CN111893040A - Mechanical mode adjustable rotary biological incubator with online operation function - Google Patents

Mechanical mode adjustable rotary biological incubator with online operation function Download PDF

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CN111893040A
CN111893040A CN202010690214.1A CN202010690214A CN111893040A CN 111893040 A CN111893040 A CN 111893040A CN 202010690214 A CN202010690214 A CN 202010690214A CN 111893040 A CN111893040 A CN 111893040A
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rotary
incubator
bracket
liquid
rotating
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CN111893040B (en
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孙树津
龙勉
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Institute of Mechanics of CAS
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Institute of Mechanics of CAS
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    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature

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Abstract

The embodiment of the invention relates to a mechanical mode adjustable rotary biological incubator with an online operation function, which comprises an incubator, a supporting base, a transmission system, three types of rotary supports, a microscopic monitoring system, a liquid conveying system, a double-way rotary seal, a collecting ring and a motor. The rotary biological incubator can utilize different installation modes of the rotary support and the incubator to adjust various mechanical loading modes, quantitatively control the centrifugal force level of the cell culture substrate of the incubator through rotating speed setting, simultaneously can utilize methods of modifying the hardness and the microstructure of the cell culture substrate and the like to adjust the physical properties of the cell growth environment, and utilizes a liquid conveying system to quantitatively adjust the fluid shearing environment. The rotary biological incubator can meet gas exchange and simultaneously avoid bubble interference, has the functions of on-line microscopic monitoring, on-line liquid supply and chemical fixation, and can carry out relatively long-term cell rotary culture experiments. Can be used for biomechanical researches such as biological effect simulation and cell adhesion at different gravity levels.

Description

Mechanical mode adjustable rotary biological incubator with online operation function
Technical Field
The invention belongs to the technical field of biological and medical equipment, and particularly relates to a mechanical mode adjustable rotary biological incubator with an online operation function.
Background
Rotary incubators generally refer to closed cell or other biological sample culture systems that rotate about a horizontal axis, and rotary incubators used in laboratories may utilize their ability to culture tissue pieces in three dimensions in suspension, or their continuous rotation about a horizontal axis to reduce or eliminate the perception of a fixed gravitational direction by an experimental subject to perform microgravity biology effect simulations. In the application of microgravity biological effect simulation, another instrument which rotates around a double axis randomly also belongs to a rotating culture device in a broad sense, and is called a Random position indicator (RPM), and the three-dimensional continuous Random rotation characteristic is mainly utilized to reduce or eliminate the perception of an experimental object to a fixed gravity direction. These applications take advantage of the special mechanical conditions of their culture systems, mainly gravity and centrifugal conditions. In the application of taking the mechanical condition as the main parameter, how to quantitatively control the mechanical parameter is a key problem. In microgravity biological effect simulation application, on one hand, the perception of a fixed gravity direction of a living sample is eliminated by utilizing continuous rotation, and meanwhile, a series of interferences of mechanical force and other conditions, such as bubbles, fluid shear, centrifugal force, gas exchange, interference of an operation mode and the like, need to be eliminated.
The rotary drum type rotary incubator is a typical incubator rotating around a horizontal shaft, adopts a suspension culture mode (when culturing adherently growing cells, the cells need to be cultured on microcarriers firstly, and then the microcarriers are suspended through the rotation of a rotary drum), and can be used for three-dimensional culture of tissue blocks and simulation of microgravity biological effect. This type of rotary incubator allows for the gas exchange requirement of the liquid tight vessel, and in some models also adds an on-line liquid supply function, and allows for the elimination of bubbles when inoculating cells. For example, a rotating drum type rotating culture device capable of supplying liquid on line supplies liquid to a rotating drum by using a rotating seal, but because the culture is in a suspension state, in order to prevent the culture from being washed away, fresh culture liquid can only pass through a columnar thin film with certain permeability from the center of the rotating drum, and the permeability of the thin film is used for exchanging with the original culture liquid in the rotating drum, so that on one hand, a large amount of culture liquid is consumed to achieve sufficient flow, and on the other hand, the osmosis necessarily has concentration gradient, so that the liquid exchange in the rotating drum is insufficient or uneven. Further, in the case of a rotary-drum type culture device using a suspension culture system, since a culture may be suspended at a position having an arbitrary radius of rotation, it is impossible to quantitatively control the centrifugal force in such a rotary culture device. Quantitative control of fluid shear is also difficult due to the relative motion between the bulk suspended particles and the liquid. In the relatively long-term culture process of the biological sample, new air bubbles are generated in the culture solution due to metabolism, liquid evaporation and the like, and the existence of the air bubbles can not only cause uncontrollable irregular fluid shearing, but also possibly damage cells. The RPM biaxial rotation incubator can be suitable for culture containers with different shapes and can be used for culture in a suspension mode and an adherence mode, but the aim is to eliminate the fixed gravity direction perception of a biological sample by utilizing random directional rotation, and the biaxial random rotation obviously cannot perform constant quantitative control on centrifugal force.
In different gravity level biological effect simulation applications, centrifugal force is the main parameter. Once the rotation stops, the mechanical conditions of the experiment change, and the experimental object may generate a corresponding response, so that the target parameters during the rotation cannot be observed in real time. This requires on-line microscopic monitoring or on-line chemical fixation during rotation. In addition, in biological experiments for a relatively long period of time, operations such as changing culture solutions or adding other biochemical reagents during the experiment are generally involved, and if the operations are performed while stopping rotation, the mechanical conditions of the experiment are also changed, and it is also necessary to perform online operations while rotating in order to maintain the mechanical conditions constant. Existing rotary biological incubators do not have these capabilities for integrated on-line operation and are therefore also difficult to support for relatively long-term experiments. This all places new demands on the performance of rotating biological incubators.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a mechanical mode adjustable rotary biological incubator with an online operation function, which can quantitatively control mechanical conditions by combining rotation speed adjustment and other auxiliary controls through different installation modes of the incubator, is used for biomechanical research of various cells, can utilize the online operation function, and meets the requirements of online microscopic monitoring, chemical fixation and relatively long-term experiments.
The technical scheme adopted by the invention is as follows:
a mechanically mode adjustable rotary biological incubator with online operation functionality, comprising: the device comprises a culture device, a supporting base, a transmission system, a rotary bracket, a microscopic monitoring system, a liquid conveying system, a double-path rotary seal, a collecting ring and a motor;
the culture device is arranged on a rotary support, the rotary support is arranged on a rotary shaft of a transmission system, the transmission system is arranged on a support base, the rotary shaft comprises a driving shaft and a driven shaft, a motor drives the driving shaft and the driven shaft to jointly support the rotary support to rotate synchronously, and the rotary support drives the culture device to synchronously rotate;
on-line liquid supply and chemical fixation are carried out on the culture device during rotation through a peristaltic pump and a double-way rotary seal of a liquid conveying system, the liquid conveying system forms culture solution circulation through the on-line liquid supply, gas exchange and bubble interference elimination are carried out on a culture chamber of the culture device, and the flowing fluid shear level of the culture solution is quantitatively regulated and controlled;
the microscopic monitoring system is arranged on the rotating support, the collecting ring is arranged at one end of the driving shaft, the collecting ring is hollow inside and is used for a liquid pipeline to pass through, and the collecting ring is powered by a CCD (charge coupled device) and an LED (light emitting diode) light source of the microscopic monitoring system which is arranged above the collecting ring from the ground when the rotating support rotates; and online microscopic observation and image acquisition operation are carried out by a microscopic monitoring system and a current collecting ring when the rotating bracket rotates.
Further, the liquid conveying system comprises a double-path rotary seal, a liquid storage bottle a, a liquid storage bottle b, a peristaltic pump, a series of pipelines and luer connectors, the two ends of the driving shaft and the driven shaft are respectively provided with the double-path rotary seal, and liquid is conveyed to the culture device in a rotating state through the double-path rotary seal.
Furthermore, when the whole rotary incubator system is placed in a carbon dioxide incubator, the bottle mouth of the liquid storage bottle is in a non-sealed state and is communicated with the environment gas environment (the liquid storage bottle is communicated with the incubator gas environment through a hose), the solution is subjected to gas exchange with the environment, and the solution subjected to gas exchange is driven by a peristaltic pump and circularly pumped into the incubator through two-way rotary sealing, so that the cell gas exchange requirement is met. Because there is no relative motion between the cell cultured by adherence and the solution, and after the rotating speed is stable, the homogeneous solution itself has no relative motion with respect to the culture device, so that the obvious flow shear can be generated only when the peristaltic pump is started to circulate or perfuse the solution, and the flow shear level is quantitatively controlled only by the driving flow of the peristaltic pump on the premise that the cross section shape and area of the culture cavity of the culture device are fixed and the bubbles are prevented from being generated.
