁离子等细胞因子，构成脱核分化体系。向该脱核分化体系加入收集的细胞，在不断向反应器中供给所需的氧等条件下进行分化培养，10天后收集得到培养的红细胞。通过Wright-Gimesa染色，并通过显微镜观察细胞形态。具体观察结果参见表1。
为了进一步验证本发明的中空纤维管有利于红系祖细胞脱核，发明人还向反应器中加入用10μg/ml丝裂霉素处理的胎儿肝脏来源的基质细胞与红系祖细胞共培养、分化。除了中空纤维管不同之外，具体条件与上述相同。10天后收集得到培养的红细胞。通过Wright-Gimesa染色，并通过显微镜观察细胞形态。具体观察结果参见表1。
另外，通过血细胞分析仪分析常规指标，结果显示红细胞平均容积为10 4±7fl，红细胞平均血红蛋白浓度为25±3％，红细胞平均血红蛋白含量为30±2pg，检测结果与正常外周血红细胞接近，显示得到成熟红细胞。
表1
Figure PCTCN2020073588-appb-000001
实施例6
本实施例为细胞仿生智能化生产系统的结构。
如图2所示，本实施例的细胞仿生智能化生产系统包括生物反应器100、培养基容器200和氧气供给器300。培养基容器200上设置有供给口210和回收口220。供给口210和回收口220分别通过管路与生物反应器100连接。 氧气供给器300通过软管与生物反应器100连接。
如图3所示，生物反应器100包括由外壳110形成的密封的腔室120和设置于腔室120内的多根中空纤维管130。外壳110为圆管结构，其两端分别设置第一封闭末端111和第二封闭末端112。在第一封闭末端设置培养基进口113，在第二封闭末端设置培养基出口114。培养基进口113用于与供给口210连接，培养基出口114用于与回收口220连接。由此使生物反应器100和培养基容器200连接形成回路。多根中空纤维管130可采用实施例1和/或2实施例的中空纤维管。多根中空纤维管130沿圆管的轴向平行设置，并通过聚氨酯结合剂固定于第一封闭末端111和第二封闭末端112之间。在外壳110的侧面还设置氧气进口115，用于与氧气供给器300连接。
在外壳110上还进一步设置有第一腔室口116和第二腔室口117，从而使腔室120与外界连通，或者通过第一腔室口116和第二腔室口117添加相应的生物因子，或者接种细胞或回收培养后的细胞。
尽管本发明已经参考示例性实施方案进行了描述，但应理解本发明不限于公开的示例性实施方案。在不背离本发明的范围或精神的情况下，可对本发明说明书的示例性实施方案做多种调整或变化。权利要求的范围应基于最宽的解释以涵盖所有修改和等同结构与功能。

Claims (10)

