CN116751680A

CN116751680A - Low-loss-rate cell culture equipment capable of automatically regulating and controlling environmental parameters

Info

Publication number: CN116751680A
Application number: CN202310475155.XA
Authority: CN
Inventors: 李慕航; 崔俊宁; 唐然
Original assignee: Harbin Institute of Technology
Current assignee: Harbin Institute of Technology
Priority date: 2023-04-28
Filing date: 2023-04-28
Publication date: 2023-09-15

Abstract

A low-loss cell culture device with automatic regulation and control of environmental parameters belongs to the technical field of biological equipment. The method comprises the steps that a control system, a mixing module, a culture module and a sample processing module are arranged on a table surface of a support frame, a circulation module and a gas introduction module are arranged in the support frame, a pressure adjusting module is fixedly arranged at the upper end part of the culture module, the circulation module is connected with the mixing module and the culture module, so that a culture medium circularly flows between the mixing module and the culture module, and the pressure adjusting module can apply a periodically-changing pressure field to the inside of a cell culture tank; the application realizes the cell culture with low loss rate in the process of uniformly mixing the cell and nutrient substances, and realizes the control of key parameters of the cell culture environment imitating the environment in human body by a decoupling method of multiple physical fields of the environment; has the characteristics of greatly improving the quantity and quality of in-vitro cultured cells, and provides technical support for the development of the cell therapy industry.

Description

Low-loss-rate cell culture equipment capable of automatically regulating and controlling environmental parameters

Technical Field

The application belongs to the technical field of biological equipment, and particularly relates to low-loss-rate cell culture equipment capable of automatically regulating and controlling environmental parameters.

Background

Cell therapy is taken as an important scientific issue with international strategic and prospective, and has obvious treatment effect on a plurality of serious diseases lacking effective treatment means, such as heart failure, neurodegenerative diseases and the like. The large-scale, high-quality, safe and stable cell culture equipment is a bottleneck problem for restricting the promotion of cell therapy. The three-dimensional cell culture technology and the three-dimensional cell culture equipment can obviously improve the production efficiency of cells, ensure the production quality and improve the application effect, so that the development of the equipment which can improve the cell product quality, improve the cell production quantity and simultaneously have the automatic production function is significant.

In cell scale culture, the cell culture volume has a direct effect on cell growth and accumulation of metabolites. In general, a larger culture volume may provide more nutrient and oxygen supply, thereby promoting cell growth and proliferation. However, too large a culture volume may also present some problems. For example, a large culture volume may result in an uneven culture environment, resulting in a difference in growth of cells at different locations. The existing solution is to raise the environmental uniformity among different positions in the equipment through a large number of stirring and mixing after the whole culture volume, for example, the problem of uneven stirring of the culture solution in the tank body of the biological reaction tank is solved by adding a spraying device and a stirring device in the 'a stem cell scale culture bioreactor system' of the patent number CN112852633A by the regional strength. However, mechanical movements such as stirring, spraying, aeration and the like inevitably generate shearing force to damage cells, so that cell loss in the cell culture process is increased, cell survival is affected, and improvement of culture density is limited, so that a cell culture device with low cell loss rate is needed.

In order to improve the production quality of cells and ensure various physiological indexes of the cells, the culture environment can be accurately controlled during the in-vitro culture of the cells. The existing cell culture equipment mainly detects and regulates the pH, temperature and dissolved oxygen concentration in the cell culture environment, for example Chen Haijia in a patent No. CN115803428A, which is a method for efficiently preparing exosomes by utilizing a stem cell scale culture device, the pH, temperature and dissolved oxygen in a reaction kettle are monitored and regulated by a culture microenvironment regulating system. However, research proves that the pressure of the environment in the human body has influence on the cell state, proliferation speed, pluripotency and stability of the cells, and in the existing cell culture process, the pressure, pH, dissolved oxygen concentration and other environmental parameters are difficult to accurately regulate and control due to coupling, so that a controllable pressure culture environment is not arranged. The existing cell culture method still has the problem that the control of environmental factors such as pressure, dissolved oxygen, pH and the like can not be simultaneously regulated, so that the cell state, proliferation speed and the like are different from those of the actual living in vivo, and the development of cell therapy is hindered. It is therefore desirable to invent a cell culture apparatus with adjustable environmental parameters including pressure.

In addition, compared with the traditional manual culture equipment, the automatic production equipment can remarkably improve the production efficiency, and continuous and high-flux culture operation is realized, so that complicated and time-consuming steps possibly occurring in manual operation are avoided. Meanwhile, by matching with an advanced control system and a sensor technology, the culture environment can be monitored and regulated in real time, and the stability and consistency of culture conditions are ensured, so that the efficiency and quality of cell culture are improved.

In summary, there is a need for a low-loss-rate, fully automatic cell culture apparatus, which can couple and regulate the pH, pressure and dissolved oxygen concentration of a culture environment in a cell culture process, achieve the purpose of simulating the cell growth environment in a human body in vitro, and promote the quantity and quality of cultured cells, thereby promoting the development of the cell therapy industry in China.

Disclosure of Invention

The application aims to solve the problems of the existing cell in-vitro culture technology, and provides a low-loss-rate full-automatic cell culture device which can be coupled to regulate and control the pH, pressure and dissolved oxygen concentration of a culture environment in the cell culture process, so that the aim of greatly improving the quality and quantity of cultured cells is fulfilled, and technical support is provided for the development of the cell therapy industry.

The application provides low-loss cell culture equipment with automatic regulation and control of environmental parameters, which is characterized in that a control system, a mixing module, a culture module and a sample processing module are sequentially arranged on the table surface of a support frame, and a circulation module and a gas introduction module are arranged in the support frame; the control system consists of a central controller and a camera module; the culture module comprises a multi-parameter transmitter, a sensor group, a shaking module, a first incubator, a cell culture tank, a temperature sensor, a temperature controller, a first heater and a cell screen; the mixing module is formed by assembling a mixing tank, a stirring paddle, a connecting rod, a stirring motor second constant temperature box and a second heater; the circulation module is formed by assembling a first peristaltic pump, a second peristaltic pump, a first one-way valve, a second one-way valve, a first pinch valve, a second pinch valve, a first long tube, a second long tube, a first short tube and a second short tube; the gas introducing module comprises an aeration disc, a first bacterial filter and a mixed gas inlet device; the sample processing module comprises a sampling tube, an automatic sampler, a reagent rack, an automatic sampler and a cell analyzer; the pressure regulating module comprises a first air pipe, a second air pipe, a pressure sensor, a pressure regulating proportional valve, a second bacterial filter and a third bacterial filter;

a pressure regulating module is assembled on the upper end part of the culture module;

the first incubator is fixedly arranged on the shaking module and can move along with the shaking module; the cell culture tank is detachably arranged in the first incubator, and a sampling tube, a first short tube, a second long tube, a first air tube and a second air tube are fixedly arranged on the top cover of the cell culture tank; the sensor group is a non-contact measuring device and is fixedly arranged in an independent small chamber in the cell culture tank, and the sensor group is connected with the multi-parameter transmitter through a signal wire; the cell screen is fixed on the periphery of the second long tube, is assembled coaxially with the second long tube, and is spaced from the bottom end of the second long tube; a temperature sensor and a first heater are arranged in the first incubator; the temperature sensor and the first heater are respectively connected with the temperature controller;

the mixing tank is detachably arranged in the second incubator, and a sampling tube, a first long tube and a second short tube are fixedly arranged on the top cover of the mixing tank; the stirring paddle is positioned in the mixing tank and is connected with a stirring motor outside the mixing tank through a connecting rod; the second heater is arranged in the second constant temperature box and is connected with the temperature controller;

in the circulation module, a first peristaltic pump is sequentially connected with a first one-way valve, a first pinch valve, a first long pipe and a first short pipe in series by adopting a silica gel pipe, and a second peristaltic pump is sequentially connected with a second one-way valve, a second pinch valve, a second long pipe and a second short pipe in series by adopting a silica gel pipe;

the mixed air inlet device in the gas introducing module is communicated with a first bacterial filter through an air pipe, the aeration disc is assembled in a mixing tank of the mixing module, and the first bacterial filter is communicated with the aeration disc through the air pipe;

the sampling tube in the sample processing module is sequentially connected with an automatic sampler and a cell analyzer through a silica gel tube; the sample injection pipe is connected with the automatic sample injector and a reagent bottle on the reagent rack through a silica gel pipe;

the pressure regulating proportional valve in the pressure regulating module is fixedly arranged on the cell culture tank cover; the air inlet port of the pressure regulating proportional valve is sequentially connected with the pressure sensor and the second bacterial filter through an air pipe, the second bacterial filter is connected with the first air pipe through a silica gel pipe and is communicated with the interior of the cell culture tank, the air return port of the pressure regulating proportional valve is connected with the third bacterial filter through an air pipe, the third bacterial filter is connected with the second air pipe through a silica gel pipe and is communicated with the interior of the cell culture tank, and the air outlet port of the pressure regulating proportional valve is directly communicated with the outside air of the module;

the central controller has the functions of signal acquisition, signal processing, data storage and the like; the central controller is respectively connected with the camera module, the multi-parameter transmitter, the temperature sensor, the cell analyzer and the pressure sensor through data lines to acquire measurement signals; the central controller is respectively connected with the shaking module, the temperature controller, the stirring motor, the first peristaltic pump, the second peristaltic pump, the first pinch valve, the second pinch valve, the mixed air inlet device, the automatic sampler and the pressure regulating proportional valve through communication wires, and the actuator is controlled to act through measurement signal feedback; the central controller controls the speed, time and the proportion of air, carbon dioxide, oxygen and nitrogen which are introduced into the mixing tank through the mixing air inlet device; the central controller controls the pressure in the cell culture tank through a pressure regulating proportional valve; the temperature sensor is communicated with the temperature controller through a data line, and feeds back the internal temperature of the first incubator to the temperature controller in real time; the temperature controller is connected with a first heater in the first incubator and a second heater in the second incubator by using communication wires, so that the temperatures inside the first incubator and the second incubator are stable.

The application has the following advantages:

(1) The application realizes cell culture capable of reducing the cell loss rate in the process of uniformly mixing cell-nutrient substances. The mixing of substances in the existing culture equipment is mainly provided by stirring paddles and aeration discs, and the shearing force damage to cells is unavoidable. If the shaking mechanism is simply adopted for mixing, as the culture system is increased, the surface of the liquid is contacted with air to transfer oxygen, and the obvious dissolved oxygen concentration gradient exists in the culture tank, so that the requirement of all cells in the tank on the dissolved oxygen is not sufficiently supported. Therefore, the aeration of gases such as oxygen and the mixing of nutrient substances are independent from the cell culture, an independent mixing module is additionally arranged, the traditional stirring and aeration are adopted to ensure that the nutrient substances in the culture medium are fully and uniformly mixed, the cell culture module ensures the uniform dispersion of cells through the gentle shaking of the shaking module, the culture medium rich in the nutrient substances after being uniformly mixed in the mixing module and the culture medium consumed by the nutrient substances in the cell culture module are subjected to the flowing replacement through the circulating module, and the shearing force damage to the cells caused by the mixed power sources such as stirring and aeration is removed on the basis of improving the transmission characteristic of the nutrient substances and enhancing the mixing stability, so that the low cell loss rate in the process of uniformly mixing the cell-nutrient substances is realized.

(2) The application realizes the control of the key environment parameters of the human body environment simulation culture based on the decoupling of multiple physical fields. The existing cell culture equipment and method can not solve the problem of regulating and controlling the pressure, pH, dissolved oxygen concentration and temperature in the culture environment at the same time; based on a feedback signal of a high-sensitivity sensor, decoupling analysis is performed on a plurality of physical fields of a cell culture environment through a built-in control model, the cell culture temperature is regulated through a heater and a temperature controller in a comprehensive regulation manner, and after the temperature is stable, the pressure, the pH value and the dissolved oxygen concentration in the cell growth environment are controlled through regulating the proportion, the speed, the time and the like of four gases of air, oxygen, nitrogen and carbon dioxide which are introduced into a cell culture tank; solves the problem that the prior art can not regulate and control the environmental pressure, pH, dissolved oxygen concentration and temperature simultaneously when culturing in vitro, so that the cells are always in the culture environment similar to human body, and the production quality of the cells is ensured to the greatest extent.

(3) The application realizes the automatic cell culture based on the application points (1) and (2). At present, a labor-intensive cell culture mode is adopted, such as manual addition of culture medium, sampling, detection, expansion of a culture system and the like, most of operation steps are easy to cause pollution and generate larger errors, so that automatic operation of equipment is changed, batch production of cells can be completed without a large number of experimenters, the repeatability and stability of a cell culture process are improved, and production requirements are met.

Drawings

FIG. 1 is a schematic diagram of an assembly structure of a low loss cell culture apparatus with automatic regulation of environmental parameters;

FIG. 2 is a schematic diagram of a connection structure of a low loss cell culture apparatus with automatic regulation of environmental parameters;

FIG. 3 is a schematic view of a connection structure of the circulation module;

FIG. 4 is a schematic diagram of a pressure regulation module connection configuration;

FIG. 5 is a schematic diagram of the cell screen assembly.

The reference numerals in the drawings:

100. a control system; 200. a culture module; 300. a mixing module; 400. a circulation module; 500. a gas introduction module; 600. a sample processing module; 700. a pressure regulating module; 800. a support frame; 110. a central controller; 120. a camera module; 210. a multi-parameter transmitter; 220. a sensor group; 230. a shaking module; 240. a first incubator; 250. a cell culture tank; 260. a temperature sensor; 270. a temperature controller; 280. a first heater; 290. a cell screen; 310. a mixing tank; 320. stirring paddles; 330. a connecting rod; 340. a stirring motor; 350. a second incubator; 360. a second heater; 410. a first peristaltic pump; 411. a second peristaltic pump; 420. a first one-way valve; 421. a second one-way valve; 430. a first pinch valve; 431. a second pinch valve; 440. a first long tube; 450. a first short tube; 460. a second long tube; 470. a second short tube; 510. an aeration disc; 520. a first bacterial filter; 530. a mixing air inlet device; 610. a sampling tube; 620. a sample inlet tube; 630. an automatic sampler; 640. a reagent rack; 650. an autosampler; 660. a cell analyzer; 710. a first air tube; 720. a pressure sensor; 730. a pressure regulating proportional valve; 740. a second bacterial filter; 750. a second air pipe; 760. and a third bacterial filter.

Detailed Description

Embodiments of the present application will be described in detail below with reference to the accompanying drawings.

The low-loss-rate cell culture equipment with automatic regulation and control of environmental parameters is characterized in that a control system 100, a mixing module 300, a culture module 200 and a sample processing module 600 are sequentially installed on the table surface of a support frame 800, and a circulation module 400 and a gas introduction module 500 are installed inside the support frame 800; the control system 100 is composed of a central controller 110 and a camera module 120; the culture module 200 comprises a multi-parameter transmitter 210, a sensor group 220, a shaking module 230, a first incubator 240, a cell culture tank 250, a temperature sensor 260, a temperature controller 270, a first heater 280, and a cell screen 290; the mixing module 300 is assembled by a mixing tank 310, a stirring paddle 320, a connecting rod 330, a stirring motor 340, a second incubator 350 and a second heater 360; the circulation module 400 is assembled by a first peristaltic pump 410, a second peristaltic pump 411, a first check valve 420, a second check valve 421, a first pinch valve 430, a second pinch valve 431, a first long tube 440, a second long tube 460, a first short tube 450 and a second short tube 470; the gas introduction module 500 includes an aeration tray 510, a first bacterial filter 520, and a mixed gas inlet 530; the sample processing module 600 includes a sampling tube 610, a sample tube 620, an autosampler 630, a reagent rack 640, an autosampler 650, a cell analyzer 660; the pressure regulating module 700 includes a first air pipe 710, a second air pipe 750, a pressure sensor 720, a pressure regulating proportional valve 730, a second bacterial filter 740, a third bacterial filter 760;

a pressure regulating module 700 is mounted on the upper end of the culture module 200;

the first incubator 240 is fixedly installed on the shaking module 230 and can move along with the shaking module; the cell culture tank 250 is detachably installed in the first incubator 240, and a sampling tube 610, a first short tube 450, a second long tube 460, a first gas tube 710 and a second gas tube 750 are fixedly installed on the top cover of the cell culture tank 250; the sensor group 220 is a non-contact measuring device, and is fixedly arranged in an independent small chamber in the cell culture tank 250, and the sensor group 220 is connected with the multi-parameter transmitter 210 through a signal wire; the cell screen 290 is fixed on the periphery of the second long tube 460, is assembled coaxially with the second long tube 460, and has a bottom spaced from the bottom end of the second long tube 460 to block the cell/cell microcarrier mixture in the cell culture tank from flowing out along with the culture medium when the circulation module is opened; the first thermostat 240 is internally installed with a temperature sensor 260 and a first heater 280; the temperature sensor 260 and the first heater 280 are respectively connected with the temperature controller 270;

the mixing tank 310 is detachably installed in the second incubator 350, and a sample inlet pipe 620, a first long pipe 440 and a second short pipe 470 are fixedly installed on the top cover of the mixing tank 310; the stirring paddle 320 is positioned inside the mixing tank 310 and is connected with a stirring motor 340 outside the mixing tank 310 through a connecting rod 330; the second heater 360 is installed inside the second thermostat 350 and connected to the temperature controller 270;

in the circulation module 400, a first peristaltic pump 410 is sequentially connected with a first check valve 420, a first pinch valve 430, a first long tube 440, and a first short tube 450 in series, and a second peristaltic pump 411 is sequentially connected with a second check valve 421, a second pinch valve 431, a second long tube 460, and a second short tube 470 in series; the circulation module 400 connects the mixing module 300 and the culture module 200, so that the nutrient-rich culture medium in the mixing module 300 flows into the culture module 200, and the nutrient-depleted culture medium in the culture module 200 flows into the mixing module 300 to be supplemented with nutrient;

the gas introducing module 500 is characterized in that the mixed gas inlet device 530 is communicated with the first bacterial filter 520 through a gas pipe, the aeration disc 510 is assembled in the mixing tank 310 of the mixing module 300, and the first bacterial filter 520 is communicated with the aeration disc 510 through a gas pipe;

the sampling tube 610 in the sample processing module 600 is sequentially connected to an automatic sampler 630 and a cell analyzer 660 through a silica gel tube; the sample injection pipe 620 is connected with the automatic sample injector 650 and the reagent bottle on the reagent rack 640 through a silica gel pipe;

a pressure regulating module 700 is mounted on the upper end of the culture module 200; the air inlet port of the pressure regulating proportional valve 730 is sequentially connected with the pressure sensor 720 and the second bacterial filter 740 through air pipes, the second bacterial filter 740 is connected with the first air pipe 710 through a silica gel pipe and is communicated with the inside of the cell culture tank 250, the air return port of the pressure regulating proportional valve 730 is connected with the third bacterial filter 760 through an air pipe, the third bacterial filter 760 is connected with the second air pipe 750 through a silica gel pipe and is communicated with the inside of the cell culture tank 250, and the air outlet port of the pressure regulating proportional valve 730 is directly communicated with the outside air of the module; an air inlet port of the pressure regulating proportional valve 730 in the pressure regulating module 700 is normally opened to maintain a normal working state of the module, and an air return port and an air exhaust port form a certain opening ratio to maintain pressure stability in the cell culture tank;

the central controller 110 is respectively connected with the camera module 120, the multi-parameter transmitter 210, the temperature sensor 260, the cell analyzer 660 and the pressure sensor 720 through data lines to obtain measurement signals; the central controller 110 is respectively connected with the shaking module 230, the temperature controller 270, the stirring motor 340, the first peristaltic pump 410, the second peristaltic pump 411, the first pinch valve 430, the second pinch valve 431, the mixing air inlet device 530, the automatic sampler 630, the automatic sampler 650 and the pressure regulating proportional valve 730 through communication wires, and the actuator is controlled to act through measurement signal feedback; wherein the central controller 110 controls the air, carbon dioxide, oxygen and nitrogen rates, time and the ratio of the air, carbon dioxide, oxygen and nitrogen introduced into the mixing tank 310 through the mixing inlet means 530; the central controller 110 controls the pressure in the cell culture tank 250 through the pressure regulating proportional valve 730; the temperature sensor 260 is communicated with the temperature controller 270 through a data line, and the temperature sensor 260 feeds back the internal temperature of the first incubator 240 to the temperature controller 270 in real time; the temperature controller 270 connects the first heater 280 in the first incubator 240 and the second heater 360 in the second incubator 350 using communication lines, so that the temperatures inside the first incubator 240 and the second incubator 350 are stable.