Further, the two-way rotary seal comprises two parts which can rotate mutually, namely a rotary seal shell and a rotary seal core, when one part is clamped by the stator clamp, the part serves as a stator, and the other part serves as a rotor; the rotary seal shell is provided with a liquid path joint a and a liquid path joint b, the rotary seal core is provided with a liquid path joint c and a liquid path joint d, the liquid path joint a is communicated with the liquid path joint c, the liquid path joint b is communicated with the liquid path joint d to form two independent liquid channels, and the two channels are sealed and isolated through two silica gel sealing rings between the rotary seal shell and the rotary seal core.
Furthermore, a liquid path joint on the two-way rotary sealing stator is connected with a solution bottle through a hose and a luer joint, a liquid path joint on the rotor is connected to an inlet and an outlet of the incubator through a hose, when the rotary incubator does not need to convey solution, the luer joint at the stator end is disconnected and sealed by a plugging cap, the stator rotates along with the rotor, and at the moment, the stator and the rotor do not move relatively; when the rotating incubator needs to convey solution, the stator is grasped by hands, the stator is clamped by the stator clamp, and then the liquid storage bottle a, the liquid storage bottle b, the hose at the peristaltic pump end and the rotary sealed hose are connected through the Ruhr connectors to carry out liquid supply operation.
Furthermore, the double-path rotary sealing stator can rotate along with the rotor when not needed, so that the abrasion is reduced, and the problems of culture solution leakage and pollution caused by abrasion due to the fact that the rotary sealing is made of a non-wear-resistant biocompatible silica gel sealing material are solved.
Further, the microscopic monitoring system comprises an LED light source, an LED light source seat, a lens focusing ring, a lens fixing screw, a CCD and a micro lens, wherein the LED light source is installed on the LED light source seat, the micro lens is installed on the lens seat, the micro lens is locked by the lens fixing screw after being focused by the lens focusing ring, cell images are collected through the CCD, and signals are transmitted to a computer through a collecting ring.
Furthermore, the collecting ring comprises a stator and a rotor, the rotor is fixed on the driving shaft through a rotor connector, the collecting ring and the rotor with rotary seal are controlled to rotate along with the driving shaft, an electric wire at the end of the rotor penetrates through the hollow driving shaft to be connected with a CCD (charge coupled device) and an LED (light-emitting diode) light source of a microscopic monitoring system on the rotary support, when power supply to the rotary support is not needed, the electric wire on the stator is disconnected with the power supply, the collecting ring and the stator with double-path rotary seal can rotate along with the rotor, at the moment, relative motion does not exist between the stator and the rotor, and abrasion of a silica gel sealing ring of the; when power needs to be supplied to the rotating bracket, the stator assembly is held by hands, the collecting ring and the rotating sealed stator are clamped by the stator clamp, and the electric wire at the stator end of the collecting ring is connected with a power supply when the rotating bracket rotates.
Further, the incubator comprises an incubator main body, a culture substrate, a sealing gasket, an upper pressing plate and a lower pressing plate, wherein the incubator main body, the sealing gasket and the culture substrate are pressed together through the upper pressing plate and the lower pressing plate from top to bottom, and a culture chamber is formed in the height range of the sealing gasket; the sealing gasket is made of airtight thermoplastic elastic rubber materials, so that bubble interference caused by liquid evaporation in a long-term experiment process is prevented; the two ends of the incubator main body are provided with an inlet end liquid path joint and an outlet end liquid path joint, and the inlet end liquid path joint and the outlet end liquid path joint are connected with a section of hose j and are blocked by a luer joint and a luer joint blocking cap, or are connected with a hose of a liquid conveying system.
Furthermore, the culture substrate is a commercial plastic culture substrate, physical properties such as substrate hardness or microstructure and the like are modified on the surface of the culture substrate according to requirements, mechanical loading or composite mechanical loading of research objects such as cells and the like in different modes is realized by quantitatively adjusting the centrifugal force and fluid shearing conditions of the culture device cell culture substrate and combining with the surface physical property modification of the cell culture substrate, the mechanical mode is controllable and is not easily interfered by air bubbles, mechanical vibration and the like, and the culture substrate can be used for various biomechanical researches such as biological effect simulation at different gravity levels, cell adhesion and the like.
Furthermore, the culture chamber is filled with culture solution, cells grow on the culture substrate in an adherent manner, the length of the culture chamber is about 6.3cm, the width of the culture chamber is adjusted within 2cm by using a sealing gasket, and the maximum culture area is about 12.5cm2. For the application that the quantitative control requirement of the mechanical condition is not strict, the cell suspension culture or the micro-tissue culture can be carried out in the culture chamber according to the requirement.
Furthermore, the incubator main part adopts transparent polycarbonate material preparation to form, top board, holding down plate adopt surperficial aluminium oxide alloy material preparation to form, and above-mentioned material satisfies the transparency of culture base vertical direction in order to be applicable to the microscopic monitoring of normal position to satisfy the demand of sterilizing with the autoclave after the integral erection, incubator and liquid conveying system can wholly put into the autoclave and sterilize.
Furthermore, the culture device and the components of the liquid conveying system are made of materials which meet the biocompatibility requirement of cell culture, and the sealing gasket is made of thermoplastic elastic rubber materials which are airtight and biocompatible, so that air bubble interference caused by liquid evaporation in the long-term experiment process is prevented.
Further, the support base comprises a base bottom plate, a front bearing seat, a middle bearing seat, a rear bearing seat, a motor base rear cover and a stator fixture seat, the motor base, the front bearing seat, the middle bearing seat and the rear bearing seat are installed on the base bottom plate, and the motor base rear cover and the stator fixture seat are installed on the motor base and the rear bearing seat respectively.
Furthermore, the transmission system comprises a driving shaft, a driven shaft, bearings, a large gear, a small gear and bearing baffles, the three bearings and the three bearing baffles are respectively arranged on a front bearing seat, a middle bearing seat and a rear bearing seat of the supporting base, the driving shaft is arranged in two bearings of the front bearing seat and the middle bearing seat, the driven shaft is arranged in a bearing of the rear bearing seat, and the motor is arranged on the motor seat and serves as a rotary driving part; the large gear and the small gear are respectively arranged on a motor shaft and a driving shaft of the motor; after the motor is started, a gear system consisting of the large gear and the small gear is driven to operate, and the driving shaft rotates to synchronously drive the driven shaft to rotate and jointly support the rotating support to rotate.
Furthermore, different gear ratios are obtained by interchanging a large gear and a small gear, the control rotating speed adjusting range is 10-100 rpm/minute, and the driving shaft and the driven shaft are both arranged as hollow shafts for a liquid pipeline and an electric wire to pass through.
Furthermore, the rotating support is a type A rotating support for microgravity effect simulation experiment research and comprises incubator supports, end supports and observation system supports, the two incubator supports and the two end supports are assembled into a frame structure, the end supports at two ends are respectively arranged on a driving shaft and a driven shaft, and the two incubators are simultaneously and symmetrically arranged on the type A rotating support and are fixed through incubator fixing bolts; the observation system bracket is arranged on the incubator bracket, and the microscope monitoring system is arranged on the observation system bracket.
Further, in the mechanical mode setting of the class a rotating holder, the inner surface of the culture substrate of the incubator faces the rotating shaft as the surface on which cells are cultured. The centrifugal force that the cell received when rotating produced pressure to the base, simulated the effect of gravity.
Furthermore, the rotary bracket is a B-type rotary bracket for variable gravity effect simulation and cell adhesion experimental study, and comprises a positioning rotary end plate, a positioning rotary end plate accessory, an incubator bracket and an observation system bracket, wherein the positioning rotary end plate is respectively arranged on a driving shaft and a driven shaft, the two incubator brackets and the two positioning rotary end plate accessories are assembled into a frame structure, a series of mounting screw holes are arranged in the middle of the positioning rotary end plate, the positioning rotary end plate accessories at the two ends of the frame structure are respectively arranged on the screw holes of the two positioning rotary end plates, which are at the same distance with the rotary shaft, the incubator is arranged on the incubator bracket, the other set of frame structure and the incubator are arranged at the positions of the two positioning rotary end plates, which are axially symmetrical to the frame structure, and the observation system bracket is arranged on the incubator bracket, the microscopic monitoring system is arranged on the observation system bracket.
