CN109825434B

CN109825434B - Cell bionic intelligent production system

Info

Publication number: CN109825434B
Application number: CN201910103065.1A
Authority: CN
Inventors: 赵涌
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-02-01
Filing date: 2019-02-01
Publication date: 2020-07-10
Anticipated expiration: 2039-02-01
Also published as: WO2020156391A1; CN109825434A

Abstract

The invention discloses a cell bionic intelligent production system which comprises a bioreactor, a culture medium container and an oxygen supplier. The bioreactor comprises a sealed cavity formed by a shell and a plurality of hollow fiber tubes arranged in the cavity, the shell is provided with a culture medium inlet, a culture medium outlet and an oxygen inlet, the inner cavity of each hollow fiber tube can be used for flowing basic culture medium and/or gas, the tail end of each hollow fiber tube can be communicated with a basic culture medium container and/or an oxygen supplier, the tube wall of each hollow fiber tube allows the components and/or the gas of the basic culture medium to pass through the tube wall from one side of the inner cavity of each hollow fiber tube to the outside, and at least the outer surface of the tube wall of each hollow fiber tube is provided with a group suitable for cell denucleation. The culture medium container is provided with a supply port, and the supply port is connected with the culture medium inlet through a pipeline; the oxygen supplier is connected with the oxygen inlet through a pipeline. The cell bionic intelligent production system is particularly suitable for the culture of erythroid progenitor cells.

Description

Cell bionic intelligent production system

Technical Field

The invention relates to a cell production system, in particular to a cell bionic intelligent production system.

Background

Data from the World Health Organization (WHO) global blood safety database shows that 9200 million units of whole blood donations are collected worldwide each year, with 3.3% of hospitals each day postponing surgery due to blood shortages, and 10.3% of hospitals each year having at least 1 day of emergency surgery without blood available. In addition, 5-10% of blood transfusions are unsafe due to HIV infection. Therefore, the demand for blood supply is very urgent worldwide.

With the research on the differentiation and development of hematopoietic stem cells, the in vitro artificial induction of the expansion of hematopoietic stem and progenitor cells to produce mature red blood cells gradually becomes a potential feasible direction for solving the dilemma faced by blood transfusion. The current 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 unidentified substances in serum and completely avoid possible pollution sources in the serum.

One of the key technologies to achieve the large-scale production of the practically required amount of erythrocytes is how to efficiently produce fully enucleated erythrocytes. At present, the research can lead the hematopoietic stem cells to be continuously expanded in vitro for 45 days in a serum-free culture system, and the cell number reaches 10⁷However, the efficiency of enucleation of erythrocytes is low and is not satisfactory for the achievement of the desired result. While complete enucleation of erythroid progenitor cells is achieved with the hematopoietic support of stromal cells, these stromal cells adversely affect later transfusions.

Disclosure of Invention

In order to solve at least part of technical problems in the prior art, the invention provides a cell bionic intelligent production system, which comprises a bioreactor, a culture medium container and an oxygen supplier;

wherein the bioreactor comprises a sealed chamber formed by a shell and a plurality of hollow fiber tubes arranged in the chamber, the shell is provided with a culture medium inlet, a culture medium outlet and an oxygen inlet, the diameter of the hollow fiber tube is 200-;

the culture medium container is provided with a supply port, and the supply port is connected with the culture medium inlet through a pipeline;

the oxygen supplier is arranged to be connected with the oxygen inlet through a pipeline.

In some embodiments, the housing is a circular tube structure, the two ends of the housing are respectively provided with a first closed end and a second closed end, thereby forming a sealed chamber, the culture medium inlet is arranged at the first closed end, the culture medium outlet is arranged at the second closed end, and the plurality of hollow fiber tubes are arranged in parallel along the axial direction of the circular tube.

In certain embodiments, the oxygen inlet is disposed on one side of the housing.

In certain embodiments, the culture medium vessel is further provided with a recovery port, and the recovery port is connected to the culture medium outlet by tubing, thereby connecting the bioreactor and the culture medium vessel to form a loop.