The application of the low-loss-rate cell culture equipment with the automatic regulation and control of the environmental parameters is as follows:

step (1) starting the equipment, and enabling the central controller 110 to command the temperature controller 270 to control the first heater 280 and the second heater 360 to heat up, and ensuring that the temperature in the first incubator 240 and the second incubator 350 is constant at 37.2 ℃;

step (2) the central controller 110 controls the autosampler 650 to add fresh medium on the reagent shelf 640 into the mixing tank 310;

step (3), the central controller 110 starts the mixed air inlet device 530, and controls the inlet ratio, the inlet rate and the inlet time of the four mixed gases of oxygen, nitrogen, carbon dioxide and air in the mixing tank 310 according to the environmental index input by the user; the central controller 110 starts the stirring motor 340 to drive the stirring paddle 320 to rotate at a high speed;

step (4) adding a cell/cell-microcarrier complex in the middle area between the cell culture tank 250 and the cell screen 290, and the central controller 110 starts the shaking module 230 to input a set rotation speed so that the cells/cell-microcarriers are uniformly distributed in the area;

step (5) central controller 110 activates first peristaltic pump 410, second peristaltic pump 411, first pinch valve 430, second pinch valve 431 in circulation module 400 to circulate the culture medium between mixing tank 310 and cell culture tank 250; the central controller 110 controls the rotational speed of the first peristaltic pump 410 and the second peristaltic pump 411 in the circulation module 210 to control the exchange rate of the culture medium in the cell culture tank 250 and the mixing tank 310;

step (6) the central controller 110 starts the pressure regulating module 700, and the central controller 110 controls the discharge amount of the mixed gas in the pressure regulating proportional valve 730 through the feedback value of the pressure sensor 720 to regulate the pressure value and the variation cycle in the cell culture tank 250;

step (7) setting sampling frequency and sampling amount, the central controller 100 controls the automatic sampler 630 to collect cells in the cell culture tank 250 and the culture medium around the cells, the samples are sent to the cell analyzer 660 through the connected silicone tube, and the cell analyzer 660 detects the cell morphology, the number/proportion of living cells, and the glucose/urea/lactic acid/inorganic salt content;

step (8), the central controller 110 judges the culturing stage of the cells according to the information obtained by the cell analyzer 660, adjusts the rotation speed of the stirring paddle 320 in the mixing tank 310 according to the environmental information required by the cells at the stage in the preset program, and adjusts the ratio of the mixed gas introduced in the gas introducing module 530; when the central controller 110 determines that the cells are in the designated culture stage, the automatic injector 650 is controlled to add the cytokines in the reagent bottles on the reagent rack 640 to the mixing tank 310, and to the cell culture tank 250 through the circulation system 400 for the cell growth requirement;

in the culturing process of step (9), the camera module 120 monitors the liquid level change in the cell culture tank 250 in the whole course, and has a plurality of preset liquid level alarm functions; sensor group 220 transmits signals to multiparameter transmitter 210, monitors temperature, dissolved oxygen concentration, pH and carbon dioxide concentration in cell culture tank 250 in real time, and transmits information to central controller 110 for viewing, processing and storage; the pressure sensor 720 monitors the pressure in the cell culture tank 250 in real time throughout the culture and transmits information to the central controller 110 for viewing, processing and storage; the pH, dissolved oxygen concentration and pressure control of the culture environment are achieved by means of the regulation and control of the pressure regulating module 700 and the mixed air inlet device 530 by the central controller 110.

The sensor group 220 includes a dissolved oxygen concentration sensor, a pH sensor, and a carbon dioxide concentration sensor.

The first, second and third bacterial filters 520, 740 and 760 can bidirectionally filter fine impurities and bacteria in the gas.

The cell analyzer 660 can detect cell morphology, number/ratio of living cells, glucose/urea/lactate/inorganic salt content.

The cell culture tank 250 of the cell culture module 200 and the mixing tank 310 of the mixing module 300 are both closed pressure-resistant containers.

When the low-loss-rate cell culture equipment with the automatic regulation and control of the environmental parameters is used, nutrient substances such as a culture medium and oxygen are uniformly mixed in the mixing module 300, and enter the cell culture tank 250 through the circulation module 400 after the mixing is finished, and the cell/cell-microcarrier compound is directly contacted with the uniformly mixed culture medium in the cell culture tank 250, so that the cell culture tank 250 does not need to be provided with modules with functions of stirring, aeration and the like, the shearing force damage to cells caused by stirring and aeration is avoided, the cell loss rate in the cell culture process is obviously reduced, the cell culture density is increased on the original basis, and the number of cultured cells is increased.