Furthermore, the culture device controlled to be installed by different installation screw holes has different rotating radiuses, and in the mechanical mode setting of the B-type rotating support, the culture device has two installation modes, one mode of the B-type rotating support is that the inner surface of a culture substrate of the culture device is one surface for culturing cells and faces to a rotating shaft, centrifugal force borne by the cells during rotation generates pressure simulation gravity effect on the substrate, and the perception of the cells to the real fixed gravity direction is counteracted through continuous rotation at a certain speed; another installation mode of the B-type rotating bracket is that the inner surface of the culture substrate of the incubator is a surface for culturing cells, which is opposite to the rotating shaft, the centrifugal force applied to the cells during rotation tends to be separated from the substrate, and the centrifugal force applied to the cells is the pulling force between the cells and the substrate.
Furthermore, the rotating bracket is a C-type rotating bracket for experimental study of substrate tangential force effect, and comprises a positioning rotating end plate, a side-mounted incubator bracket, a positioning rotating end plate side-mounted accessory and an observation system bracket, wherein the positioning rotating end plate is respectively mounted on a driving shaft and a driven shaft, the side-mounted incubator bracket and the two positioning rotating end plate side-mounted accessories are assembled into a frame structure, a plurality of mounting screw holes are formed in two sides of the positioning rotating end plate, the positioning rotating end plate side-mounted accessories at two ends of the frame are respectively mounted on the mounting screw holes of the two positioning rotating end plates, the distance between the positioning rotating end plate side-mounted accessories and the rotating shaft is equal, the incubator is mounted on the side-mounted incubator bracket, another set of frame structure and the incubator are mounted at the positions of the two positioning rotating end plates, which are axially symmetrical to the frame structure, and the incubators controlled to be mounted by, the observation system support is arranged on the incubator support, and the microscopic monitoring system is arranged on the observation system support.
Furthermore, in the mechanical mode setting of the C-type rotating bracket, the inner surfaces of the culture substrates of the two incubators are the surfaces for culturing cells, and the rotating shafts are in a plane, and the centrifugal force applied to the cells during rotation is along the tangential direction of the substrate plane.
Furthermore, the direction of centrifugal force applied to the culture substrate can be regulated and controlled quantitatively through different installation modes of the A-type rotating bracket, the B-type rotating bracket, the C-type rotating bracket and the incubator, the centrifugal force can be regulated and controlled quantitatively through surface modification of the culture substrate, the substrate hardness and the microstructure geometric dimension can be regulated and controlled quantitatively through surface modification of the culture substrate, the flowing shear of the culture solution can be controlled quantitatively through the online liquid conveying function of the liquid conveying system and the flow regulation of the peristaltic pump, and various composite mechanical loading modes can be formed through different condition combinations to meet the requirements of different purposes of biomechanical research. In addition, when carrying out online culture solution circulation through liquid conveying system, can be driven to the stock solution bottle along with the culture solution flows if the bubble appears in the culture apparatus to avoid the interference of bubble to experimental conditions.
Furthermore, the on-line microscopic monitoring and on-line liquid conveying functions of the device can support relatively long-term cell culture experiments, and do not interfere with the mechanical state of cells during microscopic observation and liquid conveying.
The invention has the beneficial effects that:
the embodiment of the application provides a mechanical mode adjustable rotary biological incubator with an online operation function, which operates by relying on the internal environment of a carbon dioxide incubator to meet the requirements of gas environment and temperature for cell culture. The rotary incubator can quantitatively control various mechanical loading modes and combinations thereof, can avoid bubble interference while meeting gas exchange, and meets the requirements of experimental research of various biomechanics. When the experimental period needs to be continuously operated for multiple days, the rotary incubator can provide gas exchange and culture solution exchange functions for the cultured biological samples. When the on-line chemical fixation of the biological sample is needed at the end of the experiment, the rotary incubator can provide the on-line injection function of the fixation liquid for the rotary incubator. The rotary incubator also has an online microscopic monitoring function.
The invention realizes the quantitative control of the mechanical parameters of the rotary incubator, and can actually utilize the determined mechanical conditions to apply the novel rotary incubator to the experimental research of various biomechanics. The quantitative mechanical condition regulation can be realized by adopting a horizontal uniaxial rotating structure, taking cell adherent culture as a main culture mode, taking quantitative control and regulation of a core parameter, namely centrifugal force as a main control target, combining the control of a fluid shearing parameter and a substrate physical microenvironment, and then controlling multiple interference factors influencing the stability of the mechanical condition, such as evaporation and bubbles, gas exchange, mechanical vibration and the like one by one. According to different conditions, experimental researches such as microgravity effect simulation, variable gravity effect simulation, cell adhesion strength test, response of cytoskeleton system and cell physiological function to single mechanical condition or composite mechanical condition can be carried out.
Drawings
FIG. 1 is a schematic view of the assembly of a rotary biological incubator core component-incubator according to an embodiment of the present invention;
FIG. 2 is an exploded view of a rotary biological incubator core component, the incubator, according to an embodiment of the present invention;
FIG. 3 is a schematic longitudinal sectional view of an incubator as a core part of a rotary biological incubator according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of the base and drive system of a rotary biological incubator according to an embodiment of the invention;
FIG. 5 is a schematic view of the installation structure of the rotary biological incubator for microgravity effect simulation according to the embodiment of the invention (overall assembly diagram);
FIG. 6 is a schematic view (exploded view) of the installation relationship between the collector ring and the rotary seal on the driving shaft side when the rotary biological incubator provided by the embodiment of the invention is used for microgravity effect simulation;
FIG. 7 is a schematic view (exploded view) of the installation relationship between the micro-monitoring system and the driven shaft side rotary seal when the rotary biological incubator provided by the embodiment of the invention is used for microgravity effect simulation;
FIG. 8 is a schematic sectional view of the upper mounting structure of the rotary biological incubator according to the embodiment of the invention, showing the mounting relationship between the collector ring on the driving shaft side and the rotary seal;
FIG. 9 is a schematic sectional view of a cross-sectional view of a micro-monitoring system of an upper mounting structure and a mounting relationship between a driven shaft side rotary seal and a rotary biological incubator for microgravity effect simulation according to an embodiment of the present invention;
FIG. 10 is a schematic view of an installation structure of a rotary biological incubator for variable gravity effect simulation according to an embodiment of the present invention and with an online operation function;
FIG. 11 is a schematic view of the installation structure of the rotary biological incubator for cell adhesion research and with on-line operation function according to the embodiment of the invention;
FIG. 12 is a schematic view of the installation structure of the rotary biological incubator for simulating the effect of substrate tangential force and with on-line operation function;
wherein, the peristaltic pump, the liquid storage bottle and the connecting pipeline thereof are not included in the figures 8-12.
Wherein, 1, the incubator main body; 2. culturing a substrate; 3. a gasket; 4. an upper pressure plate; 5. a lower pressing plate; 6. an inlet end liquid path joint; 7. an outlet port fluid line connector; 8. a luer fitting; 9. a plugging cap; 10. a culture device hold-down bolt; 11. a hose j; 12. an inlet end liquid guide groove; 12a, an outlet end liquid guide groove; 13. a culture chamber; 14. a base bottom plate; 15. a front bearing seat; 16. a middle bearing seat; 17. a rear bearing seat; 18. a motor base; 19. a motor base; 20. a motor; 21. a drive shaft; 22. a driven shaft; 23. a bearing; 24. a bull gear; 25. a pinion gear; 26. a bearing baffle; 27. an incubator fixing bolt; 28. an incubator stand; 29. an end bracket; 30. mounting a screw hole; 31. positioning the rotating end plate; 32. positioning a rotating end plate accessory; 33. laterally installing a culture device bracket; 34. positioning a lateral installation accessory of the rotating end plate; 35. a motor base rear cover; 36. a stator fixture seat; 37. an observation system support; 38. an LED light source; 39. an LED light source seat; 40. a lens mount; 41. a lens focusing ring; 42. a lens fixing screw; 43. a CCD; 44. a micro-lens; 45. a liquid storage bottle a; 46. a liquid storage bottle b; 47. a peristaltic pump; 48. a liquid path joint a; 49. a liquid path joint b; 50. a liquid path joint c; 51. a liquid path joint d; 52. a binding post; 53. a slip ring stator; 54. a rotor; 55. a rotor connector; 56. rotating the sealing connecting sleeve; 57. a rotating sealed stator sleeve; 58. a stator attachment; 59. a hose a; 60. a peristaltic pump hose; 61. a hose b; 62. a hose c; 63. a hose d; 64. a hose e; 65. a stator fixture; 66. a hose f; 67. a hose g; 68. a hose h; 69. a hose i; 70. a wiring terminal; 71. a wiring terminal; 72. rotating the seal housing; 73. rotating the seal core;
101. a culture device; 102. a support base; 103. a transmission system; 104. rotating the bracket; 105. a microscopic measuring system; 106. a liquid delivery system; 107. a collector ring; 108. and (4) performing double-path rotary sealing.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
For the convenience of understanding of the embodiments of the present invention, the following description will be further explained with reference to specific embodiments, which are not to be construed as limiting the embodiments of the present invention.
