CN110551854A - method for testing and regulating in-vitro function of myocardial cells by adopting force stimulation mode - Google Patents
method for testing and regulating in-vitro function of myocardial cells by adopting force stimulation mode Download PDFInfo
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Abstract
The invention relates to the field of medical crossing, in particular to a method for testing and regulating the in-vitro function of a myocardial cell by adopting a force stimulation mode, which comprises the following steps: step S1, applying force stimulation to the whole gel wrapping the myocardial cells to acquire extension and migration data of the myocardial cells; step S2, taking local part of the whole gel, and applying force-electric coupling type stimulation to the local gel to collect the myocardial cell contractility and contraction frequency data; and step S3, degrading the gel to obtain cell suspension, so as to collect parameter data of the cell suspension; the method of the invention applies strain or stress to the whole gel through the force stimulation loading device, and after the growth observation of the cells, the local gel is simultaneously applied with tensile stress, extrusion stress and torsional stress through selection, so that the myocardial cells can sense the static and dynamic mechanical stimulation in the microenvironment of the cells through the force sensitive ion channels on the cell membranes, and the electrophysiology on the cell membranes and the biochemical response in the cells are activated, which plays an important role in controlling the structure and the function of the myocardial cells.
Description
Technical Field
The invention belongs to the field of medical crossing, in particular to the field of biomechanics and mechanics biology, and particularly relates to a method for testing and regulating the in-vitro function of a myocardial cell by adopting a force stimulation mode.
background
Cardiovascular disease is currently the leading cause of human death worldwide, and the development of myocardial tissue engineering provides the most potential solution for the treatment of cardiovascular disease. During the occurrence and development of cardiovascular diseases, it is closely related to the change of cell force-electric microenvironment. In recent ten years, with the development of advanced biomaterials and micro-nano biological manufacturing technologies, more and more researches show that the regulation and control of the cell force-electric microenvironment is crucial to the maturation and the functionalization of engineered myocardial tissues and the regeneration and repair of the myocardial tissues. The mechanical microenvironment in the body of the myocardial cells can affect the growth and signal transmission of the myocardial cells in various ways, and the change of the mechanical microenvironment caused by diseases can also cause the myocardial cells to generate abnormal physiological states. Therefore, the research on the influence of the mechanical microenvironment on cells is of great significance for researching basic theories and diagnosing and treating diseases.
At present, the study on the aspect of the regulation and control of the cellular mechanics microenvironment mainly simulates the mechanical microenvironment of cells under normal physiological or pathological states by controlling the hardness or rigidity and the like of a two-dimensional or three-dimensional substrate material, or regulates and controls the stress state of the cells under microscale by performing bionic mechanical tensile stimulation on a scaffold material wrapping the cells so as to promote the functions of the myocardial cells. The common electrical stimulation loading is mainly realized by designing various forms of electrodes to perform pulse stimulation on cells. Research shows that the response of the myocardial cells inoculated on the conductive composite material support to electric stimulation is obviously improved, and the applied electric signals can be better conducted so as to promote the synchronous beating function of the myocardial cells. Therefore, the bionic force-electric stimulation is loaded to reconstruct cells in the in-vitro culture process, the force-electric microenvironment is beneficial to improving the preparation process and the functional simulation of the engineered myocardial tissue, and the design and the method optimization of a force signal stimulation or force-electric coupling signal stimulation device are important contents for realizing the mature engineered myocardial tissue.
In the study of the physiological response of cardiomyocytes in the force-electric coupling environment, a specific excitation application and cell function test device is usually required, but most of the current devices are single-type devices, and the problems of non-uniform mechanical excitation application, separation of a sample clamp and a force loading device, difficulty in realizing slow variable-radius twist load and ultra-small twist strain and the like exist in the aspect of cell culture in gel.
Disclosure of Invention
the invention aims to provide a method for testing and regulating the in-vitro function of a myocardial cell by adopting a force stimulation mode.
in order to solve the technical problems, the invention provides a method for testing and regulating the in-vitro function of a myocardial cell by adopting a force stimulation mode, which comprises the following steps: step S1, applying force stimulation to the whole gel wrapping the myocardial cells to acquire extension and migration data of the myocardial cells; step S2, taking local part of the whole gel, and applying force-electric coupling type stimulation to the local gel to collect the myocardial cell contractility and contraction frequency data; and step S3, degrading the gel to obtain cell suspension, so as to collect parameter data of the cell suspension.
further, the method for acquiring extension and migration data of the cardiomyocytes by applying force stimulation to the whole gel enclosing the cardiomyocytes in step S1 includes:
And applying preset strain force loading including one or more of tensile stress, extrusion stress, shearing force or shearing force to the whole gel by adopting a force stimulation loading device, continuously loading the force for 4-72 h, detecting the stretching and migration conditions of the myocardial cells by a timing interval microscopic imaging technology, and recording stretching and migration data of the myocardial cells.
