CN116042373A

CN116042373A - System for continuous production and separation and purification of extracellular body and use method thereof

Info

Publication number: CN116042373A
Application number: CN202211723931.5A
Authority: CN
Inventors: 杨展; 张朝军
Original assignee: Shijiazhuang Bikalin Biotechnology Co ltd
Current assignee: Shijiazhuang Bikalin Biotechnology Co ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-05-02

Abstract

The invention provides a system for producing, separating and purifying cell exosomes, which comprises 5 subsystems, namely a 1 cell culture fluid supply subsystem, a 2 detection subsystem, an 8 cell culture subsystem, a 13 exosome purification subsystem and a 17 cell culture fluid ventilation subsystem, wherein the 5 subsystems form a closed cycle, an inlet is the cell culture fluid supply subsystem, and an outlet is the exosome purification subsystem. The invention also provides a method for continuously producing, separating and purifying the extracellular body by using the system.

Description

System for continuous production and separation and purification of extracellular body and use method thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a process for efficiently and continuously producing, separating and purifying extracellular exosomes and application thereof.

Background

Exosomes (Exosomes) are small single membrane secretory organelles with a diameter of about 30 to 200nm, with the same topology as the cells, enriched in specific proteins, lipids, nucleic acids and glycoconjugates. Exosomes comprise a series of membrane-associated higher order oligomeric protein complexes, exhibit pronounced molecular heterogeneity, and are produced by budding on plasma and endosomal membranes. The production of exosomes is a mechanism of protein quality control, once released, the exosomes have a variety of activities, such as remodeling of extracellular matrix and transmission of signals and molecules to other cells. This intercellular vesicle transport pathway plays an important role in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. In addition, viruses select for exosome biosynthetic pathways to assemble infectious particles and establish host permissions. In the field of regenerative medicine, exosomes derived from mesenchymal stem cells can be used to treat a variety of diseases, for example: in skin wound healing, it can promote angiogenesis, reduce inflammation, promote proliferation and migration of skin cells, etc. The exosomes derived from mesenchymal stem cells are more stable than mesenchymal stem cells, and can reduce the safety risk of administering living cells, for example, have low immunogenicity, avoid the risk of microvascular blockage, etc. In addition to the above mentioned effects, exosomes can also be isolated as a nanoscale lipid bilayer vesicle and stored for a long period of time at low temperature, without considering survival rate, with small volume, easy circulation, and good stability of the contents. However, the number of exosomes produced by cells such as mesenchymal stem cells is small, and the existing separation and purification process of the exosomes is imperfect, so that the application of the exosomes is greatly limited.

Under normal physiological and in vitro culture conditions, mesenchymal stem cells secrete less exosomes, and the ratio of the number of cultured cells to the number of exosomes secreted is about 1:5. in order to obtain a large number of exosomes in the industry today, cell-wall culture in microcarrier-based bioreactors is commonly used. The method has the advantages of greatly increasing the density of cells and further improving the number of exosomes. However, the disadvantages of the reactor for producing exosomes are obvious, firstly, the price of equipment and microcarriers is high, the cost of products is greatly increased, and the production popularization and application are limited; secondly, in order to realize suspension culture, microcarriers are often degradable biological or chemical materials which are easy to degrade and incorporate into exosomes; thirdly, the cells and exosomes often need to be collected simultaneously, and some exosomes are produced for too long and are broken, so that the quality of the whole exosomes is affected. The ideal method is that enough cells grow in a limited space (generally three-dimensional culture), the supporting material is required to be reusable, environmental-friendly and nondegradable, the culture solution which is continuous in the production process brings away the exosomes secreted by the cells when the cells are provided with enough nutrients, and the culture solution after the exosomes are separated is filtered and recycled.

Another difficulty in obtaining high concentrations, high purity, high quality, standardized exosomes is isolation and purification. At present, the separation and purification of exosomes have no unified standard and mature industrial process, and the following methods are commonly used in laboratories: ultracentrifugation, density gradient centrifugation, ultrafiltration, column membrane chromatography, immunocapture, precipitation, etc., which each have advantages. (1) Ultracentrifugation is a well-known method for extracting exosomes, but has high equipment cost, time and labor consuming operation, high labor dependence, low recovery rate, different exosomes, and difficult mass production due to damage to exosomes caused by high-speed centrifugation. (2) Although the density gradient centrifugation method can obtain a very pure exosome, the method has the advantages of complex operation, poor repeatability, long time consumption and low recovery rate, and is not suitable for extracting the exosome in a large scale. (3) The ultrafiltration method can conveniently and rapidly extract exosomes, but the exosomes extracted by the method contain large-particle impurity pollution, and the low purity seriously affects downstream application. (4) The exosome obtained by the column membrane chromatography has high purity, but is time-consuming and labor-consuming, and has low recovery rate and difficult mass production. The immune capturing method can specifically capture exosomes, and the obtained exosomes have high purity, but the method has high cost and low yield, can not extract all exosomes in a sample, and can only obtain exosomes positive to a certain surface antigen. (5) The polymer precipitation method is simple and convenient to operate, can be used for extracting exosomes of large-volume samples, but the exosomes extracted by the method are more in impurity protein pollution, and the particle morphology is non-uniform, so that downstream analysis is influenced. Therefore, from the current difficulty, the preparation method reduces the cost, improves the purity, reduces the pollution and realizes the mass production, thereby achieving the fundamental conditions of industrialized and large-scale exosome production and preparation.

In view of the above, in order to realize industrial and clinical transformation applications of exosomes, there is a need to solve the problems of integrated preparation systems and processes from continuous cell culture to continuous exosome production.

Disclosure of Invention

The invention provides a system for producing, separating and purifying cell exosomes, which comprises 5 subsystems, namely a 1 cell culture fluid supply subsystem, a 2 detection subsystem, an 8 cell culture subsystem, a 13 exosome purification subsystem and a 17 cell culture fluid ventilation subsystem, wherein the 5 subsystems form a closed cycle, an inlet is the cell culture fluid supply subsystem, and an outlet is the exosome purification subsystem.

Preferably, the 1-cell culture fluid supplementing subsystem comprises a recovery culture fluid storage tank, a 2-trace element and amino acid supplementing tank and a 3-fresh culture fluid supplementing tank.

Preferably, the 13 exosome purification subsystem comprises: primary filtration, secondary filtration, envelope filtration, and column chromatography.

The invention also provides a method for continuously producing, separating and purifying the cell exosomes, which utilizes the system to supplement fresh culture solution from an inlet cell culture solution supplementing subsystem and collect concentrated exosome mixed solution from an outlet exosome purifying subsystem.

The method comprises the steps of using a three-dimensional cell culture mode and a device, wherein the supporting material of the device is recycled, does not pollute the environment and is not degradable; and the culture process takes away the exosomes secreted by the cells when the continuous culture solution is conveyed into the cells for sufficient nutrients, and the culture solution after the exosomes are separated is mixed with part of fresh culture solution by an oxygen ventilation device, and then conveyed to the cell culture device again. The process solves the problems that the existing exosome separation and purification process cannot be sustainable, is time-consuming and labor-consuming, has low recovery rate, serious pollution in the middle process, low yield, incapability of industrialized mass production and the like.