The mechanical mode adjustable rotary biological incubator with the online operation function is mainly used in the research field of cell biology and biomechanics experiments, and can be used for simulating the microgravity effect and the variable gravity effect, so that the mechanical mode adjustable rotary biological incubator with the online operation function can be suitable for ground simulation and contrast research of space microgravity experiments.
FIG. 1 and FIG. 2 are diagrams provided in the present inventionA structure diagram of a core incubator part of a mechanical mode adjustable rotary biological incubator with an online operation function is shown in figure 3, and a longitudinal section of the core incubator part is schematically shown. As shown in FIGS. 1, 2 and 3, the incubator 101 comprises an incubator main body 1, a culture substrate 2, a gasket 3, an upper platen 4 and a lower platen 5, wherein the incubator main body 1 is provided at both ends thereof with an inlet port liquid line connector 6 and an outlet port liquid line connector 7 for connecting hoses, the inlet port liquid line connector 6 and the outlet port liquid line connector 7 are connectable to a hose j11 and plugged with a luer connector 8 and a luer connector plug cap 9, or connected to a hose of a liquid transport system 106, the inlet port liquid line connector 6 is communicated with an inlet port liquid guide tank 12, the outlet port liquid line connector 7 is communicated with an outlet port liquid guide tank 12a, the culture substrate 2 is a commercial plastic culture substrate, and physical properties such as substrate hardness or microstructure can be modified on the surface thereof as required, and when the culture substrate 2, the gasket 3 and the incubator main body 1 are compressed by the upper platen 4 and the lower platen 5 by an incubator compression bolt 10, a culture chamber 13 is formed within the height of the gasket 3 (see FIG. 3). The length of the culture chamber 13 is about 6.3cm, the width thereof can be adjusted within 2cm by using the gasket 3, and the maximum culture area is about 12.5cm2. The cells may be grown adherently on the culture substrate 2 or suspended in the culture chamber 13.
For applications where quantitative control of mechanical conditions is not critical, cell suspension culture or micro-tissue culture may be performed in the culture chamber 13 as desired.
The use of a thermoplastic elastomer material that is impermeable to air and biocompatible for the gasket 3 prevents the generation of air bubbles by evaporation of the liquid in the culture chamber 13. Incubator main part 1 is transparent polycarbonate material, top board 4, holding down plate 5 are surface aluminium oxide alloy material, and the transparency of 2 vertical directions of above-mentioned material satisfaction culture base is in order being applicable to the microscopic monitoring of normal position to satisfy the demand of using the sterilization of autoclave after the integral erection.
FIG. 4 is a schematic diagram of the structure of the support base 102 and the transmission system 103 of the rotary biological incubator according to the embodiment of the invention. As shown in fig. 4, the supporting base 102 specifically includes: the motor base 18, the motor base 19, the front bearing seat 15, the middle bearing seat 16 and the rear bearing seat 17 are mounted on the base bottom plate 14. A motor mount back cover 35 and a stator fixture mount 36 are mounted to the motor mount 14 and the back bearing mount 17.
The transmission system 103 specifically comprises: two transmission gears (a large gear 24 and a small gear 25), three bearings 23, three bearing baffles 26, a driving shaft 21 and a driven shaft 22. The three bearings 23 (the bearing positions can be seen in fig. 8 and 9) and the three bearing baffles 26 are respectively installed on the front bearing seat 15, the middle bearing seat 16 and the rear bearing seat 17 of the supporting base 10, the driving shaft 21 is installed in the two bearings 23 of the front bearing seat 15 and the middle bearing seat 16, the driven shaft 22 is installed in the bearing 23 of the rear bearing seat 17, the two transmission gear bull gears 24 and the pinion gears 25 are respectively installed on the motor shaft and the driving shaft 21 of the direct current motor 20, the installation positions of the two gears 24 and the pinion gears 25 can be interchanged to obtain different transmission ratios according to requirements, and the final rotating speed adjusting range is 10-100rpm (revolutions per minute). The direct current motor 20 drives the gear after starting, drives the driving shaft 21 through the gear system and rotates, the driving shaft 21 drives the driven shaft 22 and supports with the driven shaft 22 jointly the runing rest 104 is rotatory, driving shaft 21 and driven shaft 22 are hollow shafts, can supply liquid pipeline and electric wire to pass.
Fig. 5, 6 and 7 are respectively a schematic diagram of an overall installation structure, an explosion schematic diagram of an installation relationship between a collector ring and a rotary seal on a driving shaft side, and an explosion schematic diagram of an installation relationship between a microscopic monitoring system and a rotary seal on a driven shaft side when the rotary biological incubator provided by the embodiment of the invention is used for microgravity effect simulation. Fig. 8 and 9 are schematic sectional views of an installation relationship between a collector ring and a rotary seal on the driving shaft side of the upper installation structure and a schematic sectional view of an installation relationship between a microscopic monitoring system and a rotary seal on the driven shaft side of the rotary biological incubator according to the embodiment of the present invention when the rotary biological incubator is used for microgravity effect simulation (fig. 8 and 9 do not include a peristaltic pump, a liquid storage bottle and a connecting pipeline thereof). As shown in fig. 5, 6, 7, 8 and 9, the dc motor 20 is installed on the motor base 19, after the dc motor 20 is started, the gear 24/the gear 25 is driven, the driving shaft 21 is driven to rotate by the gear system, and the driving shaft 21 drives and supports the rotation of the rotating bracket 104 together with the driven shaft 22. The driving shaft 21 and the driven shaft 22 are hollow shafts, and liquid pipelines and electric wires can pass through the hollow shafts.
In this application, two incubators 101 will be symmetrically mounted on a class a rotating bracket 104 supported by a support base 102 and a transmission system 103, as shown in fig. 5, 6, 7, 8, 9, and fixed with incubator fixing bolts 27. The A-type rotating bracket is composed of a culture device bracket 28, an end part bracket 29 and an observation system bracket 37 and is mainly used for microgravity effect simulation experiment research, the two end part brackets 29 are respectively installed on the driving shaft 21 and the driven shaft 22, and the two culture device brackets 28 are installed on two sides of the two end part brackets 29 so as to form a frame structure. Two incubators 101 may be simultaneously and symmetrically mounted on the class a swivel bracket and fixed by an incubator fixing bolt 27. The vision system mount 37 is mounted to the incubator mount 28 for mounting the microscopy monitoring system 105.
In the mechanical mode setting of the class a rotating holder, the inner surface of the culture substrate 2 of the incubator 101, i.e., the surface on which cells are cultured, faces the rotating shaft. The centrifugal force to which the cells are subjected during rotation causes them to exert a pressure on the substrate, simulating the effect of gravity. The center of the inner surface of the culture substrate 2 is about 0.9cm away from the rotating shaft, the set rotating speed range is 10-32rpm (revolutions per minute), and continuous rotation at a certain speed is used for offsetting the perception of cells to the real fixed gravity direction. The centrifugal force is proportional to the square of the angular velocity and the radius of rotation, the center of the inner surface of the culture substrate 2 is about 0.9cm away from the rotation axis, and when the rotation speed adjusting range is 10-32rpm, the centrifugal force level of the inner surface of the culture substrate 2 can be quantitatively set at 10 according to the rotation speed-3-10-2g (g represents the acceleration of gravity of the earth by 9.81m/s2) Thereby being used for microgravity effect simulation research of biological samples.
As shown in fig. 5, 6, 7, 8 and 9, two observation system brackets 37 are respectively installed on two sides of the incubator bracket 28 for installing a set of microscopic observation system 105, and the microscopic observation system 105 is installed on the observation system brackets 37 through the lens mount 40. Incubator 101 and microscopic observation system 105 are mounted in pairs about the axis of rotation to distribute rotational weight symmetrically. The microscopic observation system 105 is composed of an LED light source 38, an LED light source seat 39, a lens seat 40, a lens focusing ring 41, a lens fixing screw 42, a CCD43 and a microscope lens 44. The microscopic observation system 105 and the observation system support 37 are installed one set on each side of the class a rotating support 104 to ensure symmetry of rotating weight.
