INCUBATOR

Provided in the present invention is an incubator capable of producing pressure fluctuations. The incubator comprises an incubator body and a variable pressure apparatus, the incubator body having a variable pressure interface, the variable pressure apparatus being connected to the incubator body by means of the variable pressure interface, and the variable pressure apparatus being used for inputting fluctuating or constant air pressure into the incubator body.

TECHNICAL FIELD

The present invention relates to an incubator.

BACKGROUND ART

An incubator is a box-like apparatus mainly used for culturing the cells of microorganisms, plants and animals. It provides stable temperature, humidity and gas concentration by simulating the growing environment of microorganisms, tissues, cells, etc. The incubator has been widely used for culturing cells and tissues and for breeding and culturing some special microorganisms. A three-gas incubator, working on the same principle as other incubators, is featured with that besides CO2, nitrogen and oxygen can also be added therein, and the content of a variety of different gases can be controlled and adjusted.

The pressure in most of the incubators is not higher than the atmospheric pressure and is largely set to a constant pressure. An invention patent application with the Publication Number “CN102978112A” discloses a high-pressure cell incubator, which maintains, in a high-pressure environment, a constant pressure required for cell culture within an incubator body and avoids excessive gas pressure occurring within the incubator body due to the fact that a gas flows therein too fast by using an ordinary valve. A first valve and a second valve are both needle valves, and a gas, within the incubator body, passing through the first valve and the atmosphere passing through the second valve sequentially go through an oxygen pressure reducer and a flow meter and then enter a gas analyzer, thereby controlling the quantity of the sampled gas. However, the inventor found that a good culture effect could be achieved by applying an additional constant pressure into the incubator, in particular the three-gas incubator, and furthermore, a better culture effect could be achieved by allowing an environment for culturing stein cells to be closer to that in a human body when pressure applied into the incubator changes sinusoidally over time.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an incubator capable of producing pressure fluctuations.

According to an embodiment of the present invention, an incubator comprises an incubator body, wherein the incubator further comprises a variable pressure apparatus, the incubator body has a variable pressure interface, the variable pressure apparatus is connected to the incubator body, via the variable pressure interface, and the variable pressure apparatus is configured to input fluctuating or constant gas pressure into the incubator body.

According to an embodiment of the present invention, the variable pressure apparatus comprises a cylinder body and a piston, the cylinder body is communicated to the incubator body via the variable pressure interface, and the piston is movably disposed in the cylinder body so as to input the gas pressure by means of the movement of the piston.

According to an embodiment of the present invention, the cylinder body is connected to the variable pressure interface directly or via a pipe.

According to an embodiment of the present invention, the cylinder body is directly connected to the variable pressure interface, and the variable pressure interface is large enough to allow the cylinder body and the incubator body to synchronously change in gas pressure along with the movement of the piston.

According to an embodiment of the present invention, the cylinder body is fixedly connected to the incubator body, and preferably, the cylinder body is fixedly connected to the top of the incubator body.

According to an embodiment of the present invention, the variable pressure apparatus further comprises one of an electric cylinder, a pneumatic cylinder, and a hydraulic cylinder; preferably, the electric cylinder comprises a servo motor and a transmission mechanism, and the transmission mechanism connects the servo motor and the piston to convert the rotation of the servo motor into the movement of the piston; and preferably, the electric cylinder is an electric push rod, which can drive the piston to move.

According to an embodiment of the present invention, the variable pressure apparatus comprises a gas storage tank, which is communicated to the incubator body via a pressurization gas circuit and a decompression gas circuit;

the pressurization gas circuit is provided with a pressurization pump and a pressurization control valve, the pressurization pump is configured to transport a gas in the gas storage tank to the incubator body, and the pressurization control valve is configured to control the on-off of the pressurization gas circuit; and
the decompression gas circuit is provided with a decompression pump and a decompression control valve, the decompression pump is configured to transport a gas in the incubator body to the gas storage tank, and the decompression control valve is configured to control the on-off of the decompression gas circuit.

According to an embodiment of the present invention, the incubator body is further provided with a safety valve, which is opened after the pressure in the incubator body exceeds a safety threshold.

According to an embodiment of the present invention, the incubator body further comprises an operable/closable gas inlet and an operable/closable gas outlet, independent of the variable pressure interface.

According to an embodiment of the present invention, a sterilization apparatus or/and a humidification apparatus or/and an electric heating apparatus is/are disposed in the incubator body.

