CN103589808A - Automatic control method and system for pH/oxygen partial pressure/partial pressure of carbon dioxide of cell and tissue culture solution - Google Patents
Automatic control method and system for pH/oxygen partial pressure/partial pressure of carbon dioxide of cell and tissue culture solution Download PDFInfo
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Abstract
本发明提供一种细胞和组织培养液的pH/氧分压/二氧化碳分压的自动控制方法和系统，该系统包括培养室，还包括中央控制单元、物质交换器和氮气、氧气、二氧化碳气源，所述培养室与物质交换器之间设有循环培养液管道，所述培养室的上游端与物质交换器的循环培养液出口之间的上游循环培养液管道上设有生化传感器，所述培养室的下游端与物质交换器的循环培养液入口之间的下游循环培养液管道上设有第一液流驱动装置，所述氮气、氧气、二氧化碳气源分别经电磁阀与物质交换器的气体入口通过气体管道连接，所述生化传感器、第一液流驱动装置、电磁阀分别与中央控制单元电连接。本发明的方法和系统在全封闭的情况下实现了组织的长期培养。
The invention provides a method and system for automatic control of pH/oxygen partial pressure/carbon dioxide partial pressure of cell and tissue culture fluid. The system includes a culture room, a central control unit, a material exchanger, and nitrogen, oxygen, and carbon dioxide gas sources , a circulating culture solution pipeline is provided between the culture chamber and the material exchanger, a biochemical sensor is provided on the upstream circulation culture fluid pipeline between the upstream end of the culture chamber and the circulation culture solution outlet of the material exchanger, and the The downstream circulation culture fluid pipeline between the downstream end of the cultivation chamber and the circulation culture fluid inlet of the material exchanger is provided with a first liquid flow driving device, and the nitrogen, oxygen, and carbon dioxide gas sources are passed through the electromagnetic valve and the material exchanger respectively. The gas inlet is connected through a gas pipeline, and the biochemical sensor, the first liquid flow driving device, and the solenoid valve are respectively electrically connected to the central control unit. The method and system of the present invention realize the long-term culture of tissue under fully enclosed conditions.
Description
Technical field
The present invention relates to biomedical engineering cell and tissue cultivating field, particularly a kind of autocontrol method and system of pH/ oxygen partial pressure/partial pressure of carbon dioxide of cell and tissue culture liquid.
Background technology
In vitro culture environment, cell and be organized in process of growth and constantly consume O
2, produce CO
2and accumulate in nutrient solution, nutrient solution pH is constantly declined.
Maintaining the ordinary method that pH is stable is to abandon old nutrient solution then to add new nutrient solution, is called and changes liquid.Change this mode of liquid and be in pH value and drop to when quite low and carry out, cause the larger fluctuation of biochemical environment, frequently changing liquid simultaneously will increase the risk of polluting greatly.
The oxygen partial pressure of maintain liquid/partial pressure of carbon dioxide needs sufficient gaseous interchange, the i.e. supply of oxygen and the discharge of carbonic acid gas.Ordinary method mainly contains: one, in carbonic acid gas incubator, by the ventilating pit on nutrient solution container, in open mode, realize passive gaseous interchange, its exchange efficiency is low, can not implement on demand regulation and control; Two, in nutrient solution, insert oxygen hose, the mouth of pipe is installed micropore gas injector, and directly to direct oxygen injection in nutrient solution, produce bubble and contact oxygenation with nutrient solution, the easy breed bacteria of this method, the bubble of generation also easily destroys cultured cells.
In addition, some cells need special culture condition with organizing, and for example under the condition of hypoxemia, the growth of cartilaginous tissue is better, and inoblast needs the environment of meta-alkalescence, and the cell strain of cultured continuously is the environment that need slant acidity more.Although hypoxemia incubator can be realized a certain constant low oxygen partial pressure environment, can not control arbitrarily pH in a certain preset range, can not keep stable.
The vitro culture of tissue engineering tissue is a long-term process, needs several months even several weeks.In this culturing process, tissue growth is to different steps, and to biochemical environment need to be also different.The method that is most commonly used to change biochemical environment is to change liquid and be transferred to different incubators.The risk of this method except increase polluting, continuous process of growth that also can disrupt tissue.Therefore, cultural method and the system of the biochemical environment of a kind of totally-enclosed and automatic change is badly in need of in this area, can realize the long-term cultured continuously of tissue.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, a kind of autocontrol method of pH/ oxygen partial pressure/partial pressure of carbon dioxide of cell and tissue culture liquid is provided, present method can realize the sensitive scope that promptly pH of nutrient solution, oxygen partial pressure and partial pressure of carbon dioxide is controlled to appointment under totally enclosed culture condition, reduce biochemical environment and occur significantly fluctuation, avoid changing the Pollution risk that liquid produces.Meanwhile, present method can, in the situation that not interrupting culturing process, by changing preset control value scope, be controlled at different levels by pH, oxygen partial pressure and partial pressure of carbon dioxide, to meet the needs of tissue growth different times to the biochemical environment of difference.
The object of the invention is for the deficiencies in the prior art, a kind of automatic control system of pH/ oxygen partial pressure/partial pressure of carbon dioxide of cell and tissue culture liquid is provided, this system has two of fresh medium and the Cyclic culture liquid relatively independent recycle system separately, form gas/liquid and liquid/liquid integrated exchange, can realize does not open a position just can upgrade nutrient solution, can realize the long-term cultured continuously of tissue, can realize under totally enclosed culture condition sensitive promptly by the pH of nutrient solution, oxygen partial pressure and partial pressure of carbon dioxide are controlled at the scope of appointment, reduce biochemical environment and occur significantly fluctuation, avoid changing the Pollution risk that liquid produces.Meanwhile, native system can, in the situation that not interrupting culturing process, by changing preset control value scope, be controlled at different levels by pH, oxygen partial pressure and partial pressure of carbon dioxide, to meet the needs of tissue growth different times to the biochemical environment of difference.
The object of the present invention is achieved like this: a kind of autocontrol method of pH/ oxygen partial pressure/partial pressure of carbon dioxide of cell and tissue culture liquid, comprises the steps:
1) preset the controlling valu scope of pH, oxygen partial pressure and the partial pressure of carbon dioxide of nutrient solution;
2) nutrient solution recycle process in detect in real time pH, oxygen partial pressure and the partial pressure of carbon dioxide of nutrient solution;
3) by step 2) in the measured value of real-time detection pH, oxygen partial pressure and the partial pressure of carbon dioxide obtained and step 1) in default controlling valu scope compare, judge that measured value is whether within the scope of preset control value;
4) if measured value, not within the scope of preset control value, according to the situation of the pH of nutrient solution or oxygen partial pressure or partial pressure of carbon dioxide, is supplemented one or more in nitrogen, oxygen, carbonic acid gas to nutrient solution, carry out gaseous interchange;
5) treat that nutrient solution carries out gaseous interchange, the measured value of pH, oxygen partial pressure and partial pressure of carbon dioxide reaches after preset control value scope, stops to nutrient solution make-up gas.
If measured value is not within the scope of preset control value described in step 4), according to the situation of the pH of nutrient solution or oxygen partial pressure or partial pressure of carbon dioxide, when an index wherein, not within the scope of preset control value, regulates this index, make this index return to preset control value scope; When wherein two or three indexs, not within the scope of preset control value, regulate partial pressure of carbon dioxide, oxygen partial pressure, the pH index of nutrient solution sequentially successively, make indices return to preset control value scope.
If measured value is not within the scope of preset control value, when the measured value of pH is prescribed a time limit lower than the lower of pH preset control value scope, to nutrient solution, supplement nitrogen and oxygen, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of pH reaches the decision-point within the scope of pH preset control value, stop to nutrient solution make-up gas; When the measured value of pH is prescribed a time limit higher than the upper of pH preset control value scope, to nutrient solution, supplement carbonic acid gas, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of pH reaches the decision-point within the scope of pH preset control value, stop to nutrient solution make-up gas.
Decision-point within the scope of described pH preset control value is the intermediate value of pH preset control value scope.
If measured value is not within the scope of preset control value, when the measured value of oxygen partial pressure is prescribed a time limit lower than the lower of oxygen partial pressure preset control value scope, to nutrient solution supplemental oxygen, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of oxygen partial pressure reaches the decision-point within the scope of oxygen partial pressure preset control value, stop to nutrient solution make-up gas; When the measured value of oxygen partial pressure is prescribed a time limit higher than the upper of oxygen partial pressure preset control value scope, to nutrient solution, supplement nitrogen, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of oxygen partial pressure reaches the decision-point within the scope of oxygen partial pressure preset control value, stop to nutrient solution make-up gas.
Decision-point within the scope of described oxygen partial pressure preset control value is the intermediate value of oxygen partial pressure preset control value scope.
If measured value is not within the scope of preset control value, when the measured value of partial pressure of carbon dioxide is prescribed a time limit lower than the lower of partial pressure of carbon dioxide preset control value scope, to nutrient solution, supplement carbonic acid gas, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of partial pressure of carbon dioxide reaches the decision-point within the scope of partial pressure of carbon dioxide preset control value, stop to nutrient solution make-up gas; When the measured value of partial pressure of carbon dioxide is prescribed a time limit higher than the upper of partial pressure of carbon dioxide preset control value scope, to nutrient solution, supplement nitrogen and oxygen, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of partial pressure of carbon dioxide reaches the decision-point within the scope of partial pressure of carbon dioxide preset control value, stop to nutrient solution make-up gas.
Decision-point within the scope of described partial pressure of carbon dioxide preset control value is the intermediate value of partial pressure of carbon dioxide preset control value scope.
A kind of automatic control system of pH/ oxygen partial pressure/partial pressure of carbon dioxide of cell and tissue culture liquid, comprise culturing room, also comprise central control unit, exchange of substance device and nitrogen, oxygen, carbon dioxide air source, between described culturing room and exchange of substance device, be provided with Cyclic culture liquid pipeline, upstream Cyclic culture liquid pipeline between the Cyclic culture liquid outlet of the upstream extremity of described culturing room and exchange of substance device is provided with biochemical sensor, downstream Cyclic culture liquid pipeline between the Cyclic culture liquid entrance of the downstream end of described culturing room and exchange of substance device is provided with the first liquid stream drive unit, described nitrogen, oxygen, carbon dioxide air source is connected by gas pipeline with the gas inlet of exchange of substance device through magnetic valve respectively, described biochemical sensor, the first liquid stream drive unit, magnetic valve is electrically connected to central control unit respectively.
Between described exchange of substance device and a fresh medium holder, be provided with fresh medium circulating line, the upstream extremity of described fresh medium holder is connected by upstream fresh medium pipeline with the fresh medium outlet of exchange of substance device, the downstream end of described fresh medium holder is connected by downstream fresh medium pipeline with the fresh medium entrance of exchange of substance device, this downstream fresh medium pipeline is provided with the second liquid stream drive unit, and described the second liquid stream drive unit is electrically connected to central control unit.
Between the gas inlet of described exchange of substance device and the downstream end of magnetic valve, be provided with the first strainer, the pneumatic outlet of described exchange of substance device is provided with check valve, between the upstream extremity of described check valve and the pneumatic outlet of exchange of substance device, is provided with the second strainer.
Owing to having adopted such scheme, between described culturing room and exchange of substance device, be provided with Cyclic culture liquid pipeline, downstream Cyclic culture liquid pipeline between the Cyclic culture liquid entrance of the downstream end of described culturing room and exchange of substance device is provided with the first liquid stream drive unit, make exchange of substance device form circulation passage with culturing room, allow the cultivation tissue that Cyclic culture liquid can circulate in ，Wei culturing room under the driving of the first liquid stream drive unit between exchange of substance Qi Yu culturing room that nutrition is provided.Upstream Cyclic culture liquid pipeline between the Cyclic culture liquid outlet of the upstream extremity of described culturing room and exchange of substance device is provided with biochemical sensor.Described nitrogen, oxygen, carbon dioxide air source are connected by gas pipeline with the gas inlet of exchange of substance device through magnetic valve respectively.Described biochemical sensor, the first liquid stream drive unit, magnetic valve are electrically connected to central control unit respectively.Described biochemical sensor can detect pH, oxygen partial pressure and the partial pressure of carbon dioxide of Cyclic culture liquid in real time, and the measured value that detects in real time pH, oxygen partial pressure and the partial pressure of carbon dioxide obtained is passed to central control unit.Described central control unit compares controlling valu scope default in the measured value of biochemical sensor detection is obtained in real time pH, oxygen partial pressure and partial pressure of carbon dioxide and central control unit, judge that measured value is whether within the scope of preset control value, if measured value is not within the scope of preset control value, according to the situation of the pH of nutrient solution or oxygen partial pressure or partial pressure of carbon dioxide, to nutrient solution, supplement one or more in nitrogen, oxygen, carbonic acid gas, carry out gaseous interchange.Described nitrogen, oxygen, carbon dioxide air source are connected by gas pipeline with the gas inlet of exchange of substance device through magnetic valve respectively, make one or more in nitrogen, oxygen, carbonic acid gas under the control of central control unit, pass into exchange of substance device and Cyclic culture liquid carries out gaseous interchange, thereby change pH, oxygen partial pressure and the partial pressure of carbon dioxide of nutrient solution.Due to for gas through and form a relatively independent gas passage with the biological semi-permeable membranes boundling of hydrophobic hollow fiber that Cyclic culture liquid carries out gas/liquid exchange, and liquid can not flow away from this passage, therefore make the chamber of housing form an enclosed space, and use first, second strainer can stop bacterium, fungi to enter exchange of substance device, be difficult for breed bacteria.And one or more in nitrogen, oxygen, carbonic acid gas can pass into exchange of substance device under the control of central control unit and Cyclic culture liquid carries out gaseous interchange, and its exchange efficiency is high, can implement on demand regulation and control.Treat that nutrient solution carries out gaseous interchange, the measured value of pH, oxygen partial pressure and partial pressure of carbon dioxide reaches after preset control value scope, controls magnetic valve stop to nutrient solution make-up gas by central control unit.Automatic control system of the present invention coordinates autocontrol method of the present invention can realize the sensitive scope that promptly pH of nutrient solution, oxygen partial pressure and partial pressure of carbon dioxide is controlled to appointment under totally enclosed culture condition, reduce biochemical environment and occur significantly fluctuation, avoid changing the Pollution risk that liquid produces.Simultaneously, automatic control system of the present invention coordinates the autocontrol method of the present invention can be in the situation that not interrupting culturing process, by changing the preset control value scope in central control unit, pH, oxygen partial pressure and partial pressure of carbon dioxide can be controlled at arbitrarily in a certain preset range, and keep stable, to meet the needs of tissue growth different times to the biochemical environment of difference, the long-term cultured continuously of favourable tissue.Automatic control system of the present invention coordinates autocontrol method of the present invention can make the preparation of tissue engineering tissue realize stdn and commercialization.
Between described exchange of substance device and a fresh medium holder, be provided with fresh medium circulating line.Because fresh medium and Cyclic culture liquid have the relatively independent separately recycle system, and can mutually exchange, therefore the circulation of nutrient solution drives the pulse mode that can adopt jerk pump simulation human heart to beat, and nutrient solution is circulated and provide nutrition for the tissue of cultivating.Automatic control system of the present invention forms gas/liquid and liquid/liquid integrated exchange, can realize does not open a position just can upgrade nutrient solution, and make oxygen and carbon dioxide, fresh medium, Cyclic culture liquid in interchanger, completes gas/liquid and the exchange of liquid/liquid is upgraded, and nutrient solution upgrades at enclosed space internal recycle and exchange, the liquid level of nutrient solution can not produce foam yet, avoid passing into the bubble destruction cultured cells of γ-ray emission, stop nutrient solution completely contaminated, can realize again the circulation of nutrient solution pulsed upgrades, thereby realize the pulsed bionic cultivation of the blood circulation of human body of real meaning.Because nutrient solution upgrades at enclosed space internal recycle and exchange, in tissue cultivating process, can not discharge nutrient solution, nutrient solution total amount can not reduce, therefore with the fresh medium of small amount, just can complete the tissue engineering tissue cultivating process of long period, both reduced the consumption of fresh medium, reduce costs, make again tissue engineering tissue cultivate and possessed the condition that industrialization is produced.This automatic control system, as the visual plant in tissue engineering tissue preparation system, can make the preparation of tissue engineering tissue form industrialization production.
Below in conjunction with drawings and Examples, the invention will be further described.
Accompanying drawing explanation
Fig. 1 is system schematic of the present invention;
Fig. 2 is method flow diagram of the present invention.
In accompanying drawing, 1 is culturing room, 2 is central control unit, 3 is exchange of substance device, 31 is Cyclic culture liquid entrance, 32 is the outlet of Cyclic culture liquid, 33 is fresh medium entrance, 34 is fresh medium outlet, 35 is gas inlet, 36 is pneumatic outlet, 37 is the biological semi-permeable membranes boundling of hydrophilic hollow fibre, 38 is the biological semi-permeable membranes boundling of hydrophobic hollow fiber, 4 is upstream Cyclic culture liquid pipeline, 5 is downstream Cyclic culture liquid pipeline, 6 is biochemical sensor, 7 is the first liquid stream drive unit, 8 is fresh medium holder, 9 is upstream fresh medium pipeline, 10 is downstream fresh medium pipeline, 11 is the second liquid stream drive unit, 12 is nitrogen, oxygen, carbon dioxide air source, 13 is magnetic valve, 14 is the first strainer, 15 is the second strainer, 16 is check valve, 17 is gas pipeline.
Embodiment
Referring to Fig. 1, a kind of automatic control system of pH/ oxygen partial pressure/partial pressure of carbon dioxide of cell and tissue culture liquid, comprises culturing room 1, also comprises central control unit 2, exchange of substance device 3 and nitrogen, oxygen, carbon dioxide air source 12.Described exchange of substance device 3 comprises the housing of the belt chamber that adopts polycarbonate material making, the chamber of housing is provided with the Cyclic culture liquid entrance and exit 31 for being connected with culturing room, 32, on housing, be provided for the fresh medium entrance and exit 33 being connected with fresh medium holder, 34, in chamber, be provided for fresh medium through and the biological semi-permeable membranes boundling 37 of hydrophilic hollow fibre that carries out the exchange of liquid/liquid with Cyclic culture liquid be communicated with the entrance of fresh medium, outlet 33, 34, on housing, arrange pneumatic outlet 36 and for nitrogen, oxygen, the gas inlet 35 that carbon dioxide air source is connected, in chamber, be provided for gas through and the biological semi-permeable membranes boundling 38 of hydrophobic hollow fiber that carries out gas/liquid exchange with Cyclic culture liquid be communicated with the entrance of gas, outlet 35, 36, make the chamber of housing form enclosed space.Gas/liquid and liquid/liquid exchange all adopts the biological semi-permeable membranes boundling of tubular fibre, and the boundling that the biological semi-permeable membranes of some tubular fibres forms can make exchange area increase, and improves exchange efficiency.It is good that the biological semi-permeable membranes of described hydrophilic hollow fibre, the biological semi-permeable membranes of hydrophobic hollow fiber all adopt poly (ether sulfone) film effect.Between described culturing room and exchange of substance device, be provided with Cyclic culture liquid pipeline.Between the Cyclic culture liquid outlet 32 of the upstream extremity of described culturing room and exchange of substance device, by upstream Cyclic culture liquid pipeline 4, be connected, between the Cyclic culture liquid entrance 31 of the downstream end of described culturing room and exchange of substance device, by downstream Cyclic culture liquid pipeline 5, be connected.Upstream Cyclic culture liquid pipeline 4 between the Cyclic culture liquid outlet of the upstream extremity of described culturing room and exchange of substance device is provided with biochemical sensor 6, downstream Cyclic culture liquid pipeline 5 between the Cyclic culture liquid entrance of the downstream end of described culturing room and exchange of substance device is provided with the first liquid stream drive unit 7, and described nitrogen, oxygen, carbon dioxide air source 12 are connected by gas pipeline 17 with the gas inlet 35 of exchange of substance device through magnetic valve 13 respectively.The upstream extremity of each magnetic valve is connected by gas pipeline with nitrogen, oxygen, carbon dioxide air source respectively by four-way, and the downstream end of each magnetic valve is all connected with the gas inlet of exchange of substance device by gas pipeline.The described nitrogen of the present embodiment, oxygen, carbon dioxide air source are respectively nitrogen, oxygen, carbon dioxide cylinder.Described nitrogen, oxygen, carbon dioxide air source can also adopt additive method known in the art to realize, as central gas supply terminal etc.Between the gas inlet of described exchange of substance device and the downstream end of magnetic valve, be provided with the first strainer 14, the pneumatic outlet of described exchange of substance device is provided with check valve 16, between the upstream extremity of described check valve and the pneumatic outlet of exchange of substance device, is provided with the second strainer 15.The object of using first, second strainer is to stop bacterium, fungi to enter exchange of substance device, and in preferred version, the filtering membrane aperture of first, second strainer is 0.22 micron.Described biochemical sensor, the first liquid stream drive unit, magnetic valve are electrically connected to central control unit respectively.Between described exchange of substance device and a fresh medium holder 8, be provided with fresh medium circulating line, the upstream extremity of described fresh medium holder is connected by upstream fresh medium pipeline 9 with the fresh medium outlet of exchange of substance device, the downstream end of described fresh medium holder is connected by downstream fresh medium pipeline 10 with the fresh medium entrance of exchange of substance device, this downstream fresh medium pipeline is provided with the second liquid stream drive unit 11, and described the second liquid stream drive unit is electrically connected to central control unit.In the present embodiment, described Cyclic culture liquid pipeline, fresh medium circulating line, gas pipeline all adopt medical silicone tube, and connect by standard Luer interface.It is good that described the first liquid stream drive unit and the second liquid stream drive unit adopt jerk pump effect.Certainly, the first liquid stream drive unit and the second liquid stream drive unit also can adopt other technologies method known in the art to drive Cyclic culture liquid and fresh medium to circulate.Measurement to the pH/ PO2/ PCO2 chemical index of Cyclic culture liquid, preferably adopts silver/silver chloride electrode sensor measurement pH, Fluorimetric Quenching Method Luminescence based Oxygen Sensor to measure oxygen partial pressure, current potential severinghous method sensor measurement partial pressure of carbon dioxide.Technician also can adopt other technologies method known in the art to measure These parameters.Described magnetic valve is air-flow magnetic valve.It is good that magnetic valve is selected closed type step direct-acting type electromagnetic valve effect, and electromagnetic valve outlet gas flow rate is set as 10mL/S.Technician can be according to actual needs, and condition as required in the total amount of Cyclic culture liquid, cell and tissue growth arranges gas flow rate, can select control damper known in the art to realize gas flow rate simultaneously and control.The function of the integrated control jerk pump of described central control unit, magnetic valve and collection biochemical sensor signal.Described central control unit is connected with a touch-screen.Described touch-screen shows real time data, and operator's operation information is passed to central control unit.It is good that described touch-screen adopts liquid crystal touch screen effect.On liquid crystal touch screen, can input preset value, respectively control parameter, and check real time data.It is good that described central control unit adopts the micro-chip effect that model is C8501F.Described micro-chip uses trapezoidal pattern programming.The hardware and software of described central control unit also can adopt additive method known in the art to realize.
The workflow of this automatic control system is: fresh medium is pumped by the second liquid stream drive unit from fresh medium holder, enter exchange of substance device, through fresh medium entrance, the biological semi-permeable membrane fibers boundling of wetting ability inside, fresh medium outlet, flow back to fresh medium holder, the circulation of formation fresh medium, indicates with solid line half arrow in accompanying drawing 1.Nitrogen, oxygen, carbon dioxide cylinder are in open state, nitrogen, oxygen, carbonic acid gas are under the control of magnetic valve, with single component gas or mixed gas, through the first strainer, gas inlet, enter the biological semi-permeable membrane fibers boundling of hydrophobicity inner, through pneumatic outlet, the second strainer, check valve, be discharged in atmosphere again, form gas passage, in accompanying drawing 1, with dotted arrow, indicate.Cyclic culture liquid is under the driving of the first liquid stream drive unit, through Cyclic culture liquid entrance, enter the airtight chamber of exchange of substance device, through the outlet of Cyclic culture liquid, biochemical sensor, get back to culturing room again, form the circulation of Cyclic culture liquid, in accompanying drawing 1, with solid arrow, indicate.Fresh medium and Cyclic culture liquid all continue to circulate under the driving of liquid stream drive unit.Gas from gas inlet, enter for gas through and to carry out the biological semi-permeable membranes boundling of hydrophobic hollow fiber of gas/liquid exchange inner with Cyclic culture liquid, carry out gaseous interchange with the Cyclic culture liquid in chamber outside the biological semi-permeable membranes film of hydrophobic hollow fiber, thereby change pH value, the PO of Cyclic culture liquid
2, PCO
2, make culture condition more stable, be beneficial to and cultivate histiocytic cultivation.Fresh medium is when flowing through the biological semi-permeable membranes boundling of hydrophilic hollow fibre, part fresh medium is from the biological semipermeable membrane of hydrophilic hollow fibre in the chamber to film, to Cyclic culture liquid, take in nutritive substance, Cyclic culture liquid also penetrates in the biological semi-permeable membranes of hydrophilic hollow fibre simultaneously, meta-bolites in Cyclic culture liquid is pulled away with the fresh medium at the biological semi-permeable membranes boundling of hydrophilic hollow fibre internal recycle, forms thus liquid/liquid exchange of fresh medium and Cyclic culture liquid.
When envrionment temperature and constant pressure, the maximum value of each partial pressure sum in nutrient solution is constant.Be filled with a certain gas, as oxygen, can make the oxygen partial pressure in nutrient solution increase until oxygen reaches maxima solubility.As before oxygen do not reach maxima solubility, each partial pressure sum has reached maximum value, continues to be filled with oxygen and can make the compositions such as nitrogen, carbonic acid gas discharge from nutrient solution.In this way, can regulate oxygen partial pressure and partial pressure of carbon dioxide to a certain composition or the mixed gas that are filled with in oxygen, carbonic acid gas, nitrogen in nutrient solution.Because carbonic acid gas is dissolved in nutrient solution, generate carbonic acid, can regulate within the specific limits pH, therefore changing partial pressure of carbon dioxide can regulate pH.
Referring to Fig. 1 and Fig. 2, a kind of autocontrol method of pH/ oxygen partial pressure/partial pressure of carbon dioxide of cell and tissue culture liquid, comprises the steps:
1) in central control unit, preset the controlling valu scope of pH, oxygen partial pressure and the partial pressure of carbon dioxide of nutrient solution.Before starting native system, according to the biochemical need for environment condition of Object of Development, at central control unit, set the preset control value scope of pH, oxygen partial pressure, partial pressure of carbon dioxide.The biochemical environment that this example is cultivated according to modal cell or tissue, default pH is 7.2-7.4, and oxygen partial pressure is 160-180mmHg, and partial pressure of carbon dioxide is 30-45mmHg.Technician can adjust the preset value of pH, oxygen partial pressure and partial pressure of carbon dioxide according to actual needs.In the normal DMEM nutrient solution adopting, its maximal regulated scope that can set is respectively pH 6.5-8.1, oxygen partial pressure 30-320mmHg, partial pressure of carbon dioxide 5-160mmHg.As change the kind of nutrient solution, because buffering salt composition changes, above maximum adjustable extent may expand or dwindle.
2) nutrient solution recycle process in Cyclic culture liquid while flowing through biochemical sensor, biochemical sensor detects the value of pH, oxygen partial pressure and the partial pressure of carbon dioxide of Cyclic culture liquid in real time, and the measured value that detects in real time pH, oxygen partial pressure and the partial pressure of carbon dioxide obtained is passed to central control unit by serial port data line.
3) central control unit is by step 2) in biochemical sensor detect in real time controlling valu scope default in the measured value of pH, oxygen partial pressure and the partial pressure of carbon dioxide obtained and step 1) and compare, whether whether the measured value that judges pH, oxygen partial pressure and partial pressure of carbon dioxide according to basic controlling principle as described below within the scope of preset control value, depart from preset control value scope.
4) if the measured value of three indexs in pH, oxygen partial pressure and partial pressure of carbon dioxide all within the scope of its preset control value, is returned to step 3).
If pH, in oxygen partial pressure and partial pressure of carbon dioxide, there is one or several measured value not within the scope of its preset control value, according to the situation of the pH of nutrient solution or oxygen partial pressure or partial pressure of carbon dioxide, central control unit continues to judge needs enter exchange of substance device for which kind of gas, central control unit sends start signal to magnetic valve, control the open corresponding gas pipeline of magnetic valve, make desired gas (nitrogen, oxygen, one or more in carbonic acid gas) enter in the biological semi-permeable membrane fibers boundling of the hydrophobicity internal chamber of exchange of substance device with the biological semi-permeable membranes film of hydrophobicity outside chamber in Cyclic culture liquid carry out gaseous interchange, thereby pH or oxygen partial pressure or the partial pressure of carbon dioxide of change nutrient solution.In gaseous interchange, biochemical sensor continues to measure pH, oxygen partial pressure and three indexs of partial pressure of carbon dioxide.Enter the gas flow rate of exchange of substance device by solenoid control, too fast for avoiding the index of Cyclic culture liquid to change, electromagnetic valve outlet gas flow rate should be controlled at lower level.The electromagnetic valve outlet gas flow rate of preferred version is set as 10mL/S.Technician can be according to actual needs, and condition as required in the total amount of Cyclic culture liquid, cell and tissue growth arranges electromagnetic valve outlet gas flow rate.Through after a while, treat that nutrient solution carries out gaseous interchange, the measured value of the pH that central control unit obtains from biochemical sensor, oxygen partial pressure and partial pressure of carbon dioxide reaches preset control value scope, and reach after the decision-point within the scope of preset control value, central control unit sends shutdown signal to magnetic valve, control the corresponding gas pipeline of closed electromagnetic valve, stop passing into gas in exchange of substance device.
The kind that passes into the gas in exchange of substance device depends on the chemical index kind that departs from preset control value scope, and the amount that passes into gas depends on the degree that departs from preset control value scope.Basic controlling principle is as follows:
(a) upper and lower bound of pH preset control value scope all needs to be set in maximal regulated scope, the measured value of the pH of the Cyclic culture liquid recording when biochemical sensor lower than pH preset control value scope lower in limited time, to nutrient solution, supplement nitrogen and the oxygen that ratio is 1:1, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of pH reaches the decision-point within the scope of pH preset control value, stop to nutrient solution make-up gas; When the measured value of pH is prescribed a time limit higher than the upper of pH preset control value scope, to nutrient solution, supplement carbonic acid gas, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of pH reaches the decision-point within the scope of pH preset control value, stop to nutrient solution make-up gas.Decision-point within the scope of described pH preset control value is that the intermediate value effect of pH preset control value scope is good.Technician can be according to actual needs, and condition as required in the total amount of Cyclic culture liquid, cell and tissue growth arranges the decision-point within the scope of pH preset control value, as long as the numerical value of this decision-point is within the scope of pH preset control value.
(b) upper and lower bound of oxygen partial pressure preset control value scope all needs to be set in maximal regulated scope, the measured value of the oxygen partial pressure of the Cyclic culture liquid recording when biochemical sensor lower than oxygen partial pressure preset control value scope lower in limited time, to nutrient solution supplemental oxygen, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of oxygen partial pressure reaches the decision-point within the scope of oxygen partial pressure preset control value, stop to nutrient solution make-up gas; When the measured value of oxygen partial pressure is prescribed a time limit higher than the upper of oxygen partial pressure preset control value scope, to nutrient solution, supplement nitrogen, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of oxygen partial pressure reaches the decision-point within the scope of oxygen partial pressure preset control value, stop to nutrient solution make-up gas.Decision-point within the scope of described oxygen partial pressure preset control value is that the intermediate value effect of oxygen partial pressure preset control value scope is good.Technician can be according to actual needs, and condition as required in the total amount of Cyclic culture liquid, cell and tissue growth arranges the decision-point within the scope of oxygen partial pressure preset control value, as long as the numerical value of this decision-point is within the scope of oxygen partial pressure preset control value.
(c) upper and lower bound of partial pressure of carbon dioxide preset control value scope all needs to be set in maximal regulated scope, the measured value of the partial pressure of carbon dioxide of the Cyclic culture liquid recording when biochemical sensor lower than partial pressure of carbon dioxide preset control value scope lower in limited time, to nutrient solution, supplement carbonic acid gas, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of partial pressure of carbon dioxide reaches the decision-point within the scope of partial pressure of carbon dioxide preset control value, stop to nutrient solution make-up gas; When the measured value of partial pressure of carbon dioxide is prescribed a time limit higher than the upper of partial pressure of carbon dioxide preset control value scope, to nutrient solution, supplement nitrogen and the oxygen that ratio is 1:1, carry out gaseous interchange, treat that nutrient solution carries out gaseous interchange, when the measured value of partial pressure of carbon dioxide reaches the decision-point within the scope of partial pressure of carbon dioxide preset control value, stop to nutrient solution make-up gas.Decision-point within the scope of described partial pressure of carbon dioxide preset control value is that the intermediate value effect of partial pressure of carbon dioxide preset control value scope is good.Technician can be according to actual needs, condition as required in the total amount of Cyclic culture liquid, cell and tissue growth arranges the decision-point within the scope of partial pressure of carbon dioxide preset control value, as long as the numerical value of this decision-point is within the scope of partial pressure of carbon dioxide preset control value.
(d) the Cyclic culture liquid recording when biochemical sensor has a wherein index not within the scope of preset control value, according to aforesaid method, to regulate this index, makes this index return to preset control value scope; The Cyclic culture liquid recording when biochemical sensor has two indexs or three indexs to exceed preset control value scope simultaneously, and common situation includes but not limited to: pH is too high, and partial pressure of carbon dioxide is too low; PH is too low, and partial pressure of carbon dioxide is too high; PH is too low, and oxygen partial pressure is too low; Partial pressure of carbon dioxide is too low, and oxygen partial pressure is too high; Partial pressure of carbon dioxide is too high, and oxygen partial pressure is too low; PH is too low, and partial pressure of carbon dioxide is too high, and oxygen partial pressure is too low.Control principle is now, according to the precedence of partial pressure of carbon dioxide, oxygen partial pressure, pH index, according to aforesaid method, regulates indices, makes indices return to preset control value scope.
In the present embodiment, the regulation and control that pH, oxygen partial pressure and partial pressure of carbon dioxide exceed after preset control value scope are specific as follows.
(a) regulation and control of pH: pH < 7.2, passes into nitrogen and oxygen mixed gas (1:1), stops ventilation when pH >=7.3; PH > 7.4, passes into carbonic acid gas, and pH≤7.3 o'clock stop ventilation.
(b) regulation and control of oxygen partial pressure: oxygen partial pressure < 160mmHg, pass into oxygen, when oxygen partial pressure >=170mmHg, stop ventilation; Oxygen partial pressure > 180mmHg, passes into nitrogen, stops ventilation during oxygen partial pressure≤170mmHg.
(c) regulation and control of partial pressure of carbon dioxide: partial pressure of carbon dioxide < 30mmHg, pass into carbonic acid gas, when partial pressure of carbon dioxide >=40mmHg, stop ventilation; Partial pressure of carbon dioxide > 45mmHg, passes into nitrogen and oxygen mixed gas (1:1), stops ventilation during partial pressure of carbon dioxide≤40mmHg.
(d) as Cyclic culture liquid has two indexs or three indexs simultaneously, exceed preset range, now according to the order of partial pressure of carbon dioxide, oxygen partial pressure, pH, regulate each index to be returned to preset range separately, each index control method as previously mentioned.
The automatic control system of the application of the invention coordinates autocontrol method of the present invention culturing cell and tissue in culturing room, and the continuous tissue in the culturing room of the present embodiment is cultivated 42 days, does not change liquid midway, does not pollute.Three actual hold-in range of index of whole Cyclic culture liquid are: pH 7.20-7.32, oxygen partial pressure 160-173mmHg, partial pressure of carbon dioxide 35-45mmHg.Visible these three indexs maintain in preset range well.That cultivates organizes after testing, and growth result is obviously better than the tissue that ordinary method (carbonic acid gas incubator+change liquid) is cultivated.The method has realized the long-term cultivation of tissue in totally enclosed situation, do not need to open the cabin and discharge old Cyclic culture liquid and add new nutrient solution, the interference that has significantly reduced manual operations and produced, has realized automated tissue engineering tissue culture truly.
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| CN106467892A (en) * | 2016-09-01 | 2017-03-01 | 奥凯（苏州）生物技术有限公司 | A kind of automatic cytological amplification system |
| CN106479889A (en) * | 2016-09-01 | 2017-03-08 | 奥凯（苏州）生物技术有限公司 | A kind of cell culture blood circulation |
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| CN106497784B (en) * | 2016-09-01 | 2019-03-22 | 奥凯（苏州）生物技术有限公司 | A kind of control pressurer system and its compress control method for realizing culture solution and waste liquid exchange |
| CN110079459A (en) * | 2019-05-15 | 2019-08-02 | 刘宝全 | The adjusting method of gas balance in a kind of stem cell culture vessel |
| CN110373324A (en) * | 2019-07-30 | 2019-10-25 | 中国人民解放军陆军军医大学第一附属医院 | Collect the device of biopsy 3D printing and histocyte culture function integration |
| CN115551989A (en) * | 2020-02-14 | 2022-12-30 | 艾克斯赛尔生物科学公司 | Gas and liquid flow regulation systems for cell culture |
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