When the low-loss cell culture apparatus with automatic regulation and control of environmental parameters is used, the central controller 110 determines the culture stage of the cells according to the information obtained by the cell analyzer 660 in the sample processing module 600, adjusts the stirring rotation speed in the mixing tank 310 according to the environmental information required by the cells in the stage in the built-in model, and adjusts the opening and the introducing time of the flow regulators of the four gases, namely air, nitrogen, carbon dioxide and oxygen, in the mixing air inlet device 530; when the central controller 110 determines that the cells are in the designated culture stage, the auto injector 650 is controlled to add the cytokines in the reagent bottles in the reagent rack 640 to the mixing tank 310, and to the cell culture tank 250 through the circulation module 400 for cell growth requirements.

When the low-loss cell culture equipment with the automatic regulation and control of the environmental parameters is used, the pH, the dissolved oxygen concentration and the pressure of the culture environment are controlled mainly by the regulation and control of the pressure regulating module 700 and the mixed air inlet device 530 by the central controller 110; firstly, the target set value is brought into a built-in model, the mass proportion coefficient of carbon dioxide, oxygen, nitrogen and air which need to be introduced into the cell culture tank 250 is calculated, the central controller 110 starts the mixed air inlet device 530 to start continuously introducing mixed gas, and meanwhile, the central controller 110 starts the pressure regulating module 700 to start the stable regulation of the pressure in the cell culture tank 250; when the sensor group 220 detects that the value in the pH and/or the dissolved oxygen concentration is changed, the central controller 110 calculates the required inlet ratio of the four gases according to the model, controls the flow regulators of the four gases in the mixed gas inlet device 530, and continuously introduces the mixed gas into the cell culture tank 250 until the value detected by the sensor returns to the set range after the inlet ratio of the four mixed gases is changed; in the process of continuously charging the air in the hybrid air intake device 530, the pressure regulating module 700 needs to adjust the opening degrees of the air intake port, the air return port and the air exhaust port of the pressure regulating proportional valve 730 at any time according to the detection value fed back by the pressure sensor 720 so as to keep the pressure in the cell culture tank 250 stable.

Claims

1. The low-loss-rate cell culture equipment capable of automatically regulating and controlling environmental parameters is characterized in that a control system (100), a mixing module (300), a culture module (200) and a sample processing module (600) are sequentially installed on the table top of a support frame (800), a circulation module (400) and a gas introduction module (500) are installed inside the support frame (800), and the control system (100) consists of a central controller (110) and a camera module (120); the culture module (200) comprises a multi-parameter transmitter (210), a sensor group (220), a shaking module (230), a first incubator (240), a cell culture tank (250), a temperature sensor (260), a temperature controller (270), a first heater (280) and a cell screen (290); the mixing module (300) is formed by assembling a mixing tank (310), a stirring paddle (320), a connecting rod (330), a stirring motor (340), a second constant temperature box (350) and a second heater (360); the circulation module (400) is formed by assembling a first peristaltic pump (410), a second peristaltic pump (411), a first one-way valve (420), a second one-way valve (421), a first pinch valve (430), a second pinch valve (431), a first long pipe (440), a second long pipe (460), a first short pipe (450) and a second short pipe (470); the gas introduction module (500) comprises an aeration disc (510), a first bacterial filter (520) and a mixed gas inlet device (530); the sample processing module (600) comprises a sampling tube (610), a sampling tube (620), an automatic sampler (630), a reagent rack (640), an automatic sampler (650) and a cell analyzer (660); the pressure regulating module (700) comprises a first air pipe (710), a second air pipe (750), a pressure sensor (720), a pressure regulating proportional valve (730), a second bacterial filter (740) and a third bacterial filter (760);

characterized in that a pressure regulating module (700) is mounted on the upper end of the culture module (200);

the first constant temperature box (240) is fixedly arranged on the shaking module (230) and can move along with the shaking module; the cell culture tank (250) is detachably arranged in the first incubator (240), and a sampling tube (610), a first short tube (450), a second long tube (460), a first gas tube (710) and a second gas tube (750) are fixedly arranged on the top cover of the cell culture tank (250); the sensor group (220) is a non-contact measuring device and is fixedly arranged in an independent small chamber in the cell culture tank (250), and the sensor group (220) is connected with the multi-parameter transmitter (210) through a signal wire; the cell screen (290) is fixed on the periphery of the second long tube (460), is coaxially assembled with the second long tube (460), and has a bottom spaced from the bottom end of the second long tube (460); the first thermostat (240) is internally provided with a temperature sensor (260) and a first heater (280); the temperature sensor (260) and the first heater (280) are respectively connected with the temperature controller (270);