Further, the method for acquiring the myocardial cell contraction force and contraction frequency data by taking a part of the whole gel and applying the force-electric coupling type stimulation to the part of the gel in the step S2 includes:
The method comprises the steps of simultaneously applying preset tensile stress, extrusion stress and torsional stress to local gel by adopting a force stimulation loading device, continuously loading force for 4-72 hours, simultaneously inserting an inserted microelectrode into the local gel to electrically stimulate cardiac muscle cells, detecting the contraction force and the contraction frequency of the cardiac muscle cells in a force-electricity coupling type loading mode, and recording the data of the contraction force and the contraction frequency of the cardiac muscle cells.
further, the method for degrading the gel to obtain the cell suspension in step S3 to collect the parameter data of the cell suspension includes:
respectively degrading the remaining parts of the local gel and the whole gel, eluting the cardiac muscle cells from the gel to obtain cell suspension, and detecting parameter data of the cell suspension according to the obtained cell suspension;
Wherein the parameter data includes: redistribution of various focal adhesion proteins on cell membranes, and expression levels of GATA-4, fi-actin, beta-MHC, NKx2.5, Cx43 and cTnT proteins.
further, the method for testing and regulating the in vitro function of the myocardial cells by adopting the force stimulation mode further comprises the following steps: and step S4, taking a plurality of whole gels, repeating the steps S1-S3 for each whole gel, and applying different preset strain forces to each whole gel and the local gels obtained from each whole gel.
Further, the force stimulation loading device comprises: the device comprises an accommodating body, a stretching mechanism and a twisting mechanism; wherein the containing body is suitable for containing gel for wrapping the myocardial cells and adopts a non-rigid material; the stretching mechanism is suitable for stretching or extruding the accommodating body from two opposite sides of the accommodating body so as to apply stretching stress, shearing stress or extrusion stress to the gel; and the twisting mechanism is adapted to twist the containment body to apply a torsional shear stress to the gel.
Further, the accommodating body includes: the upper cover plate and the lower cover plate are connected through a clamping cover respectively at two sides; the upper cover plate and the lower cover plate are both made of elastic rubber materials; a plurality of first protruding parts are arranged on the inner surface of the upper cover plate at intervals; and a plurality of second convex parts are arranged on the inner surface of the lower cover plate at intervals.
further, the accommodating body includes: the device comprises an upper cover plate and a lower cover plate, wherein one side of the upper cover plate is connected with a stretching mechanism through a connecting clamping cover, and one side of the lower cover plate is connected with the stretching mechanism through another connecting clamping cover; the upper cover plate and the lower cover plate are both made of elastic rubber materials; a plurality of first protruding parts are arranged on the inner surface of the upper cover plate at intervals; and a plurality of second convex parts are arranged on the inner surface of the lower cover plate at intervals.
Further, the stretching mechanism includes: the screw rod mechanisms are respectively and symmetrically arranged on two opposite sides of the accommodating body; the screw mechanism includes: the screw rod motor, the transmission shaft, the screw rod and the nut; the screw rod penetrates through the nut, and one end of the screw rod is connected with the screw rod motor through the transmission shaft; the other end of the screw rod is connected with the clamping cover; each screw rod motor is suitable for respectively driving the corresponding screw rod to move in the direction far away from or towards the clamping cover so as to stretch or extrude the upper cover plate and the lower cover plate from two opposite sides of the gel, and the upper cover plate and the lower cover plate deform so as to stretch or extrude the gel through the micro-protrusion groove structure in contact with the surface of the gel; wherein, the shearing stress is realized by respectively pulling the upper cover plate and the lower cover plate by the screw rod at one side and the screw rod at the other side so that the upper part and the lower part of the gel are respectively elongated towards opposite directions.
Further, the torsion mechanism is located on the upper cover plate through an upper clamp plate, and includes: a torsion motor and a torsion assembly; wherein the torsion assembly comprises: the device comprises a shell, a central gear, a plurality of planetary gears meshed with the central gear, and peripheral rims meshed with the planetary gears; an output shaft of the torsion motor is connected with the central gear; the peripheral rim is fixed on the upper clamping plate; the gear shaft of the sun gear and the gear shaft of each planetary gear are fixed on the shell; the torsion motor is suitable for driving the central gear to drive the planet wheels to rotate so as to drive the peripheral rim to rotate, so that the upper cover plate is driven to rotate through the upper clamping plate, and torsion stress is applied to the gel.
further, the torsion mechanism is located on the upper cover plate through an upper clamp plate, and includes: a torsion motor and a torsion assembly; wherein the torsion assembly comprises: the device comprises a shell, a central gear, a plurality of planetary gears meshed with the central gear, and peripheral rims meshed with the planetary gears; an output shaft of the torsion motor is connected with the central gear; the gear shaft of each planetary gear is fixed on the upper clamping plate; the gear shaft and the peripheral rim of the central gear are fixed on the shell; the torsion motor is suitable for driving the central gear to drive each planetary gear to revolve to drive the shaft bracket of each planetary gear to move, and then the upper cover plate is driven to rotate through the upper clamping plate, so that torsion stress is applied to the gel; and the diameter of the upper clamping plate is smaller than that of the peripheral rim.
