CN117143725B - A human-like large-scale stem cell automatic culture device with low loss rate - Google Patents
A human-like large-scale stem cell automatic culture device with low loss rate Download PDFInfo
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Abstract
An automatic culture device for human-like large-scale stem cells with low loss rate belongs to the field of biological equipment, and comprises a plurality of culture modules, a circulation module, a shaking module and a pressure regulating module; the circulation module is connected with the mixing module and the culture module, so that the culture medium circularly flows between the mixing module and the culture module; the shaking module is connected with the culture module and used for driving the culture module to shake; the pressure regulation module may apply a periodically varying pressure field within the cell culture tank. The invention realizes the low loss rate and the expandable large-scale cell culture in the process of uniformly mixing the cell and the nutrient substances, realizes the control of key parameters of the cell culture environment imitating the environment in human body by a multi-physical field decoupling method, has the characteristic of greatly improving the quantity and the quality of the cells cultured in vitro, and provides technical support for the development of the cell treatment industry.
Description
Technical Field
The invention belongs to the field of biological equipment, and particularly relates to low-loss-rate automatic human-simulated large-scale stem cell culture equipment.
Background
Stem cell therapy is taken as an important scientific issue with international strategic and prospective property, and has obvious therapeutic effect on a plurality of serious diseases which lack effective therapeutic 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 popularization of stem cell treatment. The three-dimensional cell culture technology and the three-dimensional cell culture equipment can obviously improve the production efficiency of stem 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 production quantity of the stem cells and have the automatic production function is significant.
In stem 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 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 influenced, and the improvement of culture density is limited, so that the invention of the expandable large-scale stem cell culture equipment with low cell loss rate is required.
In order to improve the production quality of stem 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 method for efficiently preparing exosomes by using a stem cell scale culture device in a patent number CN115803428A, 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 an influence on the cell state, proliferation speed, pluripotency and stability of stem cells, and in the existing stem cell culture process, the pressure, pH, dissolved oxygen concentration and other environmental parameters are difficult to accurately regulate and control, so that a controllable pressure culture environment is not arranged. The existing stem 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 stem cell treatment is hindered. It is therefore desirable to invent a stem cell culture apparatus that mimics the in vivo environment of a human including blood 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, the culture environment can be monitored and regulated in real time by matching with an advanced control system and a sensor technology, and the stability and consistency of culture conditions are ensured, so that the efficiency and quality of stem cell culture are improved.
In summary, there is a need for a low-loss, expandable, fully automatic and large-scale stem cell culture device, which can couple and regulate the pH, pressure and dissolved oxygen concentration of a culture environment in a cell culture process, so as to achieve the purpose of simulating the cell growth environment in a human body in vitro, and promote the quantity and quality of the cultured stem cells, thereby promoting the development of the cell therapy industry in China.
Disclosure of Invention
The invention aims to solve the problems of the existing cell in-vitro culture technology, and provides low-loss-rate, extensible and fully-automatic large-scale stem cell culture equipment, 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 automatic human-like large-scale stem cell culture equipment with low loss rate, which comprises a fixed table, wherein a culture module is arranged on the fixed table; the fixed table is provided with a sterile cover for covering the culture module, and a sterilizing device is arranged in the sterile cover; a control cabinet is arranged on one side of the fixed table; the device cabinet is arranged at the left side of the mixing module and is connected with the mixing module through an air pipe, a silicone tube, a data wire, a communication wire and the like; the device also comprises a plurality of circulation modules, a shaking module and a pressure regulating module; the circulation module is connected with the mixing module and the culture module, so that the culture medium rich in the mixing module flows into the culture module, and the culture medium lean in the culture module flows into the mixing module; the shaking module is connected with the culture module and used for driving the culture module to shake; the pressure regulating module is connected with the culture module and used for regulating the air pressure in the culture module.
In some possible embodiments of the application, the circulation module comprises 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; the circulation die hole is provided with two pairs of ports, the first peristaltic pump, the first one-way valve and the first pinch valve are sequentially connected in series between one pair of ports, the second peristaltic pump, the second one-way valve and the second pinch valve are sequentially connected in series between the other pair of ports, one port of each pair of ports is connected with the culture module, and the other port of each pair of ports is connected with the mixing module.
In some possible embodiments of the application, the pressure regulating module comprises a first air tube, a second bacterial filter, a third bacterial filter, a pressure sensor, a pressure regulating proportional valve; the second air pipe, the third bacterial filter, the pressure sensor and one port of the pressure regulating proportional valve are sequentially connected, the first air pipe, the second bacterial filter and the other port of the pressure regulating proportional valve are sequentially connected, and the first air pipe and the second air pipe are respectively connected with the culture module.
In some possible embodiments of the application, the culture module comprises a culture table fixedly mounted on a fixed table and a plurality of independently operable cell culture tanks detachably mounted inside the culture table; the shaking module comprises a plurality of shaking tables; the shaking tables are fixed at the bottom end inside the culture table; a flexible semi-wrapped heater is detachably arranged on the table top of the cradle; the culture tank of the culture module is detachably arranged in the flexible semi-wrapped heater, and the culture tank of the culture module and the flexible semi-wrapped heater can move along with the movement of the table top of the shaking table.
In some possible embodiments of the application, the cell culture tank is internally provided with a sensor group and a cell screen; a communicating pipe is fixed on the tank cover of the cell culture tank, one end of the communicating pipe is positioned outside the cell culture tank, the other end of the communicating pipe is positioned in the cell culture tank, the communicating pipe comprises a long pipe A, a short pipe A and a sampling pipe, and one end of the sampling pipe is positioned in the cell culture tank; the cell screen is fixed on the periphery of the long tube A and is assembled with the long tube A coaxially, and a space is reserved between the bottom of the cell screen and the bottom end of the long tube A; the sensor group is a non-contact measuring device and is fixedly arranged in an independent small chamber in the cell culture tank.
In some possible embodiments of the application, the mixing module comprises a mixing tank, a flexible surrounding heater, a first stirring paddle, a second stirring paddle, a first connecting rod, a second connecting rod, a first stirring motor, a second stirring motor, an aeration disc, a first bacterial filter, a long tube group, a short tube group, a sample injection tube, and a liquid discarding tube;
The first bacterial filter is connected with the aeration disc;
A central controller and a multi-parameter transmitter are arranged in the control cabinet; the sensor group is connected with the multi-parameter transmitter through a signal line and a communication line, can transmit information to the multi-parameter transmitter and receives the regulation and control of the multi-parameter transmitter;
A gas introduction module, a sample processing module and a waste liquid module are arranged in the device cabinet; the gas introducing module comprises an air flow regulator, a nitrogen flow regulator, a carbon dioxide flow regulator, an oxygen flow regulator and a four-mixing-one air inlet valve;
One end of the air flow regulator, one end of the nitrogen flow regulator, one end of the carbon dioxide flow regulator and one end of the oxygen flow regulator are respectively connected with one end of the four-mixing air inlet valve, and the other end of the four-mixing air inlet valve is connected with the first bacterial filter;
the sample processing module comprises an automatic sampler, a cell analyzer, an automatic sampler and a reagent rack;
The cell analyzer is connected with the automatic sampler, and the automatic sampler is connected with the sampling tube; the reagent rack is connected with the automatic sample injector, and the automatic sample injector is connected with the sample injection pipe;
the waste liquid module comprises a waste liquid barrel and a waste liquid pump;
the waste liquid barrel is connected with the liquid discarding pump, and the liquid discarding pump is connected with the liquid discarding pipe.
In some possible embodiments of the application, the flexible circumferential heater is circumferentially fixed around the mixing tank, so that the temperature in the mixing tank is kept constant; a long pipe clamp and a short pipe clamp are fixed on the tank cover of the mixing tank; a plurality of long pipes are detachably arranged in the long pipe clamp to form a long pipe group together and used for connecting the circulation module to guide the culture medium out of the mixing tank, and a plurality of short pipes are detachably arranged in the short pipe clamp to form a short pipe group together and used for connecting the circulation module to guide the culture medium into the mixing tank; the left side of the top of the mixing tank is detachably provided with a first stirring motor, one end of a first connecting rod is connected with the power output end of the first stirring motor, the other end of the first connecting rod is connected with a first stirring paddle, and the first stirring paddle is positioned at a position, close to the left side of the aeration disc, in the mixing tank; the right side of the top cover of the mixing tank is detachably provided with a second stirring motor, one end of the second connecting rod is connected with the power output end of the second stirring motor, the other end of the second connecting rod is connected with a second stirring paddle, and the second stirring paddle is positioned in the mixing tank and close to the right side of the aeration disc; the aeration disc is positioned in the mixing tank and close to the bottom of the mixing tank, and is connected with one end of the first bacterial filter through a silica gel tube.
In some possible embodiments of the application, the circulation module comprises a first circulation unit and a second circulation unit, one end of the first circulation unit is connected with the long tube a in the cell culture tank, and the other end is connected with the short tube in the short tube group in the mixing tank; one end of the second circulation unit is connected with a long pipe in the long pipe group in the mixing tank, and the other end of the second circulation unit is connected with a short pipe A in the cell culture tank; the first circulation unit comprises a first peristaltic pump, a first check valve and a first pinch valve, one end of the first peristaltic pump is connected with the long pipe A, the other end of the first peristaltic pump is communicated with the first check valve, the first check valve is connected with the first pinch valve, and one end of the first pinch valve, which is not connected with the first check valve, is connected with the short pipe of the short pipe group; the second circulation unit comprises a second peristaltic pump, a second one-way valve and a second pinch valve, one end of the second peristaltic pump is connected with the short pipe A, the other end of the second peristaltic pump is communicated with the second one-way valve, the second one-way valve is connected with the second pinch valve, and one end of the second pinch valve, which is not connected with the second one-way valve, is connected with the long pipes in the long pipe group.
In some possible embodiments of the present application, a four-mixing air inlet valve in the gas introducing module is connected with one end of the first bacterial filter through a silicone tube, the other end of the four-mixing air inlet valve is connected with an air flow regulator, a nitrogen flow regulator, a carbon dioxide flow regulator and an oxygen flow regulator, and the other ends of the air flow regulator, the nitrogen flow regulator, the carbon dioxide flow regulator and the oxygen flow regulator are connected with corresponding gas cylinders through gas pipes;
The sample outlet of the automatic sampler in the sample processing module is connected with a detection cell in the cell analyzer through a sample conveying pipe, so that a sample taken out of the automatic sampler can be directly conveyed into the cell analyzer for observation and detection; the sampling port of the automatic sampler is connected with a sampling tube on the tank cover of the cell culture tank through a silica gel tube; the reagent rack is arranged in the device cabinet and is used for placing reagent bottles so as to facilitate sample injection of the automatic sample injector; the automatic sample injector is connected with a sample injection pipe on the tank cover of the mixing tank through a silica gel pipe, and new reagent is added into the mixing tank;
The waste liquid module is characterized in that one end of the waste liquid pump is connected with the waste liquid pipe on the tank cover of the mixing tank through the silica gel pipe, and the other end of the waste liquid pump, which is not connected with the waste liquid pipe, is connected with the silica gel pipe and is directly inserted into the waste liquid barrel.
In some possible embodiments of the application, the sensor set comprises a temperature sensor, a dissolved oxygen concentration sensor, a pH sensor, and a carbon dioxide concentration sensor; and/or the first bacterial filter, the second bacterial filter and the third bacterial filter are used for bidirectionally filtering fine impurities and bacteria in the gas; and/or the cell culture tank of the culture module and the mixing tank in the mixing module are both airtight pressure-resistant containers.
The invention has the following advantages:
(1) The invention 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 a shaking table, 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 replaced through the circulation system, 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 invention realizes the scalable large-scale cell culture based on low loss rate of cells. The existing method for expanding the culture scale of stem cells mainly comprises the steps of increasing the number of culture dishes or increasing the culture volume of a single pot, wherein the cells can only grow in an adherence way, the number of the cells is far lower than the number of the cells attached to microcarriers to proliferate in a cell culture pot, and the problem of uneven distribution of nutrients in the pot exists in the latter, and if mixed power sources such as stirring, spraying and the like are added, the damage of shearing force to the cells/the cell microcarriers is increased, the cells die, and the loss rate in the cell culture process is increased. The invention is based on the method of the invention (1), a plurality of cell culture tanks are added in the culture module, each culture tank can independently work and operate, and can be combined with the mixing module to perform cell culture, and the number of the cell culture tanks is selected to realize the expandable large-scale cell culture.
(3) The invention 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.
(4) The invention realizes the automatic cell culture based on the invention points (1), (2) and (3). 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 showing the structure of an automatic human-like large-scale stem cell culturing apparatus with a low loss rate according to an exemplary embodiment of the present application.
Fig. 2 is a schematic diagram showing the connection structure of each module of a low-loss-rate human-like large-scale stem cell automatic culture apparatus according to an exemplary embodiment of the present application.
FIG. 3 is a schematic diagram showing the structure of a culture module according to an exemplary embodiment of the present application.
FIG. 4 is a schematic diagram showing the structure of the connection of the culture module, the pressure regulating module and the sample processing module according to an exemplary embodiment of the present application.
FIG. 5 shows a schematic diagram of the connection of a culture module, a circulation module and a mixing module according to an exemplary embodiment of the present application.
Fig. 6 is a schematic diagram showing the structure of the connection of the mixing module, the gas introduction module, and the waste liquid module according to an exemplary embodiment of the present application.
Fig. 7 illustrates a schematic diagram of the structure of a long tube and a cell screen provided in an exemplary embodiment of the present application.
The reference numerals in the drawings:
1. A control cabinet; 2. a culture module; 3. a mixing module; 4. a circulation module; 5. an apparatus cabinet; 6. an aseptic cover; 7. a pressure regulating module; 8. a fixed table; 1.1, a central controller; 1.2, a multi-parameter transmitter; 2.1, a cell culture tank; 2.2, a culture table; 2.1.1, a flexible semi-wrap heater; 2.1.2, sensor group; 2.1.3, shaking table; 2.1.4, cell screen; 2.1.5, long tube A;2.1.6, short tube A;2.1.7, sampling tube; 3.1, a mixing tank; 3.2, a flexible surrounding type heater; 3.3, a first stirring paddle; 3.4, a first connecting rod; 3.5, a first stirring motor; 3.6, an aeration disc; 3.7, a first bacterial filter; 3.8, a long tube group; 3.8.1, long tube clamp; 3.9, short tube group; 3.9.1 short pipe clamp; 3.10, a sample injection tube; 3.11, a liquid discarding pipe; 3.12, a second stirring paddle; 3.13, a second connecting rod; 3.14 a second stirring motor; 4.1, a first peristaltic pump; 4.2, a first one-way valve; 4.3, a first pinch valve; 4.4, a second peristaltic pump; 4.5, a second one-way valve; 4.6, a second pinch valve; 5.1, a gas introduction module; 5.2, a sample processing module; 5.3, a waste liquid module; 5.1.1 air flow regulator; 5.1.2, a nitrogen flow regulator; 5.1.3, a carbon dioxide flow regulator; 5.1.4, an oxygen flow regulator; 5.1.5, four-mixing one air inlet valve; 5.2.1, an automatic sampler; 5.2.2, cell analyzer; 5.2.3, an autosampler; 5.5.4, reagent rack; 5.3.1, a waste liquid barrel; 5.3.2, a liquid discarding pump; 7.1, a first air pipe; 7.2, a second air pipe; 7.3, a second bacterial filter; 7.4, a third bacterial filter; 7.5, a pressure sensor; and 7.6, regulating the pressure ratio valve.
Detailed Description
Various exemplary embodiments, features and aspects of the application will be described in detail below with reference to the drawings. In the drawings, like reference numbers indicate identical or functionally similar elements. Although various aspects of the embodiments are illustrated in the accompanying drawings, the drawings are not necessarily drawn to scale unless specifically indicated. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. In addition, for the purposes of better illustrating the application, it will be apparent to one skilled in the art that numerous specific details are set forth in the various embodiments that follow. The application may be practiced without some of these specific details. In some embodiments, methods, means and elements well known to those skilled in the art have not been described in detail in order to highlight the gist of the present application.
Referring to fig. 1 to 6, the embodiment of the application provides an automatic culture device for human-like large-scale stem cells with low loss rate, which comprises a fixed table 8; the culture module 2 is arranged on the fixed table 8; the fixed table 8 is provided with a sterile cover 6 for covering the culture module 2, and a sterilizing device is arranged in the sterile cover 6; a control cabinet 1 is arranged on one side of the fixed table 8; the mixing module 3 and the device cabinet 5 are fixedly arranged in the fixed table 8, wherein the device cabinet 5 is arranged at the left side of the mixing module 3 and is connected with the mixing module through an air pipe, a silicone tube, a data wire, a communication wire and the like; the device also comprises a plurality of circulation modules 4, a shaking module and a pressure regulating module 7; the circulation module 4 is connected with the mixing module 3 and the culture module 2, so that the culture medium rich in the mixing module 3 flows into the culture module 2, and the culture medium lean in the culture module 2 flows into the mixing module 3; the shaking module is connected with the culture module 2 and used for driving the culture module 2 to shake; the pressure regulating module 7 is connected with the culture module 2 and is used for regulating the air pressure in the culture module 2.
In some exemplary implementations of the present example, the circulation module 4 includes a first peristaltic pump 4.1, a second peristaltic pump 4.4, a first one-way valve 4.2, a second one-way valve 4.5, a first pinch valve 4.3, a second pinch valve 4.6; the circulation module 4 is provided with two pairs of ports, the first peristaltic pump 4.1, the first one-way valve 4.2 and the first pinch valve 4.3 are sequentially connected in series between one pair of ports, the second peristaltic pump 4.4, the second one-way valve 4.5 and the second pinch valve 4.6 are sequentially connected in series between the other pair of ports, one port of each pair of ports is connected with the culture module 2, and the other port of each pair of ports is connected with the mixing module 3.
In some exemplary implementations of the present example, the pressure regulation module 7 includes a first air tube 7.1, a second air tube 7.2, a second bacterial filter 7.3, a third bacterial filter 7.4, a pressure sensor 7.5, a pressure regulating proportional valve 7.6; one port of the second air pipe 7.2, the third bacterial filter 7.4, the pressure sensor 7.5 and the pressure regulating proportional valve 7.6 is sequentially connected, the other port of the first air pipe 7.1, the second bacterial filter 7.3 and the pressure regulating proportional valve 7.6 is sequentially connected, and the first air pipe 7.1 and the second air pipe 7.2 are respectively connected with the culture module 2; the pressure regulating proportional valve 7.6 controls the ratio of exhaust gas and return gas to maintain the pressure in the cell culture tank stable.
In some exemplary implementations of the present example, the culture module 2 comprises a culture table 2.2 fixedly mounted on a fixed table 8 and a number of independently operable cell culture tanks 2.1 detachably mounted inside the culture table 2.2; the shaking module comprises a plurality of shaking tables 2.1.3; the shaking tables 2.1.3 are fixed at the bottom end inside the culture table 2.2; the table top of the table bed 2.1.3 is detachably provided with a flexible semi-wrapped heater 2.1.1; the culture tank of the culture module 2 is detachably arranged in the flexible semi-wrapped heater 2.1.1, and the culture tank of the culture module 2 and the flexible semi-wrapped heater 2.1.1 can move along with the table top movement of the table top 2.1.3; the function of the flexible semi-wrapped heater 2.1.1 is to keep the temperature in the cell culture tank constant.
In some exemplary implementations of the present example, the cell culture tank 2.1 is internally provided with a sensor set 2.1.2 and a cell screen 2.1.4; a communicating pipe is fixed on the cover of the cell culture tank 2.1, one end of the communicating pipe is positioned outside the cell culture tank 2.1, the other end of the communicating pipe is positioned in the cell culture tank 2.1, the communicating pipe comprises a long pipe A2.1.5, a short pipe A2.1.6 and a sampling pipe 2.1.7, and one end of the sampling pipe 2.1.7 is positioned in the cell culture tank 2.1; the cell screen 2.1.4 is fixed on the periphery of the long tube A2.1.5, is coaxially assembled with the long tube A2.1.5, and has a bottom spaced from the bottom end of the long tube A2.1.5, and is of a barrel-shaped reticular structure for blocking the flow of the cell/cell microcarrier mixture in the cell culture tank along with the culture medium when the circulation system is started; the sensor group 2.1.2 is a non-contact measuring device and is fixedly arranged in a separate small chamber in the cell culture tank 2.1.
In some exemplary implementations of the present example, the mixing module 3 includes a mixing tank 3.1, a flexible circumferential heater 3.2, a first stirring paddle 3.3, a second stirring paddle 3.12, a first connecting rod 3.4, a second connecting rod 3.13, a first stirring motor 3.5, a second stirring motor 3.14, an aeration disc 3.6, a first bacterial filter 3.7, a long tube set 3.8, a short tube set 3.9, a sample injection tube 3.10, and a reject tube 3.11; the first bacterial filter 3.7 is connected with the aeration disc 3.6;
In some exemplary implementations of the present embodiment, the control cabinet 1 is provided with a central controller 1.1 and a multi-parameter transmitter 1.2; the central controller 1.1 has the functions of signal acquisition, signal processing, data storage and the like; the sensor group 2.1.2 is connected with the multi-parameter transmitter 1.2 through a signal line and a communication line, can transmit information to the multi-parameter transmitter 1.2, and receives the regulation and control of the multi-parameter transmitter 1.2;
In some exemplary implementations of the present example, the apparatus cabinet 5 has a gas introduction module 5.1, a sample processing module 5.2, and a waste liquid module 5.3 installed therein; wherein the gas introducing module 5.1 comprises an air flow regulator 5.1.1, a nitrogen flow regulator 5.1.2, a carbon dioxide flow regulator 5.1.3, an oxygen flow regulator 5.1.4 and a four-mixing air inlet valve 5.1.5; one end of the air flow regulator 5.1.1, the nitrogen flow regulator 5.1.2, the carbon dioxide flow regulator 5.1.3 and the oxygen flow regulator 5.1.4 are respectively connected with one end of the four-mixing air inlet valve 5.1.5, and the other end of the four-mixing air inlet valve 5.1.5 is connected with the first bacterial filter 3.7; the four gases are mixed in advance through the four-mixing-one air inlet valve 5.1.5, then impurities are filtered through the first bacterial filter 3.7, and finally the mixture is introduced into the mixing tank 3.1 through the aeration disc 3.6;
in some exemplary implementations of the present example, the sample processing module 5.2 includes an autosampler 5.2.1, a cell analyzer 5.2.2, an autosampler 5.2.3, a reagent rack 5.2.4; the cell analyzer 5.2.2 is connected with the automatic sampler 5.2.1, and the automatic sampler 5.2.1 is connected with the sampling tube 2.1.7; the reagent rack 5.2.4 is connected with the automatic sampler 5.2.3, and the automatic sampler 5.2.3 is connected with the sampling tube 3.10;
in some exemplary implementations of the present example, the waste module 5.3 includes a waste tank 5.3.1 and a waste pump 5.3.2; the waste liquid barrel 5.3.1 is connected with the waste liquid pump 5.3.2, and the waste liquid pump 5.3.2 is connected with the waste liquid pipe 3.11.
In some exemplary implementations of the present example, the flexible circumferential heater 3.2 is secured circumferentially around the mixing tank 3.1, keeping the temperature within the mixing tank 3.1 constant; a long pipe clamp 3.8.1 and a short pipe clamp 3.9.1 are fixed on the cover of the mixing tank 3.1; a plurality of long pipes are detachably arranged in the long pipe clamp 3.8.1 to form a long pipe group 3.8 together, the long pipe clamp is used for connecting the circulation module 4 to guide the culture medium out of the mixing tank 3.1, a plurality of short pipes are detachably arranged in the short pipe clamp 3.9.1 to form a short pipe group 3.9 together, and the short pipe clamp is used for connecting the circulation module 4 to guide the culture medium into the mixing tank 3.1; the left side of the top of the mixing tank 3.1 is detachably provided with a first stirring motor 3.5, one end of a first connecting rod 3.4 is connected with the power output end of the first stirring motor 3.5, the other end of the first connecting rod is connected with a first stirring paddle 3.3, and the first stirring paddle 3.3 is positioned at a position, close to the left side of an aeration disc 3.6, in the mixing tank 3.1; a second stirring motor 3.14 is detachably arranged on the right side of the top of the cover of the mixing tank 3.1, one end of a second connecting rod 3.13 is connected with the power output end of the second stirring motor 3.14, the other end of the second connecting rod is connected with a second stirring paddle 3.12, and the second stirring paddle 3.12 is positioned at a position, close to the right side of the aeration disc 3.6, in the mixing tank 3.1; the aeration disc 3.6 is positioned in the mixing tank 3.1 and is close to the bottom of the mixing tank 3.1, and one end of the aeration disc is connected with one end of the first bacterial filter 3.7 through a silica gel pipe; the function of the first stirring paddles 3.3 and the second stirring paddles 3.12 is to quickly and evenly mix all the substances in the mixing tank through high-speed rotation.
In some exemplary implementations of the present example, the circulation module 4 comprises a first circulation unit and a second circulation unit, one end of the first circulation unit being connected to the long tube A2.1.5 within the cell culture tank 2.1 and the other end being connected to a short tube in the short tube set 3.9 within the mixing tank 3.1; one end of the second circulation unit is connected with a long pipe in the long pipe group 3.8 in the mixing tank 3.1, and the other end of the second circulation unit is connected with a short pipe A2.1.6 in the cell culture tank 2.1; the first circulation unit comprises a first peristaltic pump 4.1, a first check valve 4.2 and a first pinch valve 4.3, one end of the first peristaltic pump 4.1 is connected with a long pipe A2.1.5, the other end of the first peristaltic pump is communicated with the first check valve 4.2, the first check valve 4.2 is connected with the first pinch valve 4.3, and one end of the first pinch valve 4.3 which is not connected with the first check valve 4.2 is connected with short pipes of the short pipe group 3.9; the second circulation unit comprises a second peristaltic pump 4.4, a second check valve 4.5 and a second pinch valve 4.6, one end of the second peristaltic pump 4.4 is connected with a short pipe A2.1.6, the other end of the second peristaltic pump is communicated with the second check valve 4.5, the second check valve 4.5 is connected with the second pinch valve 4.6, and one end of the second pinch valve 4.6, which is not connected with the second check valve 4.5, is connected with long pipes in the long pipe group 3.8.
In some exemplary embodiments of the present embodiment, the air intake valve 5.1.5 of the air intake module 5.1 is connected to one end of the first bacterial filter 3.7 through a silicone tube, the other end of the air intake valve 5.1.5 is connected to the air flow regulator 5.1.1, the nitrogen flow regulator 5.1.2, the carbon dioxide flow regulator 5.1.3, the oxygen flow regulator 5.1.4, and the other ends of the air flow regulator 5.1.1, the nitrogen flow regulator 5.1.2, the carbon dioxide flow regulator 5.1.3, and the oxygen flow regulator 5.1.4 are connected to corresponding air cylinders through air tubes;
In some exemplary implementations of the present examples, the sample outlet of the autosampler 5.2.1 in the sample processing module 5.2 is connected to the detection chamber in the cell analyzer 5.2.2 through a sample delivery tube, and the sample taken out of the autosampler 5.2.1 can be directly delivered to the cell analyzer 5.2.2 for observation and detection; the sampling port of the automatic sampler 5.2.1 is connected with a sampling tube 2.1.7 on the tank cover of the cell culture tank 2.1 through a silica gel tube; the reagent rack 5.2.4 is installed in the device cabinet 5, and the reagent rack 5.2.4 is used for placing reagent bottles so as to facilitate sample injection of the automatic sample injector 5.2.3; the automatic sampler 5.2.3 is connected with a sample injection pipe 3.10 on the tank cover of the mixing tank 3.1 through a silica gel pipe, and new reagent is added into the mixing tank 3.1;
In some exemplary implementations of this embodiment, one end of the waste liquid pump 5.3.2 in the waste liquid module 5.3 is connected to the waste liquid pipe 3.11 on the tank cover of the mixing tank 3.1 through a silicone pipe, the other end of the waste liquid pump 5.3.2, which is not connected to the waste liquid pipe 3.11, is connected to the silicone pipe, and the silicone pipe is directly inserted into the waste liquid tank 5.3.1.
In some exemplary implementations of the present example, the sensor set 2.1.2 includes a temperature sensor, a dissolved oxygen concentration sensor, a pH sensor, and a carbon dioxide concentration sensor; and/or the first bacterial filter 3.7, the second bacterial filter 7.3 and the third bacterial filter 7.4 are used for bidirectionally filtering fine impurities and bacteria in the gas; and/or, the cell culture tank 2.1 of the culture module 2 and the mixing tank 3.1 of the mixing module 3 are both closed pressure-resistant containers.
When the automatic human-simulated large-scale stem cell culture equipment with low loss rate is used, the culture medium, gases such as oxygen and the like and nutrient substances are uniformly mixed in the mixing module 3, and enter the cell culture tank 2.1 through the circulating module 4 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 2.1, so that the cell culture tank 2.1 does not need to be provided with a module with functions of stirring, aeration and the like, the shearing force damage of the cells caused by stirring blades 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.
In some exemplary implementations of the present example, the central controller 1.1 controls the air, carbon dioxide, oxygen and nitrogen rates, time and the rate of introduction into the mixing tank 3.1 via the gas introduction module 5.1; the central controller 1.1 controls the pressure in the cell culture tank 2.1 through the pressure regulating proportional valve 7.6; the central controller 1.1 controls the heating temperatures of the flexible semi-wrapped heater 2.1.1 and the flexible surrounding heater 3.2 through the feedback information of the multi-parameter transmitter 1.2, so that the internal temperatures of the culture module 2 and the mixing module 3 are stable; the central controller 1.1 judges the culture stage of the cells according to the information obtained by the cell analyzer 5.2.2 in the sample processing module 5.2, adjusts the rotation speeds of two stirring paddles in the mixing tank 3.1 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 air flow regulator 5.1.1, the nitrogen flow regulator 5.1.2, the carbon dioxide flow regulator 5.1.3 and the oxygen flow regulator 5.1.4 in the air introducing module 5.1; when the central controller 1.1 determines that the cells are in the designated culture stage, the automatic sampler 5.2.3 is controlled to add the cytokines in the reagent bottles in the reagent racks 5.2.4 to the mixing tank 3.1 and to the cell culture tank 2.1 through the circulation system 4 for cell growth requirements.
In some exemplary implementations of the present example, the pH, dissolved oxygen concentration and pressure control of the culture environment is mainly achieved by means of the regulation of the pressure regulation module 7 and the gas introduction module 5.1 by the central controller 1.1; firstly, taking a target set value into an internal model, calculating mass proportion coefficients of carbon dioxide, oxygen, nitrogen and air which need to be introduced into a cell culture tank 2.1, starting a gas introduction module 5.1 by a central controller 1.1 to continuously introduce mixed gas, starting a pressure regulation module 7 by the central controller 1.1, and starting stable regulation of the pressure in the cell culture tank 2.1; when the sensor group detects that the pH value and/or the value in the dissolved oxygen concentration change, the central controller 1.1 calculates the required introducing proportion of the four gases according to the model, controls the flow regulators of the four gases in the gas introducing module 5.1, continuously introducing the mixed gases into the cell culture tank 2.1 after changing the introducing proportion of the four mixed gases until the value detected by the sensor returns to the set range; in the process of continuously feeding air into the air introducing module 5.1, the pressure regulating module 7 needs to adjust the opening degrees of the air inlet port, the air return port and the air exhaust port of the pressure regulating proportional valve 7.6 at any time according to the detection value fed back by the pressure sensor 7.5 so as to keep the pressure in the cell culture tank 2.1 stable.
The application of the low-loss-rate human-like large-scale stem cell automatic culture equipment in the embodiment is as follows:
Step ①, starting the equipment, starting the semi-wrapped heater 2.1.1 and the flexible surrounding heater 3.2 by the central controller 1.1, and regulating the temperature in each cell culture tank 2.1 and the mixing tank 3.1 to be constant at 37.2 ℃;
Step ② the central controller 1.1 controls the autosampler 5.2.3 to add fresh culture medium above the reagent shelf 5.2.4 into the mixing tank 3.1;
Step ③, the central controller 1.1 starts the gas introducing module 5.1, controls the opening and the introducing time of the air flow regulator 5.1.1, the nitrogen flow regulator 5.1.2, the carbon dioxide flow regulator 5.1.3 and the oxygen flow regulator 5.1.4 according to the environmental index input by the user;
Step ④, setting the rotating speed, and starting the first stirring motor 3.5 and the second stirring motor 3.14 by the central controller 1.1 to drive the first stirring paddle 3.3 and the second stirring paddle 3.12 to rotate at a high speed;
step ⑤ the central controller 1.1 starts the pressure regulating module 77, and the central controller 1.1 controls the discharge amount of the mixed gas in the pressure regulating proportional valve 7.6 through the feedback value of the pressure sensor 7.5 to regulate the pressure value and the change period in the cell culture tank 2.1;
Step ⑥, adding a cell/cell-microcarrier compound into the middle area between the inner wall of the cell culture tank 2.1 and the cell screen 2.1.4, and starting the shaking table 2.1.3 by the central controller 1.1 to input a set rotating speed so as to uniformly distribute the cell/cell-microcarrier in the area;
Step ⑦ the central controller 1.1 starts the circulation module 4 to circulate the culture medium between the mixing module 3 and the cell culture tank 2.1 in the culture module 2, and the central controller 1.1 regulates the rotation speed of the first peristaltic pump 4.1 and the second peristaltic pump 4.4 in the circulation module 4.1 to control the exchange speed of the culture medium in the cell culture tank 2.1 in the mixing module 3 and the culture module 2;
Step ⑧, setting sampling frequency and sampling amount, and controlling an automatic sampler 5.2.1 by a central controller 1.1 to collect cells in a cell culture tank 2.1 and culture mediums around the cells, and sending the collected samples to a cell analyzer 5.2.2 through a connected sample conveying pipe, wherein the cell analyzer 5.2.2 detects cell morphology, number/proportion of living cells and glucose/urea/lactic acid/inorganic salt content;
Step ⑨, the central controller 1.1 judges the culture stage of the cells according to the information obtained by the cell analyzer 5.2.2, adjusts the rotation speeds of two stirring paddles in the mixing tank 3.1 according to the environmental information required by the cells at the stage in a preset program, and adjusts the opening and the introducing time of the air flow regulator 5.1.1, the nitrogen flow regulator 5.1.2, the carbon dioxide flow regulator 5.1.3 and the oxygen flow regulator 5.1.4 in the air introducing module 5.1; when the central controller 1.1 judges that the cells are in a designated culture stage, controlling the automatic sampler 5.2.3 to add the cytokines in the reagent bottles in the reagent racks 5.2.4 into the mixing tank 3.1, and adding the cytokines into the cell culture tank 2.1 through the circulating system 4 for cell growth requirements;
Step ⑩, monitoring the temperature, the dissolved oxygen concentration, the pH value and the carbon dioxide concentration in the cell culture tank 2.1 in real time by the sensor group 2.1.2 in the whole culture process, transmitting signals to the multi-parameter transmitter 1.2, and transmitting collected information to the central controller 1.1 by the multi-parameter transmitter 1.2 for checking, processing and storing; the pressure sensor 7.5 monitors the pressure in the cell culture tank 2.1 in real time in the whole culture process, and transmits information to the central controller 1.1 for checking, processing and storage; the pH, dissolved oxygen concentration and pressure control of the culture environment are realized by means of the regulation and control of the pressure regulating module 7 and the gas introducing module 5.1 by the central controller 1.1; after the cell culture is completed, the central controller 1.1 starts the liquid discarding pump 5.3.2 in the waste liquid module 5.3, and extracts the culture medium from the mixing tank 3.1 to the waste liquid barrel 5.3.1 through the liquid discarding pipe 3.11, and discards the culture medium after treatment.
The foregoing description of embodiments of the application has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the improvement of technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.
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| CN108384714A (en) * | 2018-04-23 | 2018-08-10 | 苏州欧飞纳米科技有限公司 | A kind of gas-dynamic control system of biological cell reactor |
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