In certain embodiments, the housing is further provided with a chamber port for communicating the chamber with the outside.

In certain embodiments, the chamber ports include a first chamber port for fluid inlet and a second chamber port for fluid outlet, and the first chamber port and the second chamber port are respectively disposed at a side of the housing.

In certain embodiments, the walls of the hollow fiber tubes are made of a porous permeable material having pore sizes less than 0.3 μm, and the outer surface of the walls of the tubes have quaternary ammonium groups.

In certain embodiments, the porous permeable material is selected from the group consisting of polysulfone, polyvinyl chloride, cellulose acetate, and acrylic copolymer.

In certain embodiments, the potential of the outer surface of the wall of the hollow fiber tube is 1.20 × 10^-3To 4.0 × 10^-3V。

In certain embodiments, the medium vessel contains a basal medium and is selected from the group consisting of SFEM medium and IMDM medium without serum albumin.

The hollow fiber system of the present invention can provide very large surface areas in a very small volume, which can reach 200cm²A/ml, so that large numbers of cells can be cultured on a scale in a very small volume range. The cells can exchange nutrients and metabolites very effectively through the tube wall, and the filtration performance of the hollow fiber tube can be controlled by known means to retain fiber-specific proteinsAnd cytokines or allow them to pass through the fibers into the circulating matrix. The production system of the present invention can obtain 10⁸The cell amount of more than L per m can only be 10 at most compared with the conventional cell culture method⁶In addition, the hollow fiber tube is more beneficial to the enucleation and differentiation of erythroid progenitor cells and is more beneficial to the production of large-scale erythrocytes in vitro.

Drawings

FIG. 1 is a schematic diagram of an exemplary production system of the present invention.

FIG. 2 is a block diagram of an exemplary bioreactor of the present invention.

FIG. 3 shows cell enucleation after 5 days of culturing of erythroid progenitor cells using the production system of example 4. The left panel shows the results of the production system of the present invention, and the right panel shows the results of the control production system.

Description of reference numerals:

100-bioreactor, 110-housing, 120-chamber, 130 hollow fiber tube, 111-first closed end, 112-second closed end, 113-culture medium inlet, 114-culture medium outlet, 115-oxygen inlet, 116-first chamber port, 117-second chamber port; 200-medium container, 210-supply port, 220-recovery port; 300-oxygen supplier.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

The invention provides a cell bionic intelligent production system (the invention is sometimes abbreviated as the production system of the invention) which comprises a bioreactor, a culture medium container and an oxygen supplier. Optionally, the device further comprises a real-time monitoring device, a peristaltic pump and a biological factor adding device. The components are described in detail below.

Bioreactor

The bioreactor of the present invention includes a sealed chamber formed by a housing and a plurality of hollow fiber tubes disposed within the chamber. The chamber part formed by the inner wall of the shell and the outer walls of the hollow fiber tubes is a space for biological reaction or cell culture/biology.

The shell of the bioreactor forms an external integral structure of the bioreactor, and the shell is provided with a culture medium inlet, a culture medium outlet and an oxygen inlet. The culture medium inlet is used for being connected with the culture medium container so as to introduce the basic culture medium into the bioreactor, and the culture medium outlet is used for enabling the culture medium in the bioreactor to flow out of the bioreactor. Preferably, the medium inlet and the medium outlet are oppositely disposed, thereby facilitating the introduction of fresh basal medium and the discharge of the medium after the reaction. The oxygen inlet is used to provide the required amount of oxygen to the bioreactor. The oxygen inlet may be provided on the side of the housing, for example, on the side of the housing near the side of the medium inlet. The oxygen inlet may also be provided at the closed end of the housing so that oxygen together with the basal medium may enter the interior of the hollow fiber tubes and pass through the walls of the hollow fiber tubes into the chamber of the bioreactor.

The material of the housing of the present invention is not particularly limited, and a plastic or glass material, preferably a transparent material, can be used. The shape of the casing is not particularly limited, and may be spherical, cubic, tubular, etc. In some embodiments, the housing is a circular tube having a first closed end and a second closed end at opposite ends thereof, thereby forming a sealed chamber. Preferably, the medium inlet is disposed at the first closed end and the medium outlet is disposed at the second closed end.