the mixing tank (310) is detachably arranged in the second constant temperature box (350), and a sampling tube (620), a first long tube (440) and a second short tube (470) are fixedly arranged on the top cover of the mixing tank (310); the stirring paddle (320) is positioned inside the mixing tank (310) and is connected with a stirring motor (340) outside the mixing tank (310) through a connecting rod (330); the second heater (360) is arranged inside the second constant temperature box (350) and is connected with the temperature controller (270);

a first peristaltic pump (410) is sequentially connected with a first one-way valve (420), a first pinch valve (430), a first long tube (440) and a first short tube (450) in series by adopting a silica gel tube, and a second peristaltic pump (411) is sequentially connected with a second one-way valve (421), a second pinch valve (431), a second long tube (460) and a second short tube (470) in series by adopting a silica gel tube;

the mixed air inlet device (530) in the air introducing module (500) is communicated with the first bacterial filter (520) through an air pipe, the aeration disc (510) is assembled in the mixing tank (310) of the mixing module (300), and the first bacterial filter (520) is communicated with the aeration disc (510) through the air pipe;

the sampling tube (610) in the sample processing module (600) is sequentially connected with an automatic sampler (630) and a cell analyzer (660) through a silica gel tube; the sample injection pipe (620) is connected with the automatic sample injector (650) and a reagent bottle on the reagent rack (640) through a silica gel pipe;

the pressure regulating proportional valve (730) in the pressure regulating module (700) is fixedly arranged on the cover of the cell culture tank (250); the air inlet port of the pressure regulating proportional valve (730) is sequentially connected with the pressure sensor (720) and the second bacterial filter (740) through an air pipe, the second bacterial filter (740) is connected with the first air pipe (710) through a silica gel pipe and is communicated with the inside of the cell culture tank (250), the air return port of the pressure regulating proportional valve (730) is connected with the third bacterial filter (760) through an air pipe, and the third bacterial filter (760) is connected with the second air pipe (750) through a silica gel pipe and is communicated with the inside of the cell culture tank (250), and the air outlet port of the pressure regulating proportional valve (730) is directly communicated with the outside air of the module;

the central controller (110) is respectively connected with the camera module (120), the multi-parameter transmitter (210), the temperature sensor (260), the cell analyzer (660) and the pressure sensor (720) through data lines; the central controller (110) is respectively connected with the shaking module (230), the temperature controller (270), the stirring motor (340), the first peristaltic pump (410), the second peristaltic pump (411), the first pinch valve (430), the second pinch valve (431), the mixed air inlet device (530), the automatic sampler (630), the automatic sampler (650) and the pressure regulating proportional valve (730) through communication lines; wherein the central controller (110) controls the rates, time and the ratio of air, carbon dioxide, oxygen and nitrogen introduced into the mixing tank (310) through the mixing air inlet device (530); the central controller (110) controls the pressure in the cell culture tank (250) through a pressure regulating proportional valve (730); the temperature sensor (260) is communicated with the temperature controller (270) through a data line, and the temperature sensor (260) feeds back the internal temperature of the first incubator (240) to the temperature controller (270) in real time; the temperature controller (270) is connected with a first heater (280) in the first incubator (240) and a second heater (360) in the second incubator (350) by using communication wires, so that the internal temperatures of the first incubator (240) and the second incubator (350) are stable.

2. The low loss cell culture apparatus according to claim 1, wherein the sensor set (220) comprises a dissolved oxygen concentration sensor, a pH sensor, and a carbon dioxide concentration sensor.

3. The apparatus of claim 1, wherein the first bacterial filter (520), the second bacterial filter (740), and the third bacterial filter (760) are configured to bi-directionally filter fine impurities and bacteria from the gas.

4. The low loss cell culture apparatus of claim 1, wherein the cell analyzer (660) is configured to detect cell morphology, number/ratio of living cells, glucose/urea/lactate/inorganic salt content.

5. The low-loss cell culture apparatus with automatic environmental parameter control according to claim 1, wherein the cell culture tank (250) of the cell culture module (200) and the mixing tank (310) of the mixing module (300) are both closed pressure-resistant containers.