According to an embodiment of the present invention, the sterilization apparatus is an ultraviolet light emitter or a steam sterilization apparatus.

According to an embodiment of the present invention, one or more types of gases are present in the incubator, preferably a three-gas incubator; and more preferably, the gases comprise carbon dioxide, nitrogen, and oxygen.

According to an embodiment of the present invention, the incubator further comprises a gas replenishing system, in which a gas relief apparatus is configured to release the gas within the incubator body; and a gas supply system is configured to supply a gas with stable process parameters to the incubator body while the pressure relief apparatus performs releasing.

According to an embodiment of the present invention, a gas mixing apparatus is disposed within the incubator body and is configured to keep the gas in the incubator body in a turbulent state so as to maintain the uniformity of the gas.

According to an embodiment of the present invention, the gas supply system comprises a premixing tank, which receives the one or more types of gases via an input pipeline and is connected to the incubator body via an output pipeline, a gas mixing apparatus is disposed within the premixing tank, the input pipeline is provided with a gas source control apparatus for a gas source, and the output pipeline is provided with an incoming gas control apparatus for the incubator body.

According to an embodiment of the present invention, a gas control apparatus is further disposed within the premixing tank, and the gas control apparatus comprises a gas concentration detecting meter, a heating apparatus, a humidification apparatus and/or a temperature/humidity detection apparatus.

According to an embodiment of the present invention, the gas source control apparatus and/or the incoming gas control apparatus comprises a heating apparatus, a humidification apparatus or/and a gas filter apparatus.

According to the foregoing solution, due to the capability of providing additionally applied pressure fluctuations, the incubator can be used to simulate a pressure environment where cells exist, thereby helping to improve the biological activity of the cells. In addition, a good culture effect is also achieved by the provision of a constant pressure environment.

DETAILED DESCRIPTION OF THE INVENTION

As illustrated inFIGS.1and2, the incubator comprises an incubator body16and a variable pressure apparatus9. The incubator body16, with a configuration similar to that of an existing incubator, provides a space and environment for culturing cells. The following embodiments will be explained by taking a three-gas incubator as an example, but not limited thereto. As illustrated inFIG.1, the incubator body16comprises a chamber3, and further comprises a gas inlet4at the upper part thereof and a gas outlet5at the bottom thereof, and the chamber3is internally provided with a holder8for holding tools such as a microbial culture dish. As illustrated inFIG.2, the variable pressure apparatus9comprises a cylinder body90and a piston91. The cylinder body90is externally connected to the incubator body16. The incubator body16has a variable pressure interface that is not illustrated in the figure. By means of the variable pressure interface, a space inside the cylinder body90is communicated to the chamber3, such that a gas within the cylinder body90may be compressed by moving the piston91, thereby, inputting gas pressure to the chamber3. Inputting the gas pressure may be inputting positive or negative pressure. As illustrated inFIG.2, when the piston91moves to the left, a positive pressure is input, and when it moves to the right, negative pressure is input. The size of variable pressure interface may be configured to be almost the same as the cross section of the cylinder body90, such that the space in the cylinder body90and a space in the chamber3are almost the same one, and with the movement of the piston91, an internal gas pressure may change synchronously. In another embodiment, the variable pressure interface is configured smaller, there is a delay in the gas pressure change between the cylinder body90and the chamber3, and the gas pressures inside the two become equal only after the piston91stops moving for a while.FIG.3illustrates another embodiment, which differs from the embodiments illustrated inFIGS.1and2in that the variable pressure interface is connected to the variable pressure apparatus9via a pipe13. In this way, the variable pressure apparatus9and the incubator body16may be arranged at a distance, and the spatial arrangement of the variable pressure apparatus9and the incubator body16is more flexible. Instead of being combined into a whole, the variable pressure apparatus9and the incubator body16may be separately manufactured by different industrial divisions, which can save costs or facilitate the productization of the apparatus. In the foregoing embodiment, the movement of the piston91is implemented by connecting of a drive apparatus92, which is an electric cylinder comprising a servo motor and a transmission mechanism in one embodiment. The servo motor rotates at a changeable output speed, and the transmission mechanism converts the rotation of the servo motor into the movement of the piston91. The transmission mechanism is, for example, a rack-and-pinion or screw-and-nut transmission mechanism. In a preferred embodiment, the drive apparatus92is an electric push rod. In another embodiment, the drive apparatus92is a pneumatic cylinder or a hydraulic cylinder. By means of the reciprocating motion of the piston9:1, the gas in the incubator body16produces a fluctuating pressure, and the change of the pressure may be configured as required, for example, configured to be a sinusoidal change. When the piston91moves to a certain position and then remains stationary, a constant pressure may be produced in the incubator body. The incubator body16is provided with a safety valve that is not illustrated in the figure, and when the pressure exceeds a certain value, the safety valve is opened to release the pressure automatically. The safety valve is also suitable for the embodiments described below.

FIG.4illustrates a yet another embodiment, Compared with the embodiments illustrated inFIG.1andFIG.2, the difference lies in that the variable pressure apparatus9comprises a gas storage tank96, a pressurization gas circuit94and a decompression gas circuit95. The gas storage tank96is connected to the incubator body16via the pressurization gas circuit94and the decompression gas circuit95respectively. The pressurization gas circuit94is provided with a pressurization pump14and a pressurization control valve17. The pressurization pump14is configured to transport the gas in the gas storage tank96to the incubator body16, and the pressurization control valve14is configured to control the on-off of the pressurization gas circuit.

The decompression gas circuit95is provided with a decompression pump15and a decompression control valve18. The decompression pump15is configured to transport the gas in the incubator body16to the gas storage tank96, and the decompression control valve18is configured to control the on-off of the decompression gas circuit.

When the pressure in the chamber3needs to be increased, the control valve17in front of the pressurization pump14is opened; the pressurization pump14starts to pump the gas from the gas storage tank96into the chamber3; the control valve18in front of the decompression pump15is closed; and the pressure in the chamber3increases to a pressure value to end the pressurization. Then, the control valve17in front of the pressurizing pump14is closed; the control valve18in front of the decompression pump15is opened; the decompression pump15pumps the gas from the chamber3into the gas storage tank96; and the pressure of the chamber3decreases to a specified value to end the decompression. The pressurization pump14, the decompression pump15, and the two control valves17and18may be connected to a controller, in which pressurization and decompression operations are controlled by means of a program, and the pressure in the chamber3fluctuates periodically over time.

More detailed configurations may also be implemented on the incubator body16. As illustrated inFIG.1, a housing1of the incubator body16of the incubator is a stainless steel tank, which may be a cylinder or a cuboid with circular arc transitions at inner walls. The stainless steel tank has a cover that is integrated with an incubator door, and may withstand a certain positive pressure after being closed. The bottom of the chamber3is provided with a humidification apparatus10, and a sensor or a tester12is arranged around the humidification apparatus10and is connected to an externally disposed display that is not illustrated in the figure. The sterilization apparatus6may be an ultraviolet light emitter arranged around the chamber3, or may provide steam sterilization. The heating apparatus7provides electric heating and is distributed around the chamber3. A cell culture environment is purified by means of the sterilization apparatus6; a more realistic culture environment is simulated by means of the heating apparatus and the humidification apparatus; furthermore, the sensor or tester12may be used to provide feedback information such as temperature, humidity, and pressure, and feed the feedback information to the controller, which then compares the feedback information with a set value to control and adjust the heating apparatus7and humidification apparatus10, etc.

An embodiment of the application of an incubator is described below.

“Different conditions” or “different culture conditions” means that cell culture conditions involved in the comparison differ from each other only by the unique characteristics of a gas environment indicated below, and such a set of different culture conditions for comparison is referred to as “four conditions” for short:

normoxia (20%): the oxygen concentration is 20%, and no additional pressure is applied beyond one atmospheric pressure;
hypoxia (5%): the oxygen concentration is 5%, and no additional pressure is applied beyond one atmospheric pressure;
hypoxic static pressure (5%+static): the oxygen concentration is an additional constant pressure of 95 mmHg is applied at one atmospheric pressure, the incubator body16is pressurized by means of the variable pressure apparatus9, when the additionally applied pressure reaches 95 mmHg, the incoming and outgoing gases of the incubator body16are allowed to reach a dynamic balance without stopping inflation and pressurization, and the gas in the incubator body16is maintained at one atmospheric pressure+95 mmHg; and
hypoxic dynamic pressure (5%+dynamic): the oxygen concentration is 5%, an additional pressure is applied at one atmospheric pressure, and the pressure in the incubator body16periodically fluctuates sinusoidally in the range of 1 atmospheric pressure+(75-115) mmHg, with the frequency of 14 times/min.

Embodiment: Comparison of Expansion Folds of Hair Follicle Mesenchymal Stem Cells Cultured Under Different Conditions

1) To obtain intact human hair follicle tissues, the hair follicle tissues were carefully placed at the bottom of a 1.5 mL EP tube by using microforceps. 5 μL of enzymolysis solution TripLE (Gibco-12604021) was added to each hair follicle; the tube was then let stand in the incubator filled with 5% CO2at 37° C. for 3 hours; the bottom of the tube was carefully flicked every hour; and the resulting mixture was gently mixed.
2) After 3 hours of enzymolysis, it could be seen under a microscope that an outer root sheath of each hair follicle was completely enzymolyzed, but the hair shaft could not be completely enzymolyzed. Without removing the unenzymolyzed part, the enzymolysis solution was blown 10 times by using a 100-1000 μL pipette to completely mix the enzymolysis solution. The enzymolysis solution was let stand for 1 minute until the unenzymolyzed hair shaft sunk to the bottom of the EP tube, and the upper enzymolysis suspension, i.e., the primary mesenchymal stem cells, was sucked.
25 μL of enzymolytic primary mesenchymal stem cell suspension obtained by combining the enzymolysis solutions of 5 hair follicles was added to one well of a six-well plate, and then 2 mL of amniotic fluid medium (purchased from Guangzhou Baiyunshan Baidi Biotechnology Co., Ltd.) was added for resuspension. The six-well plate was cultured under the four conditions including normoxia (20%), hypoxia (5%), hypoxic static pressure (5%+static), and hypoxic dynamic pressure (5%+dynamic) at 37° C., and the medium was changed every 3 days.
3) After culturing for 10-12 days, the culture medium could be discarded when the cell density in the six-well plate reached more than 80%; 0.5 mL of TryplE digestion solution was added to the bottom of the six-well plate, which was then placed in a 37° C. incubator for 3-minute digestion; then, 1 mL of amniotic fluid medium was added to the six-well plate to terminate the digestion; the supernatant was removed and placed in a 15 mL centrifuge tube; the well plate was rinsed once with 1 mL of amniotic fluid medium; the rinse solution was added to the centrifuge tube. The centrifuge tube was centrifuged at 1500 rpm for 5 minutes in a centrifuge; the supernatant was discarded; 1 mL of amniotic fluid medium was added for resuspension; the cells were counted and inoculated into a T25 culture flask, and cultured to obtain P1-generation hair follicle mesenchymal stem cells,
4) The cells were continuously subcultured to the P12 generation, and the cells under the four conditions were always maintained under their respective culture conditions. Expansion fold=count of cells harvested per passage/count of inoculated cells. In this embodiment, the cells were passaged strictly according to the density of 5000 cells/cm2. The area of a T25 culture flask was 25 cm2, that is, a T25 culture flask was inoculated with 1.25×105cells each time, and the calculation formula of the expansion fold is “expansion fold=count of cells per harvest/(1.25×105)”.

The results were shown inFIG.5andFIG.6. The results showed that the expansion fold of mesenchymal stem cells cultured in hypoxia was higher than that in normoxia, the expansion fold was further increased by pressurization, and the expansion fold of mesenchymal stem cells was the highest under the hypoxic dynamic pressure condition,

FIG.7shows an incubator in still another embodiment. The incubator comprises an incubator body16, a variable pressure apparatus9and a gas premixing tank21. This embodiment follows the reference signs of elements and some of the content of the previous embodiment, in which like reference signs are used to represent the same or similar elements, and the explanation of the same technical content is selectively omitted. For the explanation of the omitted part, reference may be made to the foregoing embodiment, and the explanation will not be repeated in this embodiment. Compared with the embodiment illustrated inFIG.2,FIG.3orFIG.4, this embodiment is different in that a cylinder body90of the variable pressure apparatus9is fixedly connected to the top of the incubator body16by means of welding as one of fixed connection methods. In this way, the overall structure is more compact, firm, and reliable, and can withstand higher gas pressure and higher-frequency gas pressure fluctuations. A variable pressure interface that is not illustrated in the figure is between the incubator body16and the variable pressure apparatus9. The space in the cylinder body90is communicated to the incubator body16via the variable pressure interface, such that the gas in the cylinder body90can be compressed by moving the piston91, thereby inputting gas pressure to the incubator body16. The input gas pressure may be a positive or negative pressure. When the piston91moves downwards, a positive pressure is input, and when it moves upwards, a negative pressure is input. The variable pressure interface may be configured to be almost the same size as the cross section of the cylinder body90, such that the space in the cylinder body90and a space in the incubator body16are almost the same space, and with the movement of the piston91, an internal gas pressure may change synchronously.

In various embodiments, the incubator may further optionally comprise a gas replenishing system, which can be used in any of the preceding embodiments. The gas replenishing system comprises a gas supply system and a pressure relief apparatus. The pressure relief apparatus is configured to release the gas in the incubator body. The gas supply system is configured to supply a gas with stable process parameters to the incubator body while the gas in the incubator body is released, to ensure the stability of the process parameters of the gas in the incubator body. The gas supply system is also configured to reduce the fluctuation of a gas atmosphere in the incubator body, to ensure a more stable culture environment. The process parameters comprise, but are not limited to, gas concentration, gas temperature, and gas humidity.

In the embodiment illustrated inFIG.7, the gas supply system comprises a gas source19, a gas premixing tank21, a gas source control apparatus20, and an incoming gas control apparatus24. The function of the gas premixing tank21is to mix various gases and adjust the process parameters of the gas, and the gas source19provides the gas (for example, the mixed gas of oxygen, carbon dioxide and nitrogen) required for the culture environment. The gas source19provides one or more gases, such as nitrogen, carbon dioxide, oxygen, air, or other gases. The gas premixing tank21has an input pipeline210and an output pipeline211, a gas source control apparatus20is disposed in the input pipeline210, and an incoming gas control apparatus is disposed in the output pipeline211. The gas source control apparatus20comprises various valves, for example, one or more of a pressure regulating valve and a control valve. The gas source control apparatus further comprises one or more of a heating apparatus, a humidification apparatus, and a filter apparatus. The control valve is configured to control the on-off of the pipeline, the pressure regulating valve is configured to control the output pressure of the pipeline, and the control valve is preferably a one-way valve. The heating apparatus is configured to preheat the gas passing through the pipeline, and the heating apparatus may be selected from a jacket heater, a coil heater, or an electric heater. The humidification apparatus is configured to add water vapor to increase the humidity of the gas within the pipeline. A method for adding the water vapor may be selected from spraying or injecting the water vapor into the pipeline. The incoming gas control apparatus24comprises, but is not limited to, one or more of a pressure regulating valve and a control valve which is preferably a one-way valve. The incoming gas control apparatus24may further optionally comprise a heating apparatus, a humidification apparatus, or a gas filter apparatus, and the gas filter apparatus is preferably a high-efficiency gas filter apparatus with a pore size not greater than 0.3 μm. A gas mixing apparatus22and a gas control apparatus23are disposed in the premixing tank21. The mixing apparatus22is activated when the gas source19provides various gases, and a fan or an air pump may be selected to produce a turbulent flow in the premixing tank21. The gas control apparatus23comprises, but is not limited to, various gas concentration detecting meters, heating apparatuses, humidification apparatuses, temperature/humidity detection apparatuses, etc.

The gas comes out from the gas source19and enters the premixing tank21after being adjusted by the gas source control apparatus20. The gas passes through the gas mixing apparatus22to allow the gas in the premixing tank21to be mixed evenly. After the gas is adjusted to a set value by the gas control apparatus23in the premixing tank21, the gas may be introduced into the incubator16by means of the incoming gas control apparatus24. According to the flow direction of the gas in the incubator body16, one or more gas outlet holes161are arranged in parts that are difficult to reach by the gas flow; automatic gas releasing is effected by means of the gas relief apparatus27; and at the same time, the incoming gas control apparatus24is started for replenishing the gas. To further improve the uniformity and cleanliness of the gas inside the incubator body16, one or more gas mixing apparatuses25are placed in the incubator16to keep the gas in the incubator body16in a turbulent state so as to maintain the gas uniformity. To precisely adjust the process parameters of the gas in the incubator body16, the incubator body16is further provided therein with a gas control apparatus26. The gas control apparatus26comprises, but is not limited to, various types of gas concentration detecting meters, heating apparatuses, humidification apparatuses, temperature/humidity detection apparatuses, etc.

Although the present invention is disclosed above with preferred embodiments, they are not intended to limit the present invention, and any person skilled in the art can make possible changes and alternations without departing from the spirit and scope of the present invention. Therefore, without departing from the content of the technical solutions of the present invention, any alternation, equivalent variation and modification made to the embodiments above based on the technical essence of the present invention shall be construed as falling within the protection scope defined by the claims of the present invention.