  1. 一种用于体外规模化生产红细胞的中空纤维管，其特征在于，所述中空纤维管的直径为200-1000μm，且设置为其内腔能够流通基础培养基和/或气体，其末端能够与基础培养基容器和/或氧气储存器连通；所述中空纤维管的管壁厚度为20-80μm，且设置为允许所述基础培养基的成分和/或气体从所述中空纤维管的内腔一侧穿过所述管壁到达外侧，且在所述中空纤维管的至少管壁的外表面具有适于细胞脱核的基团。
  2. 根据权利要求1所述的用于体外规模化生产红细胞的中空纤维管，其特征在于，所述管壁由孔径小于0.3μm的多孔可渗透材料制成，且所述管壁的外表面具有季铵基团或壳聚糖。
  3. 根据权利要求2所述的用于体外规模化生产红细胞的中空纤维管，其特征在于，所述多孔可渗透材料选自聚砜、聚氯乙烯、醋酸纤维素和丙烯酸共聚物组成的组。
  4. 根据权利要求1所述的用于体外规模化生产红细胞的中空纤维管，其特征在于，所述管壁的外表面的电位为1.20×10 -3至4.0×10 -3V。
  5. 根据权利要求1所述的用于体外规模化生产红细胞的中空纤维管，其特征在于，所述基础培养基选自SFEM培养基和IMDM培养基，且不含血清白蛋白。
  6. 一种用于体外规模化生产红细胞的方法，其特征在于，包括以下步骤：
    (1)使至少部分基础培养基和/或氧气连续地从中空纤维管的内腔通过管壁进入并填充至脱核分化室；
    (2)使红系祖细胞在所述脱核分化室内与所述中空纤维管的管壁的外表面接触，其中在所述脱核分化室内添加有分化因子；
    (3)使所述脱核分化室内产生的废物随至少部分培养基一起通过管壁进入至所述中空纤维管的内腔，并流出至所述脱核分化室外；
    其中，所述中空纤维管为根据权利要求1-5任一项所述的中空纤维管。
  7. 根据权利要求6所述的用于体外规模化生产红细胞的方法，其特征在 于，所述分化因子包括Epo、铁饱和的转铁蛋白和IGF-l。
  8. 根据权利要求6所述的用于体外规模化生产红细胞的方法，其特征在于，所述基础培养基为含有L-谷氨酰胺、2-巯基乙醇和铁离子的IMDM培养基，且在所述脱核分化室内进一步补充选自BSA、人胰岛素、Epo、转铁蛋白和IGF-l组成的组中的至少之一。
  9. 根据权利要求6所述的用于体外规模化生产红细胞的方法，其特征在于，所述基础培养基为SFEM培养基，且在所述脱核分化室内进一步补充选自BSA、Epo、铁饱和的转铁蛋白和IGF-l组成的组中的至少之一。
  10. 一种细胞仿生智能化生产系统，其特征在于，包括生物反应器、培养基容器和氧气供给器；
    其中所述生物反应器包括由外壳形成的密封的腔室和设置于所述腔室内的多根根据权利要求1-5任一项所述的中空纤维管，所述外壳设置有培养基进口、培养基出口和氧气进口；
    所述培养基容器设置有供给口，并且所述供给口通过管路与所述培养基进口连接；
    所述氧气供给器设置为其通过管路与所述氧气进口连接。
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101081182A (zh) * 2007-07-05 2007-12-05 南京大学医学院附属鼓楼医院 新型生物人工肝细胞反应器
CN103194389A (zh) * 2013-04-16 2013-07-10 叶永清 体外高密度培养红细胞的装置和方法
CN103328625A (zh) * 2010-11-24 2013-09-25 帝国创新有限公司 生物反应器
CN107400633A (zh) * 2017-08-17 2017-11-28 上海白泽医疗器械有限公司 中空纤维交换器及中空纤维交换式培养系统
CN107904170A (zh) * 2017-12-18 2018-04-13 上海白泽医疗器械有限公司 细胞培养模块及细胞培养系统
CN109679834A (zh) * 2019-01-07 2019-04-26 赵涌 用于体外规模化生产红细胞的中空纤维管和方法
CN109825434A (zh) * 2019-02-01 2019-05-31 赵涌 细胞仿生智能化生产系统
CN209669249U (zh) * 2019-02-01 2019-11-22 赵涌 细胞仿生智能化生产系统

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101081182A (zh) * 2007-07-05 2007-12-05 南京大学医学院附属鼓楼医院 新型生物人工肝细胞反应器
CN103328625A (zh) * 2010-11-24 2013-09-25 帝国创新有限公司 生物反应器
CN103194389A (zh) * 2013-04-16 2013-07-10 叶永清 体外高密度培养红细胞的装置和方法
CN107400633A (zh) * 2017-08-17 2017-11-28 上海白泽医疗器械有限公司 中空纤维交换器及中空纤维交换式培养系统
CN107904170A (zh) * 2017-12-18 2018-04-13 上海白泽医疗器械有限公司 细胞培养模块及细胞培养系统
CN109679834A (zh) * 2019-01-07 2019-04-26 赵涌 用于体外规模化生产红细胞的中空纤维管和方法
CN109825434A (zh) * 2019-02-01 2019-05-31 赵涌 细胞仿生智能化生产系统
CN209669249U (zh) * 2019-02-01 2019-11-22 赵涌 细胞仿生智能化生产系统

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