In certain embodiments, the housing of the present invention is further provided with a chamber port for communicating the chamber with the outside. The chamber port is also used for the addition (seeding) or removal or recovery (collection) of the cells to be cultured. Preferably, the chamber ports include a first chamber port for fluid inlet or addition of cells to be cultured and a second chamber port for fluid outlet or removal of cells after completion of culturing. The first chamber port and the second chamber port may be respectively disposed at ends of the housing.

In the present invention, the plurality of hollow fiber tubes are preferably arranged in parallel in one direction of the chamber. In certain embodiments, the plurality of hollow fibers are arranged in parallel along the axial direction of the round tube. One end of the plurality of hollow fibers may be secured to the first closed end and the other end may be secured to the second closed end. Preferably, the ends of the plurality of hollow fibers are secured to the first closed end and/or the second closed end by a bonding agent. Any material having an adhesive function can be used as the binder as long as the binder is capable of permeating components and/or gases of small molecular substances such as a medium (particularly, a basic medium according to the present invention) into the hollow fiber while blocking passage of large molecular substances such as cells. Examples of binders include, but are not limited to, various porous permeable materials, such as polyurethane.

In the present invention, the number of the hollow fiber tubes is not particularly limited and can be freely determined by those skilled in the art as needed as long as the number of the hollow fiber tubes is sufficient to provide a desired outer surface. In an exemplary embodiment, the number of hollow fiber tubes is 10 to 10000, preferably 10 to 1000, more preferably 20 to 100.

The diameter of the hollow fiber tube of the invention is 200-. The "diameter" herein refers to the outer diameter of the hollow fiber tube. The above range of diameters is very advantageous for the culture and enucleation differentiation of erythrocytes and for providing an appropriate surface area for cell culture. If the diameter is too small, permeation of a prescribed amount of the basic medium components or gas through the tube wall is not facilitated, or a higher pressure needs to be provided in the lumen of the hollow fiber tube to promote permeation of the basic medium components through the tube wall, whereas an excessively high pressure is not conducive to growth of cells adhering to the tube wall of the hollow fiber tube, or, although cell adhesion may not be substantially affected, it is extremely disadvantageous to enucleation of erythroid progenitor cells, affecting the efficiency of enucleation.

The length of the hollow fiber tube of the present invention is not particularly limited, but preferably the length of the hollow fiber tube is 10 to 50cm, more preferably 20 to 45cm, in view of uniformity of pressure distribution in the tube diameter direction at the time of infiltration of the basic medium and/or gas (e.g., oxygen).

The thickness of the wall of the hollow fiber tube of the present invention is not particularly limited, and is generally 20 to 80 μm, preferably 25 to 70 μm, and more preferably 35 to 50 μm. The tube wall thickness arrangement described above allows the components and/or gases of the base media to pass through the tube wall from one side (inside) of the lumen of the hollow fiber tube to the other side (outside) under pressure, preferably further allowing waste products generated during cell culture or differentiation to pass from the outside of the hollow fiber tube to the inside and exit with the flow of the base media of the lumen. If the thickness of the tube wall is too small, the hollow fiber tube cannot withstand the pressure required for the base medium to enter, and is easily broken. If the thickness of the tube wall is too large, permeation of the substance is not facilitated, and growth or differentiation of cells is also affected. Preferably, the ends of the hollow fiber tubes can be in communication with a basal media container and/or an oxygen supply. Such a design is advantageous for providing nutrients and/or oxygen to the cells, thereby simulating the growth environment in the cells and further facilitating the growth of the cells.