Preferably, the method comprises the steps of:

step one, a cell culture fluid supplementing subsystem comprises a recovered culture fluid reserve tank, a microelement and amino acid supplementing tank and a fresh culture fluid supplementing tank, peristaltic pumps at all positions are automatically regulated according to the real-time monitoring of the cell culture process by a detection subsystem to realize the automatic intelligent supplement of the culture fluid,

step two, the detection subsystem monitors the nutrient of the cells in real time, including dissolved oxygen, carbon dioxide, pH and the like,

step three, the cell culture subsystem is a group of parallel connected cell three-dimensional culture devices, each culture device is provided with an inlet and two outlets, one outlet is connected with the detection subsystem, the other outlet is connected with the outlet of the other device, each culture device is filled with glass microspheres treated by strong alkali, the cell culture devices are firstly singly planted with cells, the cells are uniformly adhered on the glass microspheres and then added into the cell culture subsystem, after the cells in the cell culture device generally grow for 6-8 days, the cell culture device is taken out from the subsystem and carries out subsequent recovery treatment,

Step four, the cell culture device taken down from the subsystem is firstly introduced with PBS, then introduced with pancreatin for digestion, then the cells are flushed out by PBS, and then the cells are collected by centrifugation for standby,

step five, along with the growth of cells in the culture subsystem, the exosomes produced continuously flow into the culture solution discharge storage tank along with the culture solution, when the culture solution in the storage tank reaches a certain volume, the exosomes purification is automatically started, and the exosome purification subsystem comprises: primary filtration, secondary filtration, envelope filtration and column chromatography, wherein the primary filtration mainly removes dead cells and cell fragments at the same time, the secondary filtration isolates subcellular structures larger than 200nm, including apoptotic bodies and the like, the envelope filtration is fully concentrated and enriched with exosomes, and finally the column chromatography obtains purer exosomes,

step six, the culture solution which is enveloped and filtered by the purification subsystem flows into a liquid storage tank to be replaced by air, the pH value, microelements and the like of the liquid are detected, the supplementing subsystem automatically supplements according to the detection result,

step seven, the key factor of recycling the culture solution is CO ₂ Is discharged and dissolved oxygen is supplemented, and the air exchanging device in the cell culture fluid air exchanging subsystem is utilized to fully enable the liquid to be in reverse contact with sterile clean air, so that redundant CO is generated ₂ The liquid dissolved oxygen is increased after the replacement, and the ventilated culture solution flows into the cell culture solution supplementing subsystem under the action of a peristaltic pump, so that the recycling of part of the culture solution is realized.

Preferably, the method comprises the steps of:

step one, a cell culture fluid supplementing subsystem comprises a recovered culture fluid storage tank, a microelement and amino acid supplementing tank and a fresh culture fluid supplementing tank, peristaltic pumps at all positions are automatically regulated according to real-time monitoring of a cell culture process by a detection subsystem, and automatic intelligent supplementing of the culture fluid is realized.

And step two, monitoring the nutrients of the cells in real time by a detection subsystem, wherein the nutrients comprise dissolved oxygen, carbon dioxide, pH and the like.

And thirdly, the cell culture subsystem is composed of a group of parallel cell three-dimensional culture devices, each culture device is provided with an inlet and two outlets, one outlet is connected with the detection subsystem, and the other outlet is connected with the outlet of the other device. Each culture apparatus was filled with glass microspheres (diameter 0.8-1.5 mm) treated with alkali, and the total area was about 1000cm ² About 10 can grow ⁹ Mesenchymal stem cells greatly improve the growth density of cells. Since the cell culture devices are connected in parallel, a certain device can be increased or decreased at any time, which is also a feature of the present invention. These cell culture devices were first seeded with cells individually and added to the cell culture subsystem after the cells were uniformly adhered to the glass microspheres. After the cells in the cell culture apparatus generally grow to 6-8 days, the cell culture apparatus is removed from the subsystem and subjected to subsequent recovery processing.

And fourthly, the cell culture device taken down from the subsystem is firstly introduced with PBS with twice the volume, then introduced with pancreatin with 1.5 times the volume for digestion for 3-5 minutes, then the cells are flushed out by PBS with twice the volume, and then the cells are collected by centrifugal force of 300g/5min for standby.

And fifthly, along with the growth of cells in the culture subsystem, continuously generated exosomes flow into the culture solution and are discharged out of the storage tank along with the culture solution, and when the culture solution in the storage tank reaches a certain volume, the exosomes are automatically purified.

The exosome purification subsystem includes: primary filtration, secondary filtration, envelope filtration, and column chromatography. Primary filtration primarily removes dead cells and more localized cell debris. Secondary filtration isolates subcellular structures greater than 200nm, including apoptotic bodies and the like. The envelope filtration is fully concentrated and enriched with exosomes, and finally the column chromatography is carried out to obtain purer exosomes.

Step six, the culture solution which is enveloped and filtered by the purification subsystem flows into a liquid storage tank to be replaced by air, the pH value, microelements and the like of the liquid are detected, and the supplementing subsystem is automatically supplemented according to the detection result.

Step seven, the key factor of recycling the culture solution is CO ₂ The invention comprises a cell culture fluid ventilation subsystem, wherein a ventilation device in the subsystem fully enables liquid to be in reverse contact with sterile clean air to enable redundant CO ₂ Displacing to increase the dissolved oxygen of the liquid. The ventilated culture solution flows into the cell culture solution supplementing subsystem under the action of the peristaltic pump, so that the recycling of part of the culture solution is realized.

The system and the method have the advantages that:

(1) The invention provides a process and a method for efficiently and continuously producing, separating and purifying a cell exosome, which innovatively comprise five subsystems, namely a cell culture fluid supplementing subsystem, a detecting subsystem, a cell culture subsystem, an exosome purifying subsystem and a cell culture fluid ventilation subsystem. The five subsystems form a closed cycle, so that pollution is reduced, the cost of the culture solution is reduced, and the yield of exosomes is improved.

(2) The method comprises a method and a device for culturing the three-dimensional cells, wherein the supporting material of the device is a glass microsphere which is recycled, does not pollute the environment and is not degradable. Each cell culture device can be independently added or removed without affecting the overall operation. In addition to isolating exosomes, digested cells can also be used.

(3) The closed circulation subsystem can continuously generate a large amount of exosomes, and meanwhile, the generated exosome solution is separated through the exosome purification subsystem. The purification process is simple to operate, the recovery rate of exosomes is high, and the exosomes have no intermediate pollution and high purity.

(4) In addition, the culture solution after the exosome separation is mixed with the oxygen ventilation device, and the recovered culture solution after ventilation is mixed with part of fresh culture solution and is conveyed to the cell culture device again, so that the recycling is realized.

(5) The process solves the problems that the existing exosome separation and purification process cannot be sustainable, is time-consuming and labor-consuming, has low recovery rate, serious pollution in the middle process, low yield, incapability of industrialized mass production and the like.

The cell culture subsystem is a cell culture device filled with microglass bead carriers and is characterized by comprising a 24-medium inlet pipe; 25 culture solution dispersion discs; 26 disc microwells; 27 disc interior space; 28 a medium conveying pipe; 29 a culture solution conveying pipe in the disc; 30 pipeline sealing valves; 31 a culture solution outlet; 32 disc opening switch; 33 cell culture microcarriers and 34 tubing disc connection loops.

Preferably, the cell culture microcarrier is a microglass bead.

Preferably, the diameter of the micro glass beads is 0.5-1.0mm.

Preferably, the microglass bead carrier is sterilized by high temperature baking and can be reused.

Preferably, the cell culture device is a closed device having a volume of 50mL to 200mL, for example 100mL.

Preferably, the cell culture device comprises a layered cylindrical bracket, the volume is unequal, for example, 100mL cases are provided, a large number of micro glass beads treated by alkali are filled in the device, the diameter is 0.5-1.0mm, the bottom of each of the micro glass beads is separated by 5 discs, a large number of 26 disc micropores are formed at the bottoms of the discs, the micro glass beads uniformly distribute cells on a microcarrier on one hand, on the other hand, the culture solution uniformly permeates into all micro glass beads in the process of conveying the culture solution, the problems that the cells are locally accumulated and the culture solution does not flow smoothly are effectively avoided, the growth of the cells is maximally satisfied, wherein the disc at the bottom is only provided with 26 disc micropores at one side close to the micro glass beads, the cells or the culture solution is ensured not to accumulate at the place without the micro glass beads, the three discs at the middle are provided with 26 disc micropores at the upper surface and the lower surface, and the diameters of the three discs are slightly smaller than the inner diameter of the cylinder, so that the micro glass beads can fully fill the whole interior and simultaneously facilitate the circulation of partial culture solution, the three discs have 27 disc inner spaces, culture solution and inoculated cells are all dispersed on the micro glass beads through 26 disc micropores in the 27 disc inner spaces, 5 discs in the interior are connected together by 28 middle shaft culture solution conveying pipes, the 28 middle shaft culture solution conveying pipes are connected with the discs by 34 pipeline disc connecting rings, the 34 pipeline disc connecting rings are provided with a plurality of outlets, each outlet is connected with the culture solution conveying pipes in 29 discs, a plurality of small outlet holes are arranged on the culture solution conveying pipes in 29 discs, the culture solution and inoculated cells can enter the 27 disc inner spaces through the small holes, finally are dispersed on the micro glass beads through 26 disc micropores, the topmost disc is a single layer, a plurality of micropores are also distributed in the interior, besides, there is a 32-disc opening switch, which can be moved, when it is opened, the micro glass beads can be injected into the cylinder, at the same time, the micro glass beads can be prevented from moving in the course of cell culture, in the upper space there is a culture solution outlet pipeline with two 31 culture solution outlets, and these two outlets can directly discharge liquid or one of them can be used as outlet, and another outlet can be connected with outlets of other culture devices so as to implement series connection.

The invention provides a method for culturing stem cells in high-density 3D, which comprises the following steps:

(1) Soaking the microglass bead carrier in sodium hydroxide, then cleaning, drying, sterilizing, cooling and then loading into the cell culture device of any one of the above-mentioned components,

(2) After the microglass bead carrier is injected, a sterile small tube is connected on a 24 culture medium inlet tube of the device, under the action of a peristaltic pump, liquid enters the device from the 24 culture medium inlet tube, passes through a 28 middle shaft culture liquid conveying tube to reach each 34 pipeline disc connecting ring, passes through a 29 inner culture liquid conveying tube to enter a 27 disc inner space, passes through 26 disc micropores to be uniformly dispersed at each part, finally enters an upper space from the micropores at the top, is discharged from an outlet, sterile normal saline or PBS is introduced for the first time, powder on the surface of the microglass bead carrier is subjected to leak-proof detection at the same time, after no leakage is ensured, an inlet and an outlet are sealed, and the microcarrier is prepared for being coated at normal temperature,

(3) Coating with different materials according to cultured cells, such as umbilical Mesenchymal Stem Cells (MSC), generally using MSC adhesion promoting agent or Vitronectin (Vitronictin), firstly diluting MSC adhesion promoting agent 100 times (1:100) with sterile PBS solution, and adding appropriate amount of 1×MSC adhesion promoting agent; introducing an adherence reagent from a culture solution inlet, keeping the carrier immersed in the liquid all the time during the coating, uniformly mixing, confirming the uniform distribution of the adherence reagent, closing and sealing the entrance, incubating, introducing sterile PBS (phosphate buffer solution) before inoculation, flushing the adherence reagent, recycling, introducing a multiple basic culture medium, sealing and preserving for standby,

(4) Each cell culture device is individually seeded with cells,

(5) The cells are subjected to temperature, pH, dissolved oxygen and CO during the culture process ₂ Monitoring, conveying the culture solution to the device under the action of a peristaltic pump, regulating pH, dissolved oxygen and the like in the culture solution according to the monitoring result,

(6) And performing flow detection on the cultured mesenchymal stem cells.

Preferably, the method comprises the steps of:

(1) Soaking the microglass bead carrier in 1mol sodium hydroxide for 48 hours, then washing with deionized water for 15 times, washing with ultrapure water for 5 times, oven drying, baking in a horse boiling furnace at 230 ℃ for 3 hours for sterilization, cooling, loading into a sterile cylindrical 3D culture device,

(2) After the micro glass bead carrier is injected, a sterile small tube is connected on a 24 culture medium inlet tube of the device, under the action of a peristaltic pump, liquid enters the device from the 24 culture medium inlet tube, enters the 27 disc inner space through a 29 disc inner culture liquid conveying tube after reaching each 34 pipeline disc connecting ring through a 28 middle shaft culture liquid conveying tube, is uniformly dispersed on each part through 26 disc micropores, enters the upper space from the micropores at the top, is discharged from an outlet, firstly passes through sterile normal saline or PBS with 3 times of cylinder volume, simultaneously performs leak-proof detection on powder on the surface of the micro glass bead carrier, ensures no leakage, seals an inlet and an outlet, is preserved at normal temperature, and is ready for coating the micro carrier,

(3) According to different cultured cells, different materials are used for coating treatment, such as umbilical Mesenchymal Stem Cells (MSC), MSC adhesion promoting agents or Vitronectin (Vitronictin) are generally used, firstly, sterile PBS solution is used for diluting the MSC adhesion promoting agents 100 times (1:100), the device has different volumes, the volumes are different from 50mL to 200mL, corresponding coating liquid is prepared according to the different volumes, and a proper amount of 1 XMSC adhesion promoting agents are added; the adherence reagent is introduced from the culture fluid inlet, notably to keep the carrier immersed in the fluid throughout the coating period! Gently inverting and mixing, confirming that the adherent reagent is uniformly distributed, closing and sealing the entrance, and incubating overnight at 2-8 ℃; or in CO ₂ Incubator, at 37 deg.C, incubating for at least 30 min, slowly introducing two times of sterile PBS to flush out the adherent reagent before inoculation, recycling, introducing 1.5 times of basic culture medium, sealing and preserving for standby, inoculating cells within 48 hr,

(4) Each cell culture apparatus was inoculated with cells individually, and in this case, 100ml of the cell culture apparatus was inoculated, 1 volume of complete medium was introduced into the apparatus before the inoculation of the cells, and 5X 10 cells were collected ⁷ The umbilical cord mesenchymal stem cells cultured until 3 rd to 4 th generation are regulated to 50mL by a culture medium, then slowly flow into a culture device through a liquid inlet under the action of a peristaltic pump, are gently inverted and uniformly mixed, are subjected to static culture at 37 ℃ for 12 hours, and are added into a cell culture system one by one after the cells are uniformly adhered to micro glass beads, wherein 10 percent of the complete culture medium is added into the basic culture medium Is used for replacing blood serum of a patient,

(5) The cells are subjected to temperature, pH, dissolved oxygen and CO during the culture process ₂ And monitoring, namely continuously conveying the culture solution into the device under the action of the peristaltic pump, regulating pH, dissolved oxygen and the like in the culture solution according to the monitoring result, conveying sufficient nutrient substances for cells and simultaneously taking away metabolic products such as CO (carbon monoxide) ₂ And the like,

(6) The mesenchymal stem cells cultured by the device of the invention are subjected to flow detection, the cell culture device taken down from the system is firstly introduced with PBS with twice the volume, then introduced with pancreatin with 1.5 times the volume for digestion for 3-5 minutes, then the cells are flushed out by PBS with twice the volume, then the cells are collected by centrifugal force of 300g/5min for standby, a certain amount of PBS is added for washing once, the supernatant is discarded, the cells are ejected, and the cells are resuspended to 5 multiplied by 10 ⁶ –1×10 ⁷ Between/ml; 100 μl of cells were stained in EP tube or flow tube, and stained in four tubes, respectively Anti-human CD44 FITC, anti-human CD73 FITC, anti-human CD90 FITC, anti-human CD105 FITC, each added with 5 μl, mouse IgG1 Isotype Control PE-Cy 7.2 μl, mouse IgG1 Isotype Control APC 1.2.2 μl; adding corresponding antibody, mixing, directly blowing without gun head, incubating at room temperature in dark place for 15min, and mixing again during dyeing; after the dyeing is finished, adding 1ml of PBS to wash the cells, and centrifuging for 300g/5min; the supernatant was discarded, the cells were pelleted, resuspended in 500. Mu.l PBS, and checked on the machine.

Aiming at the defects in the prior stem cell culture, the method provided by the invention grows enough cells in a limited space, the supporting material is a non-degradable material which can be recycled and does not pollute the environment, the continuous culture solution in the production process brings away the secreted metabolites of the cells when the cells are provided with enough nutrients, and the isolated and filtered culture solution is recycled by supplementing a proper amount of fresh culture medium or trace elements and increasing dissolved oxygen.

The invention comprises layered cylindrical supports, with unequal volumes, such as 100mL cases, and a large amount of micro glass beads (with the diameter of 0.5-1.0 mm) which are subjected to alkali treatment and are filled in, and the specific structure is shown in figures 5-1 to 5-4, wherein the figure 5-1 is a cell culture device without filling, the figure 5-2 is a cell culture device filled with microcarriers, and the figure 5-3 is a cross-sectional view of the cell culture device.

The method specifically comprises the following steps: 25 culture solution dispersion discs; 26 disc microwells; 27 disc interior space; 28 a medium conveying pipe; 29 a culture solution conveying pipe in the disc; 30 pipeline sealing valves; 31 a culture solution outlet; 32 disc opening switch; 33 cell culture microcarriers; 34 pipe disk connection ring.

Firstly, in order to increase the surface area of cell growth, a large amount of micro glass beads (with the diameter of 0.5-1.0 mm) treated by strong alkali are filled in a layered cylindrical bracket, and the glass material is the most common and safer material in the aspect of traditional cell culture, is not dissolved, is high-temperature resistant and can be recycled, and no harmful substances are exuded in the final process of harvesting cells or cell products.

As shown in the figure 5-1, a cell 3D culture device with 100mL is divided by 5 discs, and a plurality of 26 disc micropores are formed at the bottoms of the discs, so that cells are uniformly distributed on microcarriers on one hand, and on the other hand, the cells uniformly permeate into all micro glass beads in the process of transferring culture solution, thereby effectively avoiding the problems of local accumulation of cells and unsmooth flow of the culture solution and maximally meeting the growth of the cells. Wherein, only the surface of the bottommost disc, which is close to the microglass beads, is provided with 26 disc micropores, so that cells or culture fluid cannot accumulate in the place without the microglass beads. The middle three discs are 26 disc micropores on the upper and lower surfaces, and the diameters of the three discs are slightly smaller than the inner diameter of the cylinder, so that the micro glass beads can fully fill the whole interior, and meanwhile, the circulation of partial culture solution is convenient. The three discs had a 27 disc interior space through which both culture medium and inoculated cells were dispersed onto the microglass beads via 26 disc microwells.

The inner 5 discs are connected in series by a 28-centre shaft culture fluid conveying pipe, the 28-centre shaft culture fluid conveying pipe is connected with the discs by a 34-pipeline disc connecting ring, the 34-pipeline disc connecting ring is provided with a plurality of outlets, each outlet is connected with a 29-disc culture fluid conveying pipe, a plurality of small outlet holes are formed in the 29-disc culture fluid conveying pipe, culture fluid and inoculated cells can enter the inner space of the 27 discs through the small holes, and finally are dispersed on micro glass beads through 26-disc micropores.

The top disc is a single layer, a plurality of micropores are distributed in the top disc, and besides, a 32-disc opening switch is also arranged, the switch can move, so that micro glass beads can be injected into a cylinder when the switch is opened, and meanwhile, the micro glass beads in the cylinder are prevented from moving in the cell culture process. In the upper space there is a culture liquid outlet pipe with two 31 culture liquid outlets, which can directly discharge liquid or one of them is used as outlet, and the other can be connected with the outlets of other culture devices to implement series connection.

Under the action of peristaltic pump, culture solution and inoculated cells enter the device from the culture medium inlet pipe (4), pass through the medium conveying pipe in the middle shaft of 28 to each 34-pipe disc connecting ring, pass through the medium conveying pipe in the 29 discs to enter the internal space of the 27 discs, and then pass through the micropores of the 26 discs to be uniformly dispersed at all positions. The culture solution finally enters the upper space from the micropores at the top and then is discharged from the outlet.

The top of the device is provided with a spiral opening, and sterilized microglass bead carriers can be injected after the device is opened in a sterile environment.

The method has the advantages that:

(1) The invention relates to a method for culturing stem cells in high-density 3D and application thereof, which firstly solve the area problem in the process of culturing cells in a 2D plane. Cell culture devices of different volumes are used according to production requirements, and the surface area of cell adsorption is greatly increased by adding a large number of microcarriers into the device.

(2) The invention uses micro glass beads (diameter is 0.5-1.0 mm) as the microcarrier of the 3D culture cells, thereby greatly reducing the cost of the current microcarrier and recycling the micro glass bead carrier. The micro glass beads with the diameter of 0.5-1.0mm have the best effect, and can easily pass through the gap when inoculating cells.

(3) The microglass bead carrier is safe, has no exudation, solves the exudation problem of some degradable biological or chemical materials as microcarriers at present, is harmless to both harvested cells and metabolites of the cells, and can also harvest cells at the same time if the cells are harvested for exosomes of the cells, which is not achieved by other carriers at present.

(4) The micro glass bead carrier is treated with alkali to strengthen the adhesion between the surface and the cell and to utilize the cell adhesion growth. The cost of the microglass bead carrier is lower through high-temperature baking sterilization and recycling. If untreated microglass beads are used, cells cannot grow up, and cell attachment cannot be achieved on the surface of the beads.

(5) The device is a closed space, the culture solution and inoculated cells enter the device from the inlet pipe, and are connected with each pipeline disc connecting ring through the middle shaft culture solution conveying pipe, so that the culture solution is uniformly dispersed at each part through the middle disc micropores, and the problems of uneven growth of the 3D cultured cells, uneven distribution of the culture solution in the flowing process and the like in the prior art are solved.

The invention provides a cell culture fluid ventilation subsystem, which is characterized by comprising a culture fluid storage tank 44 and a gas-liquid exchange liner 46 fixedly suspended in the culture fluid storage tank 44, wherein the culture fluid storage tank 44 is vertically arranged; the top of the culture solution storage tank 44 is provided with an air inlet, a culture solution input pipe 41 and a culture solution output pipe 42; the inner pipe opening of the culture solution input pipe 41 positioned in the culture solution storage tank 44 is communicated with the top of the inner cavity of the gas-liquid exchange liner 46, and the top and the bottom of the gas-liquid exchange liner 46 are respectively provided with a liner air outlet 43 and a culture solution dropping opening; a plurality of net-shaped spherical gas-liquid exchange membranes 47 are fixedly arranged in the gas-liquid exchange liner 46, and the net-shaped spherical gas-liquid exchange membranes 47 are connected in series from top to bottom by vertically arranged inner ventilation pipes 48 at equal intervals; the inner pipe mouth of the culture solution output pipe 42 positioned in the culture solution storage tank 44 is connected with a culture solution output pipe 45, and the culture solution output pipe 45 vertically extends downwards to the bottom of the inner cavity of the culture solution storage tank 44; an air inlet pipe 35 is inserted in the air inlet, an air pump 37 is arranged on the outer end part of the air inlet pipe 35 positioned outside the culture solution storage tank 44, and the inner end part of the air inlet pipe 35 positioned inside the culture solution storage tank 44 extends downwards to the bottom of the gas-liquid exchange liner 46; the inner air outlet end of the air inlet pipe 35 is connected with two air inlet branch pipes inserted at the bottom of the air-liquid exchange liner 46, and the two air inlet branch pipes are respectively led to the inner part and the outer bottom part of the lowest reticular spherical air-liquid exchange membrane 47.

Preferably, in the subsystem, the mesh-type spherical gas-liquid exchange membrane 47 and the inner vent pipe 48 are each made of a 40 mesh stainless steel mesh with a pore diameter of about 0.5mm.

Preferably, in the subsystem, an air gauge 40 is provided on top of the broth storage tank 44.

Preferably, in the subsystem, an exhaust pipe 39 is provided at the top of the culture fluid storage tank 44, and an exhaust valve is provided on the exhaust pipe 39.

Preferably, in the subsystem, a front filter 36 and a rear filter 38 are respectively disposed on the outer end of the air inlet pipe 35 and on the front and rear sides of the air pump 37.

The cell culture fluid ventilation subsystem can effectively improve the dissolved oxygen in the cell culture fluid and simultaneously reduce the content of CO 2. A plurality of netlike spherical gas-liquid exchange membranes are vertically arranged in the gas-liquid exchange liner, a layer of water film is formed on the surface of the stainless steel netlike spherical membrane by the culture solution, the gas exchange efficiency can be improved, and meanwhile, a large number of bubbles generated by directly connecting gas into the liquid are avoided. The culture solution flowing into the gas-liquid exchange liner from the culture solution input pipe falls onto the net-shaped spherical gas-liquid exchange membrane and flows downwards along the surface of the spherical membrane. The air or pure oxygen entering the bottom of the gas-liquid exchange liner is led to the inner part and the outer bottom part of the lowest reticular spherical gas-liquid exchange membrane respectively, the inner gas flows from bottom to top along the reticular inner ventilation pipe, the outer gas flows from bottom to top along the spherical surface, the culture solution to be ventilated is slowly injected into the gas-liquid exchange liner from the culture solution input pipe, and flows from top to bottom from the uppermost reticular spherical gas-liquid exchange membrane, and forms mutual flushing with the gas from bottom to top, thereby improving the gas exchange efficiency.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. The drawings in the following description are merely exemplary and other implementations drawings may be derived from the drawings provided without inventive effort for a person of ordinary skill in the art.

The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the ambit of the technical disclosure.

FIG. 1 is a system diagram of a production process flow for continuous production and separation and purification of extracellular exosomes according to the present invention. Wherein, 1 cell culture fluid supplying subsystem; 2 microelements and amino acid supply tanks; 3, a fresh culture solution supplementing tank; 4 peristaltic pump; 5, monitoring a conveying pipe by the culture solution; 6 a culture solution detection subsystem; 7, detecting an instrument; 8 cell culture subsystem; 9, discharging the cell culture solution from the storage tank; 10 electromagnetic valve; 11 a culture medium filter; 12 three-way joint of pipeline; 13 exosome purification subsystem; 14, a bevel filter membrane bag; 15, replacing the liquid storage tank with air; 16 an air filter; 17 cell culture fluid flow ventilation subsystem; 18 an air pressure gauge; 19 an air vent valve; 20 air pump;

FIG. 2 is a flow assay of mesenchymal stem cells cultured by the system of the present invention, for the expression of positive marker genes, CD44 (99.8%), CD73 (99.1%), CD90 (98.4%) and CD105 (99.7%) of mesenchymal stem cells, respectively;

FIG. 3 is an electron microscope image of exosomes extracted by the large-scale continuous production purification system of the present invention;

FIG. 4Western Blot detection of marker proteins from exosomes extracted by the large-scale continuous production purification system of the present invention;

FIG. 5-1 is a schematic diagram of a cell culture subsystem without a filled microglass bead carrier, in which a medium inlet tube 24 is shown; 25 culture solution dispersion discs; 26 disc microwells; 27 disc interior space; 28 a medium conveying pipe; 29 a culture solution conveying pipe in the disc; 30 pipeline sealing valves; 31 a culture solution outlet;

FIG. 5-2 is a schematic diagram of a cell culture apparatus filled with a microglass bead carrier, in which a 32-disc opening switch; 33 cell culture microcarriers (microglass beads).

FIG. 5-3 is a cross-sectional view of a cell culture apparatus without a packed microglass bead carrier, showing a 31 culture outlet.

FIGS. 5-4 are exploded views of a cell culture apparatus disc, in which 25 medium disperses the disc; 27 disc interior space; 29 a culture solution conveying pipe in the disc; 34 pipe disk connection ring.

The cell culture fluid ventilation subsystem of FIG. 6 is shown as 35 inlet pipe, 36 front end filter, 37 air pump, 38 rear end filter, 39, exhaust pipe, 40 air pressure gauge, 41 culture fluid input pipe, 42 culture fluid output pipe, 43 inner container air outlet, 44 culture fluid storage tank, 45 culture fluid output pipe, 46 gas-liquid exchange inner container, 47 net ball type gas-liquid exchange membrane, 48 inner ventilation pipe, 49 ventilated culture fluid.

Detailed Description

The present invention provides a process and method for efficient and continuous production and isolation of purified extracellular material, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein, wherein the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The invention comprises five subsystems, namely a cell culture fluid supplementing subsystem, a detecting subsystem, a cell culture subsystem, an exosome purifying subsystem and a cell culture fluid ventilation subsystem.

The method for continuous production and separation and purification of the extracellular body comprises the following steps:

1. inoculation of cells

FIG. 1 shows a three-dimensional culture apparatus for a plurality of cells 28The cell culture subsystem is composed of the glass microspheres which are repeatedly used as the supporting material, do not pollute the environment and are not degradable, and the treated glass microspheres greatly improve the surface area contacted by cells. First, each cell culture apparatus was inoculated individually to obtain 2X10 ⁷ The umbilical cord mesenchymal stem cells cultured to 3 rd-4 th generation are inoculated into a cell culture device through a liquid inlet, are subjected to static culture at 37 ℃ for 12 hours, and are added into a cell culture subsystem one by one after being uniformly adhered to glass microspheres.

2. System operation and access cell culture device

The cell culture fluid is added into a fresh culture fluid supplementing tank 3, a corresponding peristaltic pump 4 is started to enable the culture fluid to run in the system, and the cell purification subsystem is in a closed state. The broth detection subsystem ensures dissolved oxygen (do=), CO in the broth ² (5-30%) and a pH level of 7.2-7.4, allowing the cell culture fluid to run in the system for at least 3 cell culture device volumes, then connecting to the cell stereoscopic culture device, and adjusting the fluid flow rate according to the number of connected cells, 10 ⁸ Cells were 10ml/min. According to the production requirement, one or more groups of cell three-dimensional culture devices can be connected at intervals to realize circulation replacement, and enough stable exosomes are generated in the system. In the process, dissolved oxygen and CO are detected according to a detection subsystem ₂ And the pH value and the like, and the cell culture fluid supplementing subsystem automatically supplements fresh culture fluid and adjusts acid-base balance. The culture fluid ventilation subsystem 17 is used for regulating CO in the culture fluid ² The air pump 20 is adjusted to increase the dissolved oxygen of the liquid, ensure in-process dissolved oxygen (do=40-70%), CO ₂ (5-30%) and a pH level of 7.2-7.4.

3. Large-scale production and purification of exosomes

When the cell culture fluid is discharged from the storage tank 9 to about 2/3 (1000 mL), the purification subsystem of the exosome automatically operates. Firstly, under the action of peristaltic pump, the culture solution passes through a double coarse filter device 11, namely a primary filter device and a secondary filter device respectively. Primary filtration primarily removes dead cells and larger cell debris. Secondary filtration isolates subcellular structures greater than 200nm, including apoptotic bodies and the like. The filtrate rich in exosomes reaches the envelope filter device 14, the molecular weight cut-off of the envelope filter device is 50KD and is far smaller than the particle size (about 100 KD) of the exosomes, the filtered cell culture fluid directly flows into the liquid storage tank 15 to be replaced by air, and enters the air interchanger for final recycling after pH and microelements are adjusted. The blocked exosomes were concentrated and liquid-replaced in the purification subsystem, and 1000mL of culture medium was concentrated to about 100 mL. Finally separating the concentrated exosomes by using a glucan chromatographic column, and collecting exosomes with different sizes according to a molecular sieve principle. Since exosomes are produced continuously in the system and the exosomes produced should be handled as soon as possible (4 ℃ for no more than 12 hours), the purification subsystem operates continuously as a process line.

4. Detection of exosomes

Firstly, we perform flow detection on the mesenchymal stem cells cultured by the system. The cell culture device removed from the system is firstly filled with PBS with double volume, then is filled with pancreatin with 1.5 times volume for digestion for 3-5 minutes, then is flushed out with PBS with double volume, and then is collected by centrifugal force of 300g/5min for standby. Washing with a certain amount of PBS once, discarding the supernatant, and re-suspending the cells to 5×10 ⁶ –1×10 ⁷ Between/ml; 100 μl of cells were stained in EP tube or flow tube, and stained in four tubes, respectively Anti-human CD44 FITC, anti-human CD73 FITC, anti-human CD90 FITC, anti-human CD105 FITC, each added with 5 μl, mouse IgG1 Isotype Control PE-Cy 7.2 μl, mouse IgG1 Isotype Control APC 1.2.2 μl; adding corresponding antibody, mixing, recommending to stretch the tube, directly blowing without gun head as much as possible, incubating at room temperature in dark place for 15min, and mixing again in the dyeing process; after the dyeing is finished, adding 1ml of PBS to wash the cells, and centrifuging for 300g/5min; the supernatant was discarded, the cells were pelleted, resuspended in 500. Mu.l PBS, and checked on the machine. As shown in fig. 2A to 2D, the positive antigens on the surface of the mesenchymal stem cells are respectively CD44 (99.8%), CD73 (99.1%), CD90 (98.4%) and CD105 (99.7%), which are all in the normal range, and the peak type is single, so that the stem cells cultured by the system are proved to have high purity and good growth state.

5. Exosome is detected to electron microscope

Firstly, fixing an exosome obtained by the implementation of the invention on a sample-carrying copper plate by using glutaraldehyde liquid, fully reacting for 5min, washing a copper mesh by using deionized water, incubating for 5min in uranium oxalate liquid and the copper mesh, finally, sucking redundant liquid on filter paper, drying the copper mesh, and then placing the copper mesh in a sample box, and shooting an electron microscope picture at 80 kV. As shown in FIG. 3, the example extracts a purified exosome electron microscope detection image, and the electron microscope can see the vesicle structure of the obvious tea tray-shaped double-layer film, the particle size is in the range of 30-150nm, the background is clean, the structure is clear, and no impurity particle interference exists.

Western blot detection of exosomes

Mu.l of the exosome mixture obtained by the present invention was taken and added to 10. Mu.l of 5 Xloading buffer, followed by thoroughly mixing, and then heating and denaturation in a boiling water bath for 5 minutes. After separation by 10% SDS-PAGE, the gel was removed and the proteins were electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% skim milk for 2 hours. Finally, the membrane was incubated with primary antibody overnight at 4 ℃. The antibodies used were as follows: anti-CD 9 (1:1000, 20597-1-AP), CD63 (1:500, 25682-1-AP), CD81 (1:1000, 27855-1-AP) and Calnexin (1:1000, 10427-2-AP); after the next day of reaction with HRP-labeled secondary antibody (1:10000, rockland), the reaction was followed with Immobilon ^TM The membrane was treated with a Western chemiluminescent HRP substrate (Millipore) and detected by ECL (enhanced chemiluminescence) Fuazon Fx (Vilber lourimat) imaging. The results of the Western Blot experiments of the CD9, CD63, CD81 and Calnexin proteins of the exosomes obtained in the example are shown in figure 4, the bands of the CD9, CD63 and CD81 of the three specific proteins of the exosomes are clear and bright, the positions of the bands are correct, and the negative protein contrast Calnexin has no visible band, so that the substance extracted and prepared by the system can be proved to be the exosomes, and the purity is higher.