As shown in fig. 5, 6, 7, 8, 9, the liquid delivery system 106 is comprised of two-way rotary seals 108, two reservoirs a 45 and b46, a peristaltic pump 47, and a series of tubing and luer fittings. According to the flowing path of the culture solution, fresh culture solution can be injected into the liquid storage bottle a 45 through the hose a 59, after the peristaltic pump 47 is started, the culture solution is sucked out of the liquid storage bottle a 45, is output through the peristaltic pump hose b 60 and then is divided into two paths of a hose c 61 and a hose d 62 through a tee joint. Both the hose c 61 and the hose d 62 can be clamped closed by a hose clamp, one of the hose c 61 and the hose d 62 is clamped closed, and the other is kept open, i.e. the passage can be supplied with liquid, and vice versa. The hoses c 61 and d 62 are respectively connected with two liquid path joints a 48 and b 49 on the rotary sealing shell 72 of the two-way rotary seal 108 through a group of luer joints 8, further respectively connected with two liquid path joints c50 and a liquid path joint d 51 on the rotary sealing core 73, and further respectively connected with the inlet end liquid path joints 6 of the two incubators 101 on the class A rotary support 104 through a pair of hoses d 63 passing through the driven shaft 22. The liquid path joints 7 at the outlet ends of the two incubators 101 penetrate through the hollow driving shaft 21 through a pair of hoses e64 to be connected with two liquid path joints a 48 and b 49 on the rotary sealing shell 72 of the double-path rotary seal 108 at one end of the driving shaft 21, and further are respectively connected with two liquid path joints c50 and d 51 on the rotary sealing core 73, and are respectively connected with a hose g 67 and a hose h 68 through a pair of hoses f66 and a luer joint 8 to be jointly merged into a liquid storage bottle b46 through a tee joint.
When the culture solution needs to circularly flow, the tee joint connected with the hose g 67 and the hose h 68 is directly connected with the hose a 59 of the liquid storage bottle a 45, and the liquid storage bottle b46 is removed.
When the rotating culture device 101 does not need to convey solution, the luer connector at the stator end can be disconnected and sealed by the luer connector plugging cap, the stator rotates along with the rotor, relative movement does not exist between the stator and the rotor, and abrasion of the sealing ring can be reduced so as to avoid leakage. When the rotating incubator 101 needs to convey solution, the stator is grasped by hands, then the stator is clamped by the stator clamp 65, and then the liquid storage bottle a 45, the liquid storage bottle b46, the hose at the peristaltic pump end and the rotary sealed hose are connected by the luer connector, so that liquid supply operation can be carried out.
When the whole rotary incubator system is placed in a carbon dioxide incubator, the bottle openings of the liquid storage bottle a 45 and the liquid storage bottle b46 are in an unsealed state and are communicated with an ambient gas environment (for example, the liquid storage bottle a 45 and the liquid storage bottle b46 can be communicated with the incubator gas environment through a hose a 59 and a hose i 69), the solution can be subjected to gas exchange with the environment, and the solution subjected to the gas exchange is driven by a peristaltic pump 47 to be circularly pumped into the incubator 101 through a double-way rotary seal 108, so that the cell gas exchange requirement can be met. Because there is no relative motion between the cells cultured by adherence and the solution, and after the rotation speed is stable, the homogeneous solution itself has no relative motion with respect to the culture device 101, so that the obvious flow shear can be generated only when the peristaltic pump 47 is started to circulate or perfuse the solution, and the flow shear level is quantitatively controlled only by the driving flow of the peristaltic pump 47 on the premise that the cross section shape and area of the culture cavity 13 of the culture device 101 are fixed and the generation of bubbles is prevented.
As shown in fig. 5, 6, 7, 8 and 9, the slip ring 107 is mounted at one end of the outer side of the driving shaft 21, is hollow inside, and allows a liquid pipeline to pass through. The collecting ring 107 is mainly used for supplying power to the CCD43 and the LED light source 38 of the microscopic monitoring system 105 facing the rotating bracket 104 when the rotating bracket is rotated, so as to meet the requirement of on-line monitoring during the rotating culture. The collecting ring 107 is divided into a stator 53 and a rotor 54, the collecting ring rotor 54 is fixed on the driving shaft 21 through a rotor connector 55, so that the collecting ring and the rotor with rotary seal rotate together with the driving shaft 21, and an electric wire at the rotor end passes through the hollow driving shaft 21 to be connected with a CCD43 and an LED38 light source of a microscopic monitoring system 105 on the rotary support 104. The rotor 54 of the slip ring 107 and the rotor-rotary seal housing 72 of the double rotary seal 108 at this end are both fixed to the rotor connector 55, the rotor connector 55 is fixed to the drive shaft 21, and the three rotate together with the drive shaft 21 as a rotor assembly. The rotary seal stator sleeve 57 passes through the middle hole of the collecting ring 107 and is fixed with the stator 53 of the collecting ring 107 by the stator accessory 58, and the rotary seal core 73 of the stator of the double-path rotary seal 108 is fixed with the rotary seal stator sleeve 57, so that a stator assembly is formed. Two hoses f66 connecting the fluid connection c50 and the fluid connection d 51 of the rotary seal core 73 are passed through the rotary seal stator sleeve 57 and the stator attachment 58, and thus also through the slip ring 107, and are connected to the hose g 67 and the hose h 68 through the luer 8, respectively. All of the stator assembly can rotate with the rotor assembly when the luer connector and wires are disconnected and the stator attachment 58 is not caught by the stator clip 65. When the stator accessory 58 is clamped by the stator fixture 65, the stator assembly stops rotating, and at the moment, a luer connector and an electric wire can be connected for liquid supply operation and image acquisition.
When the power supply to the rotating support 104 is not needed, the electric wires on the collecting ring stator 53 can be disconnected with the power supply, the collecting ring 107 and the stator of the double-path rotating seal 108 can rotate together with the rotor, and at the moment, the stator and the rotor do not move relatively, so that the abrasion of a silica gel sealing ring of the rotating seal can be reduced to avoid liquid leakage. When power needs to be supplied to the rotating bracket 104, the stator assembly is held by hands, then the stator fixture 65 is used for clamping the collecting ring 107 and the stator of the double-path rotating seal 108, and the electric wires at the stator end of the collecting ring 107 can be connected with a power supply during operation.
Because the driving shaft 21 is hollow, the power line and the signal line of the binding post 52 of the CCD43 and the power line of the LED light source 38 can pass through the hollow driving shaft 21 and pass through the rotor connector 55 to be connected with the lines of the connection terminal 70 of the slip ring rotor 54, and when the lines of the connection terminal 71 of the slip ring stator 53 are connected with the ground equipment, the CCD 36 and the LED light source 38 can be supplied with power while rotating and the signal of the CCD43 can be transmitted out through the slip ring 107.
As shown in fig. 5, 6, 7, 8 and 9, at one end of the driven shaft 22, there is only one two-way rotary seal 108, the rotor-rotary seal core 73 is fixed with the driven shaft 22 through the rotary seal connecting sleeve 56, and the stator-liquid connector on the rotary seal shell 72 is connected with the hose b61 and the hose c 62 through the luer connector 8. When the luer fitting is disconnected and the rotary seal housing 72 is not captured by the stator fixture 65, the stator and rotor of the two-way rotary seal 108 may rotate together. When the rotary seal housing 72 is clamped by the stator clamp 65, a luer connector can be connected to perform a liquid supply operation.
FIG. 10 is a schematic view of the installation structure of the rotary biological incubator for the variable gravity effect simulation according to the embodiment of the invention (excluding the liquid storage bottle, the peristaltic pump and the hose connected thereto). As shown in fig. 10, in this application, the support base 102 and the drive train 103 are the same as in fig. 5, 6, 7. The B-type rotating supports 104 are arranged on the driving shaft 21 and the driven shaft 22, namely, a positioning rotating end plate 31 is respectively arranged on the driving shaft 21 and the driven shaft 22, a series of screw holes 30 are arranged on the center line of the positioning rotating end plate 31, two positioning rotating end plate accessories 32 are symmetrically arranged on the screw holes with the same distance with the rotating shaft on the two positioning rotating end plates 31, and two incubator supports 28 are respectively arranged at the two ends of each positioning rotating end plate accessory 32. The two positioning and rotating end plate attachments 32 and the two incubator stands 28 form a frame structure, the two frame structures are symmetrically distributed on two sides of the rotating shaft, and the two incubators 101 are also symmetrically mounted on the incubator stands 28 of each frame respectively. A symmetrical rotary structure is easy to keep rotationally stable. Two sets of microscopic monitoring systems 105 are symmetrically and respectively arranged on the two B-type rotating supports 104 and are used for acquiring microscopic images of the corresponding incubators 101. The mounting of the liquid delivery system 106 and the current collector ring 107 is the same as in fig. 5, 6, 7. In the mechanical mode of this application, the inner surface of culture substrate 2 of incubator 101, i.e., the surface on which cells are cultured, faces the rotation axis.
FIG. 11 is a schematic diagram of the installation structure of the rotary biological incubator for cell adhesion study (excluding the liquid storage bottle, the peristaltic pump and the hose connected thereto). As shown in fig. 11, in this application, the support base 102 and the drive train 103 are the same as in fig. 5, 6, 7. The mounting of the class B swivel bracket 104 is the same as in fig. 8 and 9. The two incubators 101 are still symmetrically mounted on the incubator mount 28. Two sets of microscopic monitoring systems 105 are symmetrically and respectively arranged on the two B-type rotating supports 104 and are used for acquiring microscopic images of the corresponding incubators 101. The installation of the liquid delivery system 106 and the current collector ring 107 is the same as in fig. 5, 6, 7 and 10. The direction of installation of the incubator 101 on the type B rotating bracket 104 in FIG. 11 is opposite to that in FIG. 10. In the mechanical mode of this application, the inner surface of culture substrate 2 of incubator 101, i.e., the side on which cells are cultured, faces away from the rotation axis.
The B-type rotating bracket consists of a positioning rotating end plate 31, a positioning rotating end plate accessory 32, a culture device bracket 28 and an observation system bracket 37, is mainly used for the simulation of variable gravity effect and the experimental study of cell adhesion, the positioning and rotating end plate 31 is respectively arranged on the driving shaft 21 and the driven shaft 22, two incubator brackets 28 and two positioning and rotating end plate accessories 32 are assembled into a frame structure, a series of mounting screw holes 30 are arranged in the middle of the positioning rotating end plate 31, positioning rotating end plate accessories 32 at two ends of the frame structure are respectively mounted on the mounting screw holes 30 of the two positioning rotating end plates 31 with the same distance with the rotating shaft, the culture device 101 is mounted on the culture device bracket 28, another set of frame and culture device 101 is installed on the two positioning rotating end plates 31 at the position which is axisymmetrical to the frame structure, and different installation screw holes enable the culture device 101 to be installed with different rotating radiuses. The vision system mount 37 is mounted to the incubator mount 28 for mounting the microscopy monitoring system 105. In the mechanical mode setting of the type B rotating holder, there are two mounting modes of the incubator 101, one is that the inner surface of the culture substrate 2 of the incubator 101, i.e., the surface on which cells are cultured, faces the rotating shaft, and the centrifugal force to which the cells are subjected during rotation causes the cells to generate pressure on the substrate, thereby simulating the action of gravity. A continuous rotation at a certain speed is used to counteract the perception of a truly fixed gravitational direction by the cells. In the B-type rotating bracket structure, the distance between the center of the inner surface of the culture substrate 2 and the rotating shaft can be adjusted between 1.4 cm and 8.6cm, the rotating speed of the motor 20 and the transmission system 103 is adjusted within the range of 10 rpm to 100rpm, and the centrifugal force level of the inner surface of the culture substrate 2 can be quantitatively set between 0.01 g and 1g by selecting a specific combination of the centrifugal radius and the rotating speed, so that the B-type rotating bracket structure can be used for simulating the variable gravity effect of different levels of a biological sample and researching the gravity response threshold of a biological system. Another way of mounting the type B rotating holder is to mount the inner surface of the culture substrate 2 of the incubator 101, i.e., the surface on which the cells are cultured, facing away from the rotating shaft, so that the cells tend to separate from the substrate due to the centrifugal force applied to the cells during rotation. The level of centrifugal force on the inner surface of the culture substrate 2 can also be quantitatively set to be between 0.01 and 1g by selecting a specific combination of the radius of centrifugation and the rotation speed, but since the centrifugal force to which the cells are subjected in this case causes them to behave as a pulling force between the cells and the substrate, it can be used for quantitative studies of the adhesion between the cells and the substrate.
FIG. 12 is a schematic view of the installation structure of the rotary biological incubator for the study of the effect of the substrate tangential force provided by the embodiment of the invention (the assembly view is removed of the liquid storage bottle, the peristaltic pump and the hose connected with the peristaltic pump). As shown in fig. 12, in this application, the support base 102 and the drive train 103 are the same as in fig. 5, 6, 7. The C-type rotating bracket consists of a positioning rotating end plate 31, a side mounting incubator bracket 33, a positioning rotating end plate side mounting accessory 34 and an observation system bracket 37, and is mainly used for experimental research of substrate tangential force effect. The C-type rotating supports 104 are arranged on the driving shaft 21 and the driven shaft 22, namely, the positioning rotating end plates 31 are respectively arranged on the driving shaft 21 and the driven shaft 22, a frame structure is formed by two positioning rotating end plate side mounting accessories 34 and two side mounting incubator supports 33, a plurality of mounting screw holes 30a are formed in the side edges of the positioning rotating end plates 31, and the positioning rotating end plate side mounting accessories 34 at the two ends of the frame are respectively arranged on the two positioning rotating end plates 31 at the mounting screw holes 30a with the same distance with the rotating shaft. Two frame structures are axisymmetrically distributed on both sides of the rotation axis, and two incubators 101 are also axisymmetrically mounted on the side-mounted incubator brackets 33 of each frame, respectively. Two sets of microscopic monitoring systems 105 are respectively arranged on the two C-type rotating supports 104 in an axisymmetric manner and are used for acquiring microscopic images of the corresponding incubators 101. The mounting of the liquid delivery system 106 and the current collector ring 107 is the same as in fig. 5, 6, 7, 10 and 11.
Different mounting screw holes 30 allow different radii of rotation of the mounted incubator 101, and the vision system mount 37 is mounted on the incubator mount 28 for mounting the microscopic monitoring system 105. In the mechanical mode setting of the C-type rotating holder, the inner surfaces of the culture substrates 2 of the two incubators 101, i.e., the surfaces on which cells are cultured, and the rotating shaft are in a plane, and the centrifugal force to which the cells are subjected during rotation is in the tangential direction to the substrate plane. The distance between the center of the inner surface of the culture substrate 2 and the rotation axis can be adjusted at two positions of 3.9cm and 5.3cm, the width of the inner surface of the culture substrate 2 can be adjusted within 2cm by using the sealing gasket 3, so that the minimum and maximum rotation radii of the inner surface of the culture substrate 2 are respectively 2.9 and 6.3cm, the rotation speed of the transmission system 103 is adjusted within the range of 10-100rpm, the centrifugal radius and the rotation speed are selected to be a specific combination, the centrifugal force level of the inner surface of the culture substrate 2 can be quantitatively set between 0.01-0.7g, and the method can be used for quantitative research of the influence of the tangential force of the substrate on the adhesion and migration of the cell substrates.
The direction of centrifugal force borne by the culture substrate can be regulated and controlled quantitatively by utilizing different installation modes of the A-type rotating bracket, the B-type rotating bracket, the C-type rotating bracket and the incubator 101, the hardness and the microstructure geometric dimension of the substrate can be regulated and controlled quantitatively by utilizing the surface modification of the culture substrate 2, the flowing shear of the culture solution can be controlled quantitatively by utilizing the online liquid conveying function of the liquid conveying system 106 through the flow regulation of the peristaltic pump 47, and various composite mechanical loading modes can be formed by combining the different conditions so as to meet the requirements of different purposes of biomechanical research. In addition, when the liquid delivery system 106 is used for on-line culture liquid circulation, if air bubbles appear in the incubator, the air bubbles can be driven to the liquid storage bottles a 45 and b46 along with the flowing of the culture liquid, so that the interference of the air bubbles on experimental conditions is avoided.
The installation operation process will be briefly described below by taking the installation and use modes of fig. 5, 6, and 7 as examples. The incubator 101 is mounted as shown in FIGS. 1, 2 and 3, and the inlet and outlet are connected to a hose d 63 and a hose e64, respectively. The fluid path joint c50 and the fluid path joint d 51 of the two-way rotary seal 108 are connected to a hose f66, respectively, and the rotary seal stator sleeve 57 is fitted over the hose f66 and the rotary seal core 73. A hose a 59, a peristaltic pump hose 60, a hose b61 and a hose c 62 are connected to the liquid storage bottle a 45, and a hose g 67, a hose h 68 and a hose i 69 are connected to the liquid storage bottle b 46. The above components together with another set of two-way rotary seal 108 are placed into an autoclave for sterilization.
The incubator 101 comprises polycarbonate material, aluminum alloy material and high temperature resistant plastic substrate, and double-circuit rotary seal 108 comprises polycarbonate and polytetrafluoroethylene material, and rotary seal stator sleeve 57 is the polytetrafluoroethylene material, and stock solution bottle a 45 and stock solution bottle b46 are the glass bottle, and the hose is the silica gel material, all can be suitable for the autoclave sterilization.
The supporting base 102 and the A-type rotating bracket 104 are assembled and placed in an ultra-clean workbench, and the inner cavities of the driving shaft 21 and the driven shaft 22 and the A-type rotating bracket 104 are wiped by 75% alcohol for later use.
Injecting cell suspension into the sterilized incubator 101 from an inlet by using an injector to fill the culture cavity 13, then plugging the inlet and the outlet by using a luer connector plugging cap 9, placing the culture substrate 2 downwards into a carbon dioxide incubator to incubate for 12 hours, and taking out and placing the culture substrate into a super clean bench after the cells adhere to the wall. Two incubators 101 are fixed to the class a rotating bracket 104 by the incubator fixing bolts 27. A class a rotating bracket 104 is mounted on the base bracket 102.
In the super clean bench, two flexible pipes d 63 at the inlet ends of the incubators 101 penetrate through the driven shaft 22, are respectively connected with a liquid path joint c50 and a liquid path joint d 51 of a set of two-way rotary seal 108, and then a rotor 73 of the two-way rotary seal 108 is fixed on the driven shaft 22 by using a rotary seal connecting sleeve 56.
Then, the rotor connector 55 is fixed to the drive shaft 21 by screws, and the outlet-end hoses e64 of the two incubators 101 are passed through the drive shaft 21 and the rotor connector 55 and connected to the liquid path joint a 48 and the liquid path joint b 49 of the other set of two-way rotary seal 108, respectively, and the rotor rotary seal case 72 of the two-way rotary seal 108 is fixed to the rotor connector 55 by screws. While a 10-way thin cable is run through the drive shaft 21 for standby. The liquid path joint c50 and the liquid path joint d 51 on the stator rotary seal core 73 of the set of two-way rotary seal 108 are connected with the hose f66 before sterilization, the rotary seal stator sleeve 57 is sleeved on the hose f66 and the rotary seal core 73, the rotary seal stator sleeve 57 penetrates through the hollow stator 53 and the rotor 54 of the collecting ring 107, the rotor 54 of the collecting ring 107 is fixed at the other end of the rotor connector 55 by screws, and each joint at one end of the spare 10-way thin cable is connected with each joint of the connecting terminal 70 of the rotor 54 of the collecting ring 107. Thus, the drive shaft 21, the rotor connector 55, the rotor rotary seal housing 72 of the dual rotary seal 108, and the rotor 54 of the slip ring 107 form a rotor assembly that rotates together. The stator 53 of the slip ring 107 and the rotary seal stator sleeve 57 are fixed together by the stator attachment 58, so that the stator rotary seal core 73 of the dual rotary seal 108, the rotary seal stator sleeve 57, the stator 53 of the slip ring 107 and the stator attachment 58 are fixed together to form a stator assembly.
After the installation operation is finished, two injectors filled with fresh culture solution are respectively connected with two joints of the rotary sealing shell 72 of the double-path rotary seal 108 on one side of the driven shaft, two empty injectors are respectively connected with two hoses f66 on one side of the driving shaft, and the culture solution in the culture cavities 13 of the two incubators 101 and dead cells which are not attached to the wall are replaced by the fresh culture solution and air bubbles are removed. The syringe is then removed and the luer of the two-end tubing is plugged with a stopcock.
Two sets of microscopic monitoring systems 105 are symmetrically arranged on two sides of an A-type rotating bracket 104 by utilizing an observation bracket 37, 4 power lines of two CCD43 binding posts 52, 4 power lines of two LEDs 38 and 2 signal lines of two CCD43 binding posts 52 are respectively connected with each line of a standby 10-path thin cable penetrating through a driving shaft, a power supply and the signal lines are connected from a wiring terminal 71 of a stator 53 of a collecting ring 107, images are observed, the images are adjusted to be clear by a lens focusing ring 41, and then the focal distance is locked by a lens fixing screw 42. And (3) disconnecting the electric wire at the stator 53 end of the collecting ring 107, putting the installed system into a carbon dioxide incubator, switching on the power supply of the direct current motor 20, and driving the A-type rotating bracket 104 to rotate according to the set rotating speed.
When liquid supply operation or image acquisition is needed, the stators at the two ends are clamped by the stator clamp 65, and the luer connectors and the electric wires are connected. Before the liquid supply operation, fresh culture solution is injected into the liquid storage bottle a 45 through the hose 59 by using a syringe, the peristaltic pump hose 60 is arranged at the bottom of the bottle, the hose b61 and the hose c 62 are filled with the culture solution and bubbles are removed by using the syringe, then the hose b61 and the hose c 62 are clamped by using hose clamps, the peristaltic pump hose 60 is clamped on the peristaltic pump 47, and the luer connector is plugged by using a plugging cap for standby. The hose g 67 and the hose h 68 at the end of the liquid storage bottle a 45 are clamped and closed by using hose clamps, and the luer connector is plugged by using a plugging cap for standby. Before the system is connected with the hose b61, the hose c 62, the hose g 67 and the hose h 68, each luer connector and the plugging cap thereof are wiped by 75% alcohol, and the luer connectors are connected after the plugging caps are removed. And (3) releasing the hose clamps of the hose b61 and the hose g 67, starting the peristaltic pump 47 to supply liquid for one incubator, after the operation is completed, locking the hose b61 and the hose g 67 again, releasing the hose clamps of the hose c 62 and the hose h 68, and starting the peristaltic pump 47 to supply liquid for the other incubator. After the liquid supply operation is completed, the luer connectors of the hose b61, the hose c 62, the hose g 67 and the hose h 68 are disconnected, and the connectors at the two ends are plugged by the plugging caps again, so that the online liquid supply operation is completed during the rotation process. The computer can be used for image acquisition and storage operation while the online liquid supply operation is carried out.
When the culture solution circulation is needed, the three-way connection of the hose g 67 and the hose h 68 is connected with the hose a 59 of the liquid storage bottle a 45, and the liquid storage bottle b46 is removed.
Other mechanical modalities may be mounted in the same manner as the microscopic monitoring system 105 and the fluid delivery system 106, except for the differences in mounting of the rotating gantry and the incubator.
The mechanical mode adjustable rotary biological incubator provided by the embodiment of the application runs depending on the internal environment of the carbon dioxide incubator, and can be used for various biomechanical researches. The online operation function of the device is particularly suitable for experimental research with relatively long period.
Those of skill would further appreciate that the various illustrative components and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both, and that the various illustrative components and steps have been described above generally in terms of their functionality in order to clearly illustrate this interchangeability of hardware and software. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the implementation. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the present invention.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. A mechanical mode adjustable rotary biological incubator with online operation function, comprising: the device comprises a culture device (101), a supporting base (102), a transmission system (103), a rotary bracket (104), a microscopic monitoring system (105), a liquid conveying system (106), a double-way rotary seal (108), a collecting ring (107) and a motor (20);
the incubator (101) is mounted on a rotary support (104), the rotary support (104) is mounted on a rotary shaft of a transmission system (103), the transmission system (103) is mounted on a support base (102), the rotary shaft comprises a driving shaft (21) and a driven shaft (22), a motor (20) drives the driving shaft (21) and the driven shaft (22) to synchronously drive the rotary support (104) to rotate together, and the rotary support (104) drives the incubator (101) to synchronously rotate;
on-line liquid supply and chemical fixation are carried out on the culture device (101) during rotation through a peristaltic pump (47) and a double-path rotary seal (108) of a liquid conveying system (106), the liquid conveying system (106) forms culture liquid circulation through the on-line liquid supply, gas exchange and bubble interference elimination are carried out on a culture chamber (13) of the culture device (101), and the flowing fluid shear level of the culture liquid is quantitatively regulated and controlled;
the microscopic monitoring system (105) is arranged on the rotary support (104), the collecting ring (107) is arranged at one end of the driving shaft (21), the interior of the collecting ring is hollow, a liquid pipeline passes through the collecting ring (107), and when the rotary support (104) rotates, the collecting ring (107) supplies power to the CCD (43) and the LED light source (38) of the microscopic monitoring system (105) on the rotary support (104) from the ground; on-line microscopic observation and image acquisition operations are performed by a microscopic monitoring system (105) and a current collecting ring (107) while a rotating support (104) rotates.
2. The mechanical mode adjustable rotary biological incubator with the online operation function as claimed in claim 1, wherein the liquid delivery system (106) comprises a double-path rotary seal (108), a liquid storage bottle a (45) and a liquid storage bottle b (46), a peristaltic pump (47), and a series of pipelines and luer connectors, the double-path rotary seal (108) is respectively installed at two ends of the driving shaft (21) and the driven shaft (22), and liquid is delivered to the incubator (101) in a rotary state through the double-path rotary seal (108).
3. The mechanical mode adjustable rotary biological incubator with the online operation function according to claim 1, wherein the support base (102) comprises a base bottom plate (14), a front bearing seat (15), a middle bearing seat (16), a rear bearing seat (17), a motor base (18), a motor base (19), a motor base rear cover (35) and a stator fixture seat (36), the motor base (18), the motor base (19), the front bearing seat (15), the middle bearing seat (16) and the rear bearing seat (17) are installed on the base bottom plate (14), and the motor base rear cover (35) and the stator fixture seat (36) are respectively installed on the motor base (14) and the rear bearing seat (17).
4. The mechanical mode adjustable rotary biological incubator with the online operation function according to claim 1, wherein the microscopic monitoring system (105) comprises an LED light source (38), an LED light source seat (39), a lens seat (40), a lens focusing ring (41), a lens fixing screw (42), a CCD (43) and a microscope lens (44), the LED light source (38) is installed on the LED light source seat (39), the microscope lens (4) is installed on the lens seat (40), the microscope lens (44) is locked by the lens fixing screw (42) after being focused by the lens focusing ring (41), cell images are collected by the CCD (43), and signals are transmitted to a computer through a current collecting ring (107).
5. The mechanical mode adjustable rotary biological incubator with the online operation function according to claim 1, the device is characterized in that the collecting ring (107) comprises a stator (53) and a rotor (54), the rotor (54) is fixed on the driving shaft (21) through a rotor connector (55), the collecting ring (107) and the rotor with rotary seal rotate together with the driving shaft (21), an electric wire at the end of the rotor (54) penetrates through the hollow driving shaft (21) to be connected with a CCD (43) of a microscopic monitoring system (105) on a rotary bracket (104) and a light source of an LED (38), when power supply to the rotating support (104) is not needed, the electric wire on the stator (53) is disconnected with a power supply, the collecting ring (107) and the stator of the two-way rotary seal (108) can rotate together with the rotor (54), and no relative motion exists between the stator and the rotor, so that the abrasion of a silica gel sealing ring of the rotary seal is reduced; when power needs to be supplied to the rotating bracket (104), the stator assembly is held by hands, the collecting ring (107) and the rotating sealed stator are clamped through the stator clamp (65), and the electric wire at the stator end of the collecting ring (107) is connected with a power supply when the rotating bracket (104) rotates.
6. The mechanical mode adjustable rotary biological incubator with the online operation function according to claim 1, wherein the incubator (101) comprises an incubator main body (1), a culture substrate (2), a sealing gasket (3), an upper pressure plate (4) and a lower pressure plate (5), wherein the incubator main body (1), the sealing gasket (3) and the culture substrate (2) are pressed through the upper pressure plate (4) and the lower pressure plate (5) from top to bottom, and a culture chamber (13) is formed within the height range of the sealing gasket (3); the sealing gasket (3) is made of airtight thermoplastic elastic rubber materials, an inlet end liquid path joint (6) and an outlet end liquid path joint (7) are arranged at two ends of the incubator main body (1), and the inlet end liquid path joint and the outlet end liquid path joint are connected with a section of hose j (11) and are plugged or connected with a pipeline of a liquid conveying system (106).
7. The mechanical mode adjustable rotary biological incubator with the online operation function is characterized in that the transmission system (103) comprises a driving shaft (21), a driven shaft (22), bearings (23), a large gear (24), a small gear (25) and bearing baffles (26), the three bearings (23) and the three bearing baffles (26) are respectively installed on a front bearing seat (15), a middle bearing seat (16) and a rear bearing seat (17) of a supporting base (102), the driving shaft (21) is installed in the two bearings (23) of the front bearing seat (15) and the middle bearing seat (16), the driven shaft (22) is installed in the bearing (23) of the rear bearing seat (17), and a motor (20) is installed on a motor seat (19) and serves as a rotary driving component; the large gear (24) and the small gear (25) are respectively arranged on a motor shaft and a driving shaft (21) of the motor (20); after the motor (20) is started, a gear system consisting of a large gear (24) and a small gear (25) is driven to operate, and the driving shaft (21) rotates to synchronously drive the driven shaft (22) to rotate and jointly support the rotating support (104) to rotate.
8. The mechanical mode adjustable rotary biological incubator with the online operation function according to claim 1, wherein the rotary support (104) is a class A rotary support for microgravity effect simulation experiment research, and comprises an incubator support (28), an end support (29) and an observation system support (37), the two incubator supports (28) and the two end supports (29) are assembled into a frame structure, the end supports (29) at two ends are respectively installed on the driving shaft (21) and the driven shaft (22), the two incubators (101) are simultaneously and symmetrically installed on the class A rotary support and fixed through incubator fixing bolts (27); the observation system bracket (37) is arranged on the incubator bracket (28), and the observation system bracket (37) is arranged on the microscopic monitoring system (105).
9. The mechanical mode adjustable rotary biological incubator with the online operation function as claimed in claim 1, wherein the rotary bracket (104) is configured as a class B rotary bracket for variable gravity effect simulation and cell adhesion experimental study, and comprises a positioning rotary end plate (31), a positioning rotary end plate attachment (32), an incubator bracket (28) and an observation system bracket (37), wherein the positioning rotary end plate (31) is respectively installed on the driving shaft (21) and the driven shaft (22), the two incubator brackets (28) and the two positioning rotary end plate attachments (32) are assembled into a frame structure, a series of installation screw holes (30) are arranged in the middle of the positioning rotary end plate (31), the positioning rotary end plate attachments (32) at the two ends of the frame structure are respectively installed on the screw holes of the two positioning rotary end plates (31) which are at the same distance from the rotating shaft, the incubator (101) is arranged on the incubator bracket (28), the other set of frame structure and the incubator (101) are arranged at the positions of the two positioning rotating end plates (31) which are axially symmetrical to the frame structure, the observation system bracket (37) is arranged on the incubator bracket (28), and the microscopic monitoring system (105) is arranged on the observation system bracket (37).
10. The mechanical mode adjustable rotary biological incubator with the online operation function as claimed in claim 1, wherein the rotary bracket (104) is configured as a class C rotary bracket for experimental study of substrate tangential force effect, and comprises a positioning rotary end plate (31), a side-mounted incubator bracket (33), a positioning rotary end plate side-mounted accessory (34) and an observation system bracket (37), the positioning rotary end plate (31) is respectively mounted on the driving shaft (21) and the driven shaft (22), the side-mounted incubator bracket (33) and the two positioning rotary end plate side-mounted accessories (34) are assembled into a frame structure, the two sides of the positioning rotary end plate (32) are provided with a plurality of mounting screw holes (30), the positioning rotary end plate side-mounted accessories (34) at the two ends of the frame are respectively mounted on the mounting screw holes (30) of the two positioning rotary end plates (31) which are at the same distance from the rotary shaft, the incubator (101) is installed on a side-mounted incubator bracket (33), another set of frame structure and the incubator (101) are installed on the positions, axially symmetrical to the frame structure, of the two positioning rotating end plates (31), different installation screw holes control the installed incubators (101) to have different rotating radiuses, the observation system bracket (37) is installed on the incubator bracket (28), and the microscopic monitoring system (105) is installed on the observation system bracket (37).
CN202010690214.1A 2020-07-17 2020-07-17 Mechanical mode adjustable rotary biological incubator with online operation function Active CN111893040B (en)

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