In certain embodiments, the walls of the hollow fiber tubes of the present invention are made of porous permeable material, and the pores in the walls have a pore size of less than 0.3 μm, preferably less than 0.2 μm. in another aspect, it is desirable that the pores in the walls have a pore size sufficient to allow the smooth passage of the components of the basal medium, for which the pore size is preferably greater than 0.005 μm, more preferably greater than 0.01 μm, and even more preferably greater than 0.05 μm²/hr/mmHg, preferably 150-²hr/mmHg. In the water permeation range, the basic culture medium components are favorably permeated, harmful waste is permeated, and an effective barrier is formed for beneficial macromolecular organic matters (such as albumin and the like) for cell growth and differentiation, so that the use amount of the components is reduced, and the cost is reduced.

In certain embodiments, the hollow fiber tubes of the present invention have an asymmetric two-layer structure in the thickness direction along the tube wall: a first layer with a distribution of nano-scale micro-pores exposed on the outside, and a second layer with relatively loose larger pore diameter facing the lumen of the hollow fiber. Such a structure is more advantageous for the permeation of the basic medium components into the differentiation chamber, while preventing the permeation of differentiation factors and the like that come into contact with the cells outside the differentiation chamber.

The hollow fiber tube of the present invention has a group suitable for cell enucleation on at least the outer wall surface of the tube wall, and can be used for efficient enucleation of cells requiring enucleation such as erythroid progenitor cells without depending on stromal cells by providing a group suitable for cell enucleation on the outer wall surface of the tube wall^-3To 4.0 × 10^-3V, preferably 1.20 × 10^-3To 3.5 × 10^-3And V. In the present invention, the potential ζ can be measured based on the principle of electrophoresis. Specifically, when the wall surface of the fiber tube is brought into contact with the solution, the solution is adsorbed or dissociatedIons present in the solution and having a surface charge opposite to that of the ions are caused to move directionally in the applied electric field. When the surface of the fiber tube has positive charges, the solvated negative ions near the fiber tube move to the positive pole under the action of the electric field. Conversely, if the surface of the fiber tube has a negative charge, the solvated positive ions will move toward the negative electrode, and this movement is associated with the amount of charge. From this, the zeta potential of the surface of the fiber tube can be determined.

The present inventors have found that when the quaternary ammonium groups are present on the outer surface of the tube wall, they are very advantageous for the differentiation and enucleation of erythroid progenitor cells to form erythrocytes and for the discharge of waste products produced by the cells. The reason for this is unclear, and the inventors speculate that it is possible that the nuclear component of the cell shows electronegativity during enucleation of the cell, and when the outer surface of the tube wall has positive electric nuclei, detachment of the nuclei is promoted due to the interaction of the positive and negative electric charges. In addition, the waste products produced by metabolism of cells during differentiation or culture mainly include small-molecule acidic substances such as lactic acid and pyruvic acid. Because the outer surface of the fiber tube has positive charges, the negative ions solvated by the acidic substances are easy to move to or be adsorbed by the tube wall of the hollow fiber with the positive charge nucleus, and are discharged through the flow and free diffusion of the culture solution in the inner cavity into the inner cavity of the hollow fiber tube.

The hollow fiber tube of the present invention can be prepared by a known method. In an exemplary method, the method of making the hollow fiber tube of the present invention comprises the steps of:

(1') step of preparing an activated hollow fiber

Activated hollow fibers can be prepared by methods known in the art. In certain embodiments, activated hollow fibers are prepared from chloromethyl ether modified polysulfones by, for example, melt extrusion methods.

In an exemplary method, a melt of chloromethyl ether modified polysulfone is extruded by a melt extrusion method for producing a plastic article, and is gradually cooled in an atmospheric environment, and the melt fluidity is gradually reduced with the decrease of the melt temperature to be solidified and molded.

In another exemplary method, activated hollow fibers having an asymmetric structure are prepared, for example, by multiple-pass profiled spinneret forming. Specifically, chloromethyl ether modified polysulfone is dissolved in organic solvent (such as DMF or DMAc), and additive (such as PEG) with porogenic effect is mixed, defoamed and extruded by a metering pump through a central liquid-passing insertion tube type nozzle. And (3) after the extruded fiber is exposed to air, carrying out a coagulating bath, carrying out double diffusion on the solvent in the unformed modified polysulfone solution and the curing agent in the coagulating bath, and curing and forming the modified polysulfone. Due to the evaporation of partial solvent in the modified polysulfone solution in the air, the diffusion effect of the solvent and the curing agent in the radial direction of the fiber, the adjustment of the phase separation speed by the additive in the polysulfone solution and the influence of the vacancy left after the water-soluble additive is eluted, the required pore canal is formed in the radial direction.