the method for testing and regulating the in-vitro function of the myocardial cells by adopting the force stimulation mode has the advantages that the upper cover plate and the lower cover plate are elastically deformed by the force stimulation loading device, and then the gel is stretched or extruded by the micro-convex groove structure in contact with the surface of the gel. Applying force to the whole gel wrapping the myocardial cells for stimulation, so that the force sensitive ion channels on the cell membranes of the population cells can sense the static and dynamic mechanical stimulation in the 3D microenvironment, to regulate the expansion, migration and other physiological functions of a large number of cell populations, then selecting partial local gel in which cells with specific forms (such as cells with high expansion and migration degrees) are positioned in the overall gel in which the cell populations are positioned according to requirements, simultaneously applying tensile stress, extrusion stress, torsion stress and coupling stimulation of probe type electrode to the local gel, so that the myocardial cell can sense specific local force-electric coupling type stimulation through various sensitive ion channels on the cell membrane to activate mechanical force and electric signal induction molecules on the cell membrane, then the regulation and control function to the structure and function of the cell is realized through signal transduction and other biochemical responses in the cell. The reason that the whole gel is stimulated by force but not electrically stimulated is that the whole gel has relatively large size and large integral characteristic dimension, and an electric field with controllable three-dimensional distribution is not easy to generate under the conventional voltage; for a specific local gel, because the characteristic dimension is obviously smaller, high voltage is not needed (low cost and safety), and the electric field distribution under the local small dimension can be considered to be approximately uniform, so that the control and analysis are convenient, and a more stable method is provided for regulating and controlling the physiological function of cells.
drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a diagram illustrating the steps of a method for testing and controlling the in vitro function of cardiomyocytes by force stimulation according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a force stimulation loading device in a method for performing in vitro myocardial cell function testing and regulation by using a force stimulation method according to an embodiment of the present invention (a part of the supporting component is omitted);
FIG. 3 is a schematic structural diagram of another angle of the force stimulation loading device (with the stretching mechanism omitted) in the method for testing and controlling the in vitro function of the cardiomyocytes by using the force stimulation method according to the embodiment of the present invention;
FIG. 4 is a schematic diagram of the embodiment of the present invention in which the upper cover plate and the lower cover plate are respectively pulled by the left lead screw and the right lead screw to respectively pull the upper part and the lower part of the gel in opposite directions, thereby achieving shear strain (a part of the support assembly is omitted);
FIG. 5 is a schematic diagram of an embodiment of the present invention operating in a manner that simultaneously loads shear stress F τ in the x-direction and tensile stress Fp in the y-direction;
FIG. 6 is a schematic structural diagram of a torsion assembly (without a housing) of the force stimulation loading device according to the method for testing and controlling the in vitro function of cardiomyocytes by using a force stimulation method in the embodiment of the present invention;
fig. 7 is a schematic view of a torsional state of the force stimulation loading device of the method for testing and controlling the in vitro function of the cardiomyocyte by using the force stimulation method according to the embodiment of the present invention.
wherein:
The upper cover plate 1, the first boss 11, the rotation center 12, the card cover 13, the gel 2, the cardiomyocyte 21, the lower cover plate 3, the second boss 31, the screw motor 40, 50, the transmission shaft 41, 51, the screw 42, 52, the bracket 43, 53, the lower clamp plate 60, the upper clamp plate 70, the torsion assembly 80, the central gear 81, the planetary gear 82, the shaft bracket 821 of the planetary gear 82, the peripheral rim 83, the torsion motor 84, the support rod 91, the cross rod 92 and the support rod 93.
Detailed Description
The structure of the present invention will now be described in further detail with reference to the accompanying drawings.
Example 1
as shown in fig. 1, this example 1 provides a method for testing and regulating the in vitro function of cardiomyocytes by using a force stimulation method, which includes: step S1, applying force stimulation to the whole gel wrapping the myocardial cells to acquire extension and migration data of the myocardial cells; a step S2 of selectively taking local parts of the whole gel according to the data collected in the step S1 and applying force-electricity coupling type stimulation to the local gel so as to collect the data of the myocardial cell contractility and contraction frequency; and step S3, degrading the gel to obtain cell suspension, so as to collect parameter data of the cell suspension.
further, the method for acquiring extension and migration data of the cardiomyocytes by applying force stimulation to the whole gel enclosing the cardiomyocytes in step S1 includes: the method comprises the steps of applying preset strain loading to the whole gel by adopting a force stimulation loading device, wherein the preset strain loading comprises one or more of tensile stress, extrusion stress, shear stress or torsional shear stress, detecting the stretching and migration conditions of the myocardial cells by a timing interval microscopic camera shooting technology after the force loading is continued for 4-72 hours, and recording stretching and migration data of the myocardial cells so as to provide data for mechanical stimulation on migration and differentiation of the myocardial fibