CN110106085B - An integrated bioreactor for adherent cell culture - Google Patents

Abstract

The invention provides an integrated bioreactor for culturing adherent cells, the bioreactor comprises a control system, a reactor body and a cell culture unit connected in sequence, the control system, the reaction still body and the cell culture unit The inside is connected; the control system is provided with a gas pressure balance hole and a drip hole, the cell culture unit is provided with a central guide hole and a liquid level monitoring hole, the gas pressure balance hole and the liquid level monitoring hole A liquid level sensor is formed by communicating with a pipeline. The bioreactor further includes a cell suspension bottle. One end of the central guide hole is connected to the drip hole through a pipeline, and the other end is connected to the cell suspension bottle through a pipeline. The invention successfully exchanges and circulates the culture medium of the cell culture unit and the control system, and successfully realizes the unified control of the pH value and dissolved oxygen value of the culture medium of each unit; Control of surface height.

Description

An integrated bioreactor for adherent cell culture

technical field

The present invention relates to the technical field of bioreactors, and more particularly, to an integrated bioreactor for adherent cell culture.

Background technique

Bioreactors are in vitro culture expansion devices that can well mimic the internal environment of organisms to provide the required nutrients, physical environment and chemical environment for the growth of different cells. Therefore, bioreactors have great development prospects and potential in drug production, tissue engineering and regenerative medicine.

In the bioreactor, the flowable culture medium makes the nutrients in the culture medium more evenly distributed, so that the cells can absorb the nutrients well for growth and excrete metabolic wastes immediately. At present, bioreactors can be divided into many types according to different culture methods. According to the flow of culture fluid, it can be divided into perfusion bioreactor and hybrid bioreactor. Among them, perfusion bioreactor has high transfer efficiency and stable flow field, so it has attracted more and more attention of researchers. Perfusion bioreactors can also be divided into fluidized bed reactors, multi-layer plate reactors and hollow fiber reactors according to their structure. In the multi-layer plate perfusion bioreactor, the parallel-arranged multi-layer plates are used as the carrier of cells, and the plates and plates are filled with culture medium, which is collected in the outflow hole through the central guide hole or flows into different layers of culture plates. It has been successfully used in clinical trials such as stem cell culture in vitro and bioartificial liver.

Chinese patent ZL 201310197609.8 discloses a perfusion bioreactor, which contains a double-layer circulation system, including an outer circulation system arranged outside the culture tank, a culture tank, a gas injection system inside the culture tank, and an inner circulation system The system is designed to solve the contradiction in the needs of cells for circulating culture medium in the process of experimental culture, and can improve the solubility and uniform distribution of oxygen in the culture medium, which is more convenient for the discharge of waste, and greatly reduces the amount of waste generated in the culture medium. Air bubbles in the cell culture area. However, this patent does not solve the technical problems of pH, dissolved oxygen and shear force controllability.

In a bioreactor, the growth of cells is closely related to the magnitude and distribution of the shear force in the flow field. Appropriate flow field shear force can effectively promote the activity of cell differentiation and cell growth. On the contrary, excessive flow field shear force will lead to the reduction of cell viability and even cell apoptosis. The problem of the perfusion bioreactor is that the shear force of the flow field at the entrance of the culture medium is large, which cannot provide a suitable growth environment for the cells. In addition, cells are subjected to shear forces where the bubbles burst, resulting in reduced cell viability. The current multi-layer plate bioreactor directly perfuses the culture solution into the multi-layer plate structure, resulting in an unevenly distributed flow field shear force environment and inhibiting the growth of cells. Therefore, it is necessary to develop a bioreactor with controllable pH, dissolved oxygen and shear force.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the deficiencies in the prior art that pH, dissolved oxygen and shear force cannot be controlled, and to provide an integrated bioreactor with controllable pH, dissolved oxygen and shear force for culturing adherent cells. The integrated bioreactor provided by the invention can realize the microenvironment regulation of the pH value, dissolved oxygen, laminar shear force and temperature of the adherent cell culture environment.

In order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is:

An integrated bioreactor with controllable pH, dissolved oxygen and shear force for adherent cell culture, the bioreactor comprises a control system, a reactor body and a cell culture unit connected in sequence, the control system , the interior of the reactor body and the cell culture unit are connected; the control system is provided with a gas pressure balance hole and a drip hole, the cell culture unit is provided with a central guide hole and a liquid level monitoring hole, the gas pressure balance hole and the drip hole are arranged on the control system The pressure balance hole and the liquid level monitoring hole are connected through a pipeline to form a liquid level sensor. The bioreactor also includes a cell suspension bottle. One end of the central guide hole is connected to the drip hole through a pipeline, and the other end is connected to the cell through a pipeline. The suspension bottle is connected; the pipe connecting the drip tube and the central guide hole is provided with an outflow peristaltic pump, and the pipe connecting the central guide hole and the cell suspension bottle is provided with an inflow peristaltic pump.

The invention connects the central guide hole of the cell culture unit and the drip hole of the control system through a pipeline, and realizes the medium exchange between the cell culture unit and the control system through a peristaltic pump, so as to realize the unified control of pH value and dissolved oxygen of each unit. In the invention, the laminar shear force in the cell culture unit is regulated by the rotational speed of the outflow peristaltic pump, the liquid level of the cell culture unit is fed back by the information of the liquid level sensor, and the liquid level of the cell culture unit is adjusted by regulating the rotational speed of the inflow peristaltic pump high. In addition, the temperature regulation can be centralized regulation, or can be separately regulated by each culture unit according to different needs.

In the present invention, the cell culture unit is a perfusion tray type bioreactor tank for adherent cell culture. The regulation system is an upper-level regulation kettle body for realizing the regulation of dissolved oxygen and pH value.

Preferably, a support for carrying the bioreactor is also included.

Preferably, the cell culture unit is further provided with air inlets for adjusting pH and dissolved oxygen.

Preferably, the pipeline is a transparent medical high temperature and high pressure silicone tube.

The present invention also protects the integrated control method of the above-mentioned integrated bioreactor's centralized control and individualized control, and the control method is as follows:

The laminar shear force in the cell culture unit is regulated by the rotational speed of the outflow peristaltic pump, the liquid level of the cell culture unit is fed back through the information of the liquid level sensor, and the liquid level of the cell culture unit is adjusted by regulating the rotational speed of the inflow peristaltic pump.

Compared with the prior art, the present invention has the following beneficial effects:

The integrated bioreactor with controllable pH, dissolved oxygen and shear force for adherent cell culture provided by the invention successfully exchanges and circulates the culture medium of the cell culture unit and the control system, and successfully realizes each unit Unified regulation of PH value and dissolved oxygen value of medium; by adjusting the speed of outflow peristaltic pump, the size and distribution of laminar shear force in the cell culture unit and the reactor body were successfully changed, and the laminar shear force was further studied. The influence of force on cell reproduction and growth morphology; successfully monitored the medium liquid level of the cell culture unit through the information of the liquid level sensor; changed the rotational speed of the inflow peristaltic pump, successfully adjusted the liquid level of the cell culture unit, thus extremely The function and performance of the system are greatly optimized.

Description of drawings

FIG. 1 is a front view of the integrated bioreactor provided in Example 1. FIG.

FIG. 2 is a schematic diagram of the control system of the integrated bioreactor provided in Example 1. FIG.

FIG. 3 is a side view of the reactor body of the integrated bioreactor provided in Example 1. FIG.

FIG. 4 is a bottom view of the cell culture unit of the integrated bioreactor provided in Example 1. FIG.

FIG. 5 is a schematic structural diagram of an inflow peristaltic pump and an outflow peristaltic pump.

Detailed ways

The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

Example 1

FIG. 1 is a front view of the integrated bioreactor provided in this embodiment. As shown in FIG. 1 , the bioreactor includes a control system 1 for realizing dissolved oxygen and pH value control, a reaction kettle body 2, and a wall for sticking to the wall. Cell culture unit 3 and scaffold 4 for cell culture.

Figures 2 and 4 are schematic diagrams of the control system and the cell culture unit, respectively, Figure 3 is a side view of the reactor body, and Figure 5 is a schematic structural diagram of the inflow peristaltic pump and the outflow peristaltic pump. As shown in Figures 2, 3, 4, and 5, during the experiment, a transparent medical high-temperature and high-pressure silicone tube was used to connect the drip hole 6 of the control system 1 to the central guide hole 7 of the cell culture unit 3 to match the outflow. The peristaltic pump 11 realizes the exchange of the medium, thereby realizing the unified regulation and cyclic exchange of the pH value and dissolved oxygen value of the medium in the cell culture unit 3 and the regulation system 1, forming a self-circulating system. The rotation speed of the outflow peristaltic pump 11 was changed to regulate the magnitude and distribution of the laminar shear force in the cell culture unit 3 and the reactor body 2, and the effect of the laminar shear force on the cell reproduction number and growth morphology was studied.

During the monitoring and control process, the liquid level monitoring hole 8 and the gas pressure balance hole 5 are connected with a transparent medical high temperature and high pressure silicone tube to form a liquid level sensor that can transmit the liquid level information in the cell culture unit 3 and the reactor body 2 . A transparent medical high-temperature and high-pressure silicone tube is used to connect the central guide hole 7 and the cell suspension bottle, and an inflow peristaltic pump 10 is set on the silicone tube. Through the use of the inflow flow pump 10, the cell suspension is pumped into the cell culture unit 3 and the reactor body 2 from the central guide hole 7, so as to monitor the actual liquid level of the medium in the cell culture unit 3 and the reactor body 2. .

FIG. 4 is a bottom view of the cell culture unit. As shown in FIG. 4 , the cell culture unit 3 is further provided with an air inlet 9 for adjusting pH and dissolved oxygen.

The operation steps of the integrated bioreactor provided by this embodiment are as follows:

(1) Put the bioreactor support into 75% alcohol solution for soaking and cleaning, then place it in the ultra-clean bench ultraviolet environment to irradiate and air dry;

(2) The integrated bioreactor was soaked in Xinjieerfen for 2 hours, washed with clean water, and then put into a 60°C blast drying oven for drying;

(3) Take out the integrated bioreactor after drying, install the bioreactor support on the bottom of the bioreactor to form a cell culture unit, put the integrated bioreactor into an autoclave for sterilization at 120°C, and then Place the integrated bioreactor in a cell culture incubator with 5% CO ₂ at 37°C;

(4) Culture human bone marrow mesenchymal stem cells in a cell culture flask to proliferate to 10 million cells, digest the cells with trypsin to prepare a cell suspension, and then pump the cell suspension into a bioreactor through a peristaltic pump. After the cells were allowed to stand for 24 hours and adhered to the scaffold, the bioreactor was connected to the BioBundles fermenter through a silicone tube for parameter adjustment and circulatory perfusion;

(5) When the cells were cultured for 10 days, the growth and reproduction of the cells were observed, and the cells reached 85% confluence. The bioreactor plate support was taken out on the ultra-clean bench, and it was found by counting that the number of cells reached 120 million, that is, the in vitro expansion of stem cells was achieved.

The integrated bioreactor with controllable pH, dissolved oxygen, and shear force for adherent cell culture provided in this embodiment successfully exchanges and circulates the culture medium of the cell culture unit and the control system, and successfully realizes the Unified regulation of pH value and dissolved oxygen value of the unit medium; by adjusting the speed of the outflow peristaltic pump, the size and distribution of the laminar shear force in the cell culture unit and the reactor body were successfully changed, and the laminar shear force was further studied. The effect of shear force on cell reproduction and growth morphology; successfully monitored the medium liquid level of the cell culture unit through the information of the liquid level sensor; changed the rotational speed of the inflow peristaltic pump and successfully adjusted the liquid level of the cell culture unit, thereby Greatly optimized the function and performance of the system.

Obviously, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims (5)

1. an integrated bioreactor for adherent cell culture, characterized in that the bioreactor comprises a control system (1), a reactor body (2) and a cell culture unit (3) connected in turn, The interior of the control system (1), the reaction kettle body (2) and the cell culture unit (3) are communicated with each other; the control system (1) is provided with a gas pressure balance hole (5) and a drip hole (6) , the cell culture unit (3) is provided with a central guide hole (7) and a liquid level monitoring hole (8), and the gas pressure balance hole (5) and the liquid level monitoring hole (8) are connected through a pipeline to form A liquid level sensor, the bioreactor further comprises a cell suspension bottle, one end of the central diversion hole (7) is communicated with the drip hole (6) through a pipeline, and the other end is communicated with the cell suspension bottle through a pipeline; the An outflow peristaltic pump (11) is arranged on the pipeline connecting the drip tube (6) and the central guide hole (7), and an inflow pump (11) is arranged on the pipeline connecting the central guide hole (7) with the cell suspension bottle. Peristaltic pump (10); the cell culture unit (3) is provided with a culture medium, the control system (1) is provided with a culture medium, and the culture medium of the control system (1) is used for mixing with the cell culture unit (3) Medium exchange and circulation. 2. The integrated bioreactor according to claim 1, further comprising a support (4) for carrying the bioreactor. 3. The integrated bioreactor according to claim 1, characterized in that, the cell culture unit (3) is further provided with an air inlet (9) for adjusting pH and dissolved oxygen. 4 . The integrated bioreactor according to claim 1 , wherein the pipeline is a transparent medical high-temperature and high-pressure silicone tube. 5 . 5. A comprehensive control method for the centralized control and individualized control of the integrated bioreactor according to any one of claims 1 to 4, wherein the control method is as follows: The laminar shear force in the cell culture unit is regulated by the rotational speed of the outflow peristaltic pump, the liquid level of the cell culture unit is fed back through the information of the liquid level sensor, and the liquid level of the cell culture unit is adjusted by regulating the rotational speed of the inflow peristaltic pump.

Images