The invention provides a process and a method for efficiently and continuously producing, separating and purifying a cell exosome, which innovatively comprise five subsystems, namely a cell culture fluid supplementing subsystem, a detecting subsystem, a cell culture subsystem, an exosome purifying subsystem and a culture medium fluid ventilation subsystem. The five subsystems form a closed cycle, the inlet is filled with fresh culture solution, and the outlet is concentrated exosome mixed solution. The method comprises a method and a device for culturing the three-dimensional cells, which increase the cell culture area. The supporting material of the device is glass microspheres which are recycled, do not pollute the environment and are not degradable; and the culture process takes away the exosomes secreted by the cells when the continuous culture solution is conveyed into the cells for sufficient nutrients, and the culture solution after the exosomes are separated is mixed with part of fresh culture solution by an oxygen ventilation device, and then conveyed to the cell culture device again. The process solves the problems that the existing exosome separation and purification process cannot be sustainable, is time-consuming and labor-consuming, has low recovery rate, serious pollution in the middle process, low yield, incapability of industrialized mass production and the like. The system realizes the integrated preparation flow from cell culture to exosome production, and is very suitable for clinical-grade exosome mass production; greatly accelerates the technical development and clinical transformation application process of the biological products related to the exosome, and has great commercial value.

While the invention has been described in detail with respect to the general description and specific embodiments thereof, it will be apparent to those skilled in the art that various modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. The system for producing, separating and purifying the cell exosomes comprises 5 subsystems, namely (1) a cell culture fluid supplementing subsystem, (2) a detection subsystem, (8) a cell culture subsystem, (13) an exosome purifying subsystem and (17) a cell culture fluid ventilating subsystem, wherein the 5 subsystems form a closed cycle, the inlet is the (8) cell culture fluid supplementing subsystem, and the outlet is the (13) exosome purifying subsystem.

2. The system of claim 1, wherein the (1) cell culture broth make-up subsystem comprises a recovery broth reserve tank, (2) a trace element and amino acid make-up tank, and (3) a fresh broth make-up tank.

3. The system of claim 1, wherein the (13) exosome purification subsystem comprises: primary filtration, secondary filtration, envelope filtration, and column chromatography.

4. A method for continuous production and isolation and purification of extracellular fluid, characterized in that the method uses the system according to any one of claims 1-3 for fresh culture fluid feeding from an inlet (8) cell culture fluid feeding subsystem and for collection of a concentrated extracellular fluid mixture from an outlet (13) extracellular fluid purification subsystem.

5. The method of claim 4, comprising the steps of:

6. The method of claim 5, comprising the steps of:

step three, the cell culture subsystem is composed of a group of parallel cell three-dimensional culture devices, each culture device is provided with an inlet and two outlets, one outlet is connected with the detection subsystem, and the other outlet is connected with the other outletThe outlet of a device was connected, and each culture device was filled with glass microspheres (diameter 0.8-1.5 mm) treated with a strong base, and the total area was about 1000cm ² About 10 can grow ⁹ The mesenchymal stem cells greatly improve the growth density of the cells, and because the cell culture devices are connected in parallel, the cell culture devices can be increased or decreased at any time, which is also one of the characteristics of the invention, the cell culture devices are firstly singly planted into the cells, the cells are uniformly adhered on the glass microspheres and then added into a cell culture subsystem, after the cells in the cell culture devices generally grow for 6-8 days, the cell culture devices are taken out from the subsystem and are subjected to subsequent recovery treatment,

Step four, the cell culture device taken down from the subsystem is firstly introduced with PBS with double volume, then introduced with pancreatin with 1.5 times volume for digestion for 3-5 minutes, then the cells are flushed out by PBS with double volume, then the cells are collected by centrifugal force of 300g/5min for standby,

step seven, the key factor of recycling the culture solution is CO ₂ The invention comprises a cell culture fluid ventilation subsystem, wherein a ventilation device in the subsystem fully enables liquid to be in reverse contact with sterile clean air to enable redundant CO ₂ Displacing, increasing dissolved oxygen of liquid, and peristaltic pump with ventilated culture solutionAs downflow into the cell culture fluid supply subsystem, and realize the recycling of part of culture fluid.

7. The system of claim 1, wherein said (8) cell culture subsystem comprises (24) a culture medium inlet tube; (25) a culture solution dispersion disc; (26) disc microwells; (27) a disc interior space; (28) a medium transport tube; (29) a culture fluid delivery tube in the disc; (30) a conduit shut-off valve; (31) a culture fluid outlet; (32) a disc opening switch; (33) Preferably, the cell culture microcarrier and the (34) pipeline disc connecting ring are microglass beads, the diameter of the microglass beads is 0.5-1.0mm, the microglass bead carrier is sterilized through high-temperature baking and can be reused, and the cell culture device is a closed device, and the volume of the cell culture device is 50-200 mL, for example 100mL.

8. The system of claim 1, wherein the cell culture subsystem (8) comprises layered cylindrical supports, the volumes of which are unequal, for example, 100mL, the inside of the cylindrical supports is filled with a large amount of micro glass beads treated by strong alkali, the diameters of the micro glass beads are 0.5-1.0mm, 5 discs are separated, the bottoms of the discs are provided with a large amount of (6) disc micropores, on the one hand, cells are uniformly distributed on microcarriers, on the other hand, culture solution uniformly infiltrates into all micro glass beads in the process of culture solution transmission, the problems of local accumulation of cells and unsmooth flow of culture solution are effectively avoided, the growth of cells is maximally satisfied, the surface of the disc at the bottommost part is only provided with (26) disc micropores, the cells or culture solution can not accumulate in places without the micro glass beads, the upper surface and the lower surface of the middle of the disc are respectively provided with (26) disc micropores, the diameters of the three discs are slightly smaller than the inner diameters of cylinders, thus the micro glass beads can fully fill the whole interior, on the other hand, the whole interior of the whole interior can be conveniently filled with part of culture solution, the three discs are provided with (27) inner spaces and the inner spaces of the culture solution are respectively connected with medium shafts (28) through medium shafts (28) and medium outlets) connected with the inner shafts (28) and medium conveying pipes (28) through the medium conveying pipes (28) and medium outlets) respectively connected with the medium shafts and the medium conveying pipes (28) in series together, the culture medium and inoculated cells can enter the inner space of the disc (27) through the small holes, finally are dispersed on the microglass beads through the micropores of the disc (26), the topmost disc is a single layer, and a plurality of micropores are distributed in the inner part, besides, the switch (32) is also provided with a disc opening switch, so that the microglass beads can be often injected into the cylinder when the switch is opened, the microglass beads in the cylinder are prevented from moving in the cell culture process, a culture medium outlet pipeline is arranged in the upper space, and the culture medium outlet pipeline is provided with two (31) culture medium outlets, wherein the two outlets can directly discharge liquid or one outlet, and the other outlet can be connected with the outlets of other culture devices to realize series connection.