In certain embodiments, the polysulfone hollow fibers are subjected to an activation treatment to produce activated hollow fibers. First, the polysulfone hollow fiber is soaked in an organic solvent (e.g., ethylene dioxide) containing chloromethyl ether, and then activated by adding an inorganic accelerator. Wherein the molar ratio of chloromethyl ether to organic solvent is generally from 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 deg.C, preferably 45-50 deg.C for 5-24 hours, preferably 8-12 hours. It is preferable to stir the organic solvent containing chloromethyl ether at the time of activation. After the activation reaction is finished, the method further comprises the steps of washing the activated hollow fiber tube by deionized water, and drying the hollow fiber tube at the temperature of 60-85 ℃ to obtain the activated hollow fiber.

(2') surface treatment step for activating hollow fiber

The surface treatment of the invention comprises that the activated hollow fiber is soaked in 20-40% trimethylamine (TM A) solution for 30-50h at room temperature.

Culture medium container

The medium vessel of the present invention is any means for containing a medium, and the size and material of the medium vessel are not particularly limited as long as they can contain a desired amount of medium. May be a flexible material such as a plastic bag; and may be a hard material such as a glass bottle, a hard plastic (e.g., ABS), etc. The shape of the medium container of the present invention is not particularly limited, and may be a bottle shape.

The culture medium container of the present invention is provided with a supply port, and the supply port is connected to a culture medium inlet of the bioreactor through a pipe, thereby allowing the culture medium in the culture medium container to enter the bioreactor through the hollow fibers. In certain embodiments, the culture medium container of the present invention is further provided with a recovery port, and the recovery port is connected to the culture medium outlet of the bioreactor through a pipeline, so that the culture medium participating in at least partial reaction in the bioreactor flows out of the bioreactor through the hollow fiber tube, thereby forming a loop between the bioreactor and the culture medium container.

In certain embodiments, the medium container contains a basal medium. The basic culture medium is mainly used for providing energy for cell growth and/or differentiation and comprises a large amount of inorganic substances and small molecular organic substances. The basal medium may use a medium known in the art. For example, SFEM medium and IMDM medium. Wherein the SFEM medium is a product produced by Stem Cell, such as product No. Cat # 09600. IMDM medium is a product known in the art and may also be referred to as an eiskov modified broth. It contains higher concentration of nutrients and is suitable for high density cell culture. Preferably, the basal medium is SFEM medium and IMDM medium without serum albumin.

The ingredients in the basal medium of the present invention can freely pass through the tube wall of the hollow fiber tube and the binding agent. In addition, waste products (such as small molecule acidic substances such as lactic acid and pyruvic acid) which are generated in the process of cell growth or differentiation and are unfavorable for cell differentiation can also be discharged out of the bioreactor through the hollow fiber tube.

Examples of such other small molecule components include L-glutamine, 2-mercaptoethanol, and iron ions the concentration of L-glutamine in the basal medium is generally from 1 mmol/L to 5 mmol/L, preferably from 2 mmol/L to 3 mmol/L based on the volume of the basal medium.the concentration of 2-mercaptoethanol is generally from 1 × 10 based on the volume of the basal medium^-4mol/L-9 × 10^-4mol/L, preferably 1 × 10^-4mol/L-5 × 10^-4mol/L radicalThe concentration of iron ions in the basal medium is not limited. Based on the volume of the basal medium, it is generally from 200. mu.g/ml to 400. mu.g/ml, preferably from 250. mu.g/ml to 300. mu.g/ml. The above iron ion concentration can be achieved by adding an appropriate amount of, for example, lronSupplement (product of Sigma company, Cat # I3153).

Oxygen supplier

The oxygen supplier of the present invention is used to supply a required amount of oxygen to the bioreactor, thereby facilitating the simulation of the living organism environment. The oxygen supplier may be an oxygen storage device or an oxygen generator. The oxygen supplier of the present invention is configured such that it is connected to the oxygen inlet through a pipe.

Other parts

To increase the level of intelligence, the production system of the present invention may optionally further comprise other components, such as real-time monitoring devices, peristaltic pumps, biological factor addition devices, and the like.

The real-time monitoring device of the present invention comprises a component selected from the group consisting of a temperature sensor, a pH monitor, a cell concentration monitor, and a controller. The components of the temperature sensor, the pH monitor and the cell concentration monitor can be arranged on the shell for monitoring various parameters in the bioreactor in real time and transmitting the data to the controller, and the controller outputs corresponding execution commands according to the data, thereby realizing intelligent control and culture.

The peristaltic pump is used for providing liquid flowing power, promoting a basic culture medium to enter a cavity of the bioreactor from the culture medium container through the hollow fiber tube, and controlling the culture medium after reaction to flow out of the bioreactor through the hollow fiber tube.

The biological agent adding device of the present invention is used to add one or more desired agents to the chamber of a bioreactor. As mentioned above, in addition to the components of the basal medium described above, some biological factors, such as one or more differentiation factors, are required for cell culture in the production of cells in vitro. These differentiation factors ensure the mature 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-l). These biological factors generally need to be added to the chamber of the bioreactor and cannot be added directly to the basal media. For this reason, the production system of the present invention needs to be provided with a biological factor adding device. The biological factor adding device can be further connected with a controller, so that the addition of the biological factors can be automatically controlled according to real-time monitoring data. In general, Epo is generally added in an amount of 8 to 10U/ml, preferably 8.5 to 9.5U/ml, based on the volume of the basal medium. The amount of iron-saturated transferrin is generally 400-600. mu.g/ml, preferably 450-550. mu.g/ml. The amount of IGF-l added is generally 40-60ng/ml, preferably 45-55 ng/ml. The amount of SCF added is generally from 50 to 100ng/ml, preferably from 60 to 80 ng/ml. Preferably, dexamethasone is further included, generally in an amount of 0.5-2. mu.M, preferably 1-1.5. mu.M.

Preferably, serum albumin is further added into the chamber of the bioreactor through the biological factor adding device during the production of the red blood cells, and it is required to be noted that the serum albumin cannot permeate into the chamber from the inner cavity of the hollow fiber. The amount of serum albumin added is 1-3%, preferably 1-2%, based on the weight of the basal medium.

In certain embodiments, the basal medium of the present invention is IMDM medium containing L-glutamine, 2-mercaptoethanol, and ferric ions, when it is desired to further supplement the chambers of the bioreactor with BSA, Epo, transferrin, and IGF-l, preferably with 5-15. mu.g/ml, preferably 6-10. mu.g/ml, human insulin by a biological factor addition device.

In certain embodiments, the basal medium of the invention is a SFEM medium, in which case the chamber of the bioreactor needs to be further supplemented with BSA, Epo, iron-saturated transferrin, and IGF-l by a biological factor addition device.

Application method of bionic intelligent production system

The use method of the bionic intelligent production system comprises the following steps:

(1) continuously feeding and filling a basal medium or further optional oxygen from the lumen of the hollow fiber tube through the tube wall to the chamber of the bioreactor;

(2) contacting the erythroid progenitor cells with the wall surface of the hollow fiber tube in a chamber, wherein a biological factor is added to the chamber in advance or during the culturing process;

(3) waste generated in the chamber enters the inner cavity of the hollow fiber tube through the tube wall along with at least part of the culture medium components and flows out of the bioreactor.

Example 1

This example is the preparation of a hollow fiber tube 1.

0.2kg of polysulfone hollow fiber C2011(fiber cell Systems, Frederick, MD) was immersed in 1000ml of an organic solvent composed of chloromethyl ether and ethylene dioxide at a volume ratio of 1:10, to which was added 8g of zinc chloride. The polysulfone hollow fibers were reacted at 40 ℃ for 12 hours. The organic solvent is intermittently stirred during the reaction to maintain the uniformity of the reaction between the hollow fiber tubes and the organic solvent. And then washing the hollow fiber tube after multiple times of activation by using deionized water. And then dried at 60 c to obtain an activated hollow fiber. The activated hollow fiber was immersed in a solution of 25% TMA at room temperature for 45 hours to obtain a hollow fiber tube 1 of the present invention.

Example 2

This example is the preparation of a hollow fiber tube 2.

Dissolving chloromethyl ether modified polysulfone in DMF organic solvent, using PEG6000 as additive, mixing and defoaming, preparing hollow fiber by using a hollow fiber membrane spinning machine comprising a metering pump (specification is 1.2ml/r), an inserted tubular spinneret, a nitrogen steel cylinder and the like, and performing coagulation bath (the temperature of the gel bath is 25 ℃, the concentration of the solvent in the gel bath is 55 percent, and the concentration of the solvent in the core solution is 78 percent) after air exposure. Thereby producing an activated hollow fiber tube having an asymmetric structure. The activated hollow fiber was immersed in a 25% TM a solution at room temperature for 45 hours to obtain a hollow fiber tube 2 of the present invention.

Example 3

This example is an example of testing the performance of a hollow fiber tube.

A plurality of hollow fiber tubes are arranged in parallel on a planar substrate to form a membrane shape. University of reference Compound DanThe membrane potential was measured by the method described in the methods of physical and chemical experiments, Shanghai, Compound denier university Press, 1982, the results of the surface potential were that the solution flowed to the positive electrode, and the hollow fiber tube 1 was 2.67 × 10^-3And the hollow fiber tube 2 was 4.21 × 10^-3。

Example 4

This embodiment is the structure of the bionic intelligent production system of cells.

As shown in fig. 1, the intelligent cell-simulated production system of the present 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.

As shown in fig. 2, bioreactor 100 includes a sealed chamber 120 formed by a housing 110 and a plurality of hollow fiber tubes 130 disposed within chamber 120. The housing 110 is a circular tube structure, and two ends of the housing are respectively provided with a first closed end 111 and a second closed end 112. 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 adapted to be connected to the supply port 210, and the medium outlet 114 is adapted to be connected to the recovery port 220. Thereby connecting bioreactor 100 and medium vessel 200 to form a loop. The plurality of hollow fiber tubes 130 may employ the hollow fiber tubes of the examples 1 and/or 2. The plurality of hollow fiber tubes 130 are arranged in parallel along the axial direction of the circular tube and fixed between the first closed end 111 and the second closed end 112 by a polyurethane cement. An oxygen inlet 115 is further provided at a side of the housing 110 for connection 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 is communicated with the outside, or corresponding biological factors are added through the first chamber port 116 and the second chamber port 117, or cells are seeded or cultured cells are recovered.

Example 5

The structure is the same as that of example 4 except that the oxygen inlet 115 is integrated with the culture medium inlet 113, and the oxygen supplier 300 supplies oxygen to the bioreactor 100 through the hose and the culture medium inlet 113 of the bioreactor 100.

Example 6

The production system of this example was the same as that of example 4 except that it further included a real-time monitoring device, a peristaltic pump, and a biological factor adding device.

In this embodiment, the real-time monitoring device includes a temperature sensor, a pH monitor, and a cell concentration monitor, which are respectively disposed on the housing 110, and the controller receives monitoring data and outputs an execution command.

A peristaltic pump is provided on the hose connecting the recovery port 210 and the medium outlet 114 for facilitating the flow of the medium.

A biological agent adding means is provided at the first chamber port 116 of the housing for adding the desired agent to the bioreactor 100.

Example 7

Culture experiments for in vitro induction of cord blood-derived HSC-produced erythroid progenitor cells were performed with the production system of example 4. The basic culture medium of the erythroid progenitor cells adopts a serum-free culture medium StemBan SFEM.

Adding desired volume of StemSpan SFEM into the production system, adding cytokines such as Epo 5U/m L, SCF100ng/m L, IGF-l 50ng/m L, Dex l/-lM, etc. to form a culture system, and culturing at 5 × 10⁵The desired erythroid progenitor cells were added at a concentration of/m L, the reactor was started, fresh StemBan SFEM was continuously added to the reactor through the wall of the hollow fiber tube, and waste products generated by cell culture were continuously discharged through the wall, thereby constituting a circulation system, while continuously supplying the desired oxygen and the like to the reactor.

The culture medium was prepared by replacing new StemBan SFEM in a bioreactor and adding thereto Epo 10U/m L, iron-saturated transferrin 500. mu.g/m L-l 50ng/m L, cytokines such as iron ion, etc., to constitute a denucleation differentiation system, adding the collected cells to the denucleation differentiation system, performing differentiation culture under the condition that the required oxygen and the like are continuously supplied to the reactor, and collecting the cultured erythrocytes after 10 days.

In addition, the conventional index was analyzed by a hemocytometer, and the result showed that the mean volume of red blood cells was 10⁴7fl, the average hemoglobin concentration of the red blood cells is 25 +/-3%, the average hemoglobin content of the red blood cells is 30 +/-2 pg, and the detection result is close to that of normal peripheral red blood cells, so that mature red blood cells are obtained.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

Claims

1. A cell bionic intelligent production system is characterized by comprising a bioreactor, a culture medium container, a biological factor adding device and an oxygen supplier;

wherein the bioreactor comprises a sealed chamber formed by a shell and a plurality of hollow fiber tubes arranged in the chamber, the shell is provided with a culture medium inlet, a culture medium outlet and an oxygen inlet arranged on one side of the shell, the diameter of each hollow fiber tube is 200-1000 μm, the hollow fiber tubes are arranged in such a way that the inner cavities of the hollow fiber tubes can flow basic culture medium and/or gas, the tail ends of the hollow fiber tubes can be communicated with a basic culture medium container, the wall thickness of the hollow fiber tubes is 20-80 μm, the hollow fiber tubes are arranged in such a way that the components and/or gas of the basic culture medium can pass through the tube walls from the inner cavity side of the hollow fiber tubes to the outside, and quaternary ammonium groups are arranged on the outer surfaces of at least the tube walls of the hollow fiber;

the oxygen supplier is arranged to be connected with the oxygen inlet through a pipeline;

the bioreactor further comprises a chamber port used for communicating the chamber with the outside, the chamber port comprises a first chamber port used as a fluid inlet and a second chamber port used as a fluid outlet, the first chamber port and the second chamber port are respectively arranged on the side surface of the shell, and the biological factor adding device is arranged at the first chamber port of the shell and used for adding required factors to the bioreactor.

2. The system of claim 1, wherein the housing is a circular tube, the two ends of the housing are respectively provided with a first closed end and a second closed end, so as to form a sealed chamber, the medium inlet is arranged at the first closed end, the medium outlet is arranged at the second closed end, and the hollow fiber tubes are arranged in parallel along the axial direction of the circular tube.

3. The system of claim 1, wherein the culture medium container is further provided with a recovery port, and the recovery port is connected with the culture medium outlet through a pipeline, so that the bioreactor and the culture medium container are connected to form a loop.

4. The system for intelligently bionic production of cells as claimed in claim 1, wherein the wall of the hollow fiber tube is made of porous permeable material with pore size less than 0.3 μm.

5. The cell biomimetic intelligent production system according to claim 4, wherein the porous and permeable material is selected from the group consisting of polysulfone, polyvinyl chloride, cellulose acetate, and acrylic acid copolymer.

6. The system for intelligently bionic and producing the cells as claimed in claim 4, wherein the potential of the outer surface of the tube wall of the hollow fiber tube is 1.20 × 10^-3To 4.0 × 10^-3V。

7. The system of claim 1, wherein the medium container contains a basal medium and is selected from the group consisting of SFEM medium and IMDM medium without serum albumin.