9. The method of claim 4, wherein step 3 comprises the steps of:

(1) The micro glass bead carrier is soaked by sodium hydroxide, then washed, dried, sterilized, cooled and then is put into the culture device according to any one of claims 1 to 6,

(2) After the microglass bead carrier is injected, a sterile small tube is connected on a culture medium inlet tube (24) of the device, under the action of a peristaltic pump, liquid enters the device from the culture medium inlet tube (24), passes through a central shaft culture liquid conveying tube (28) to reach a disc connecting ring of each pipeline (34), passes through a culture liquid conveying tube (29) in the disc and enters the disc inner space (27), then uniformly distributes at each part through a disc micropore (26), the culture liquid finally enters an upper space from the micropore at the top, then is discharged from an outlet, sterile normal saline or PBS is introduced for the first time, powder on the surface of the microglass bead carrier is subjected to leak-proof detection at the same time, after no leakage is ensured, an inlet and an outlet are sealed, and the microcarrier is prepared for coating at normal temperature,

(4) Each cell culture device is individually seeded with cells,

(6) And performing flow detection on the cultured mesenchymal stem cells.

Preferably, the method comprises the steps of:

(2) After the microglass bead carrier is injected, a sterile small tube is connected on a culture medium inlet tube (24) of the device, under the action of a peristaltic pump, liquid enters the device from the culture medium inlet tube (24), enters a pipeline disc connecting ring (34) through a medium shaft culture liquid conveying tube (28), enters the disc inner space (27) through a medium liquid conveying tube (29) in the disc, is uniformly dispersed at each part through a disc micropore (26), the culture liquid finally enters an upper space from the micropore at the top, is discharged from an outlet, sterile physiological saline or PBS with 3 times of cylinder volume is introduced for the first time, powder on the surface of the microglass bead carrier is subjected to leak-proof detection at the same time, after no leakage is ensured, an inlet and an outlet are sealed, and the microcarrier is prepared for being coated at normal temperature,

(3) Coating with different materials according to cultured cells, such as umbilical Mesenchymal Stem Cells (MSC), oneMSC adherence promoting reagent or Vitronectin (Vitronectin) is generally used, firstly, the MSC adherence promoting reagent is diluted 100 times (1:100) by using sterile PBS solution, the device has different volumes from 50mL to 200mL, corresponding coating liquid is prepared according to the different volumes, and a proper amount of 1 XMSC adherence promoting reagent is added; introducing an adherence reagent from a culture solution inlet, notably, keeping the carrier immersed in the liquid all the time during the coating period, gently reversing and uniformly mixing, confirming uniform distribution of the adherence reagent, closing and sealing the entrance, and incubating overnight at 2-8 ℃; or in CO ₂ Incubating at 37deg.C for at least 30 min, slowly introducing two times of sterile PBS before inoculation, washing out the adherent agent, recycling, introducing 1.5 times of basic culture medium, sealing, preserving for use, inoculating cells within 48 hr,

(4) Each cell culture apparatus was inoculated with cells individually, and in this case, 100ml of the cell culture apparatus was inoculated, 1 volume of complete medium was introduced into the apparatus before the inoculation of the cells, and 5X 10 cells were collected ⁷ The umbilical cord mesenchymal stem cells cultured until 3-4 generations are obtained, the volume of the umbilical cord mesenchymal stem cells is regulated to 50mL by a culture medium, the umbilical cord mesenchymal stem cells slowly flow into a culture device through a liquid inlet under the action of a peristaltic pump, are gently and reversely mixed uniformly, are subjected to static culture at 37 ℃ for 12 hours, the cells are uniformly adhered to micro glass beads and then added into a cell culture system one by one, 10 percent of generation serum is added into the complete culture medium serving as a basic culture medium,

(6) The mesenchymal stem cells cultured by the device of the invention are subjected to flow detection, the cell culture device taken down from the system is firstly introduced with PBS with twice the volume, then introduced with pancreatin with 1.5 times the volume for digestion for 3-5 minutes, then the cells are flushed out by PBS with twice the volume, then the cells are collected by centrifugal force of 300g/5min for standby, a certain amount of PBS is added for washing once, the supernatant is discarded, the cells are ejected, and the cells are obtainedRe-suspended to 5 x 10 ⁶ –1×10 ⁷ Between/ml; 100 μl of cells were stained in EP tube or flow tube, and stained in four tubes, respectively Anti-human CD44 FITC, anti-human CD73 FITC, anti-human CD90 FITC, anti-human CD105 FITC, each added with 5 μl, mouse IgG1Isotype Control PE-Cy 7.2 μl, mouse IgG1Isotype Control APC 1.2.2 μl; adding corresponding antibody, mixing, directly blowing without gun head, incubating at room temperature in dark place for 15min, and mixing again during dyeing; after the dyeing is finished, adding 1ml of PBS to wash the cells, and centrifuging for 300g/5min; the supernatant was discarded, the cells were pelleted, resuspended in 500. Mu.l PBS, and checked on the machine.

10. The system of claim 1, wherein the (17) cell culture fluid ventilation subsystem comprises a vertically disposed culture fluid storage tank (44) and a gas-liquid exchange liner (46) fixedly suspended in the culture fluid storage tank (44); an air inlet, a culture solution input pipe (41) and a culture solution output pipe (42) are arranged at the top of the culture solution storage tank (44); the culture solution input pipe (41) is positioned in the culture solution storage tank (44) and is communicated with the top of the inner cavity of the gas-liquid exchange liner (46), and the top and the bottom of the gas-liquid exchange liner (46) are respectively provided with a liner air outlet (53) and a culture solution dropping opening; a plurality of net-shaped spherical gas-liquid exchange membranes (47) are fixedly arranged in the gas-liquid exchange liner (46), and the net-shaped spherical gas-liquid exchange membranes (47) are connected in series from top to bottom by inner ventilation pipes (48) which are vertically arranged in an equidistant manner; the inner pipe mouth of the culture solution output pipe (52) positioned in the culture solution storage tank (44) is connected with a culture solution discharge pipe (45), and the culture solution discharge pipe (45) vertically extends downwards to the bottom of the inner cavity of the culture solution storage tank (44); an air inlet pipe (35) is inserted into the air inlet, an air pump (37) is arranged at the outer end part of the air inlet pipe (35) positioned outside the culture solution storage tank (44), and the inner end part of the air inlet pipe (35) positioned inside the culture solution storage tank (44) extends downwards to the bottom of the gas-liquid exchange liner (46); the inner air outlet end of the air inlet pipe (35) is connected with two air inlet branch pipes inserted at the bottom of the air-liquid exchange liner (46), and the two air inlet branch pipes are respectively led to the inner part and the outer bottom part of the lowest reticular spherical air-liquid exchange membrane (47),

Preferably, in the subsystem, the net-shaped spherical gas-liquid exchange membrane (47) and the inner ventilation pipe (48) are both made of a 40-mesh stainless steel net, the pore diameter of the net is about 0.5mm,

preferably, in the subsystem, an air pressure gauge 40 is provided at the top of the culture fluid storage tank (44),

preferably, in the subsystem, an exhaust pipe (39) is arranged at the top of the culture solution storage tank (44), and an exhaust valve is arranged on the exhaust pipe (39),

preferably, in the subsystem, a front end filter (36) and a rear end filter (38) are respectively arranged on the outer end part of the air inlet pipe (35) and positioned on the front side and the rear side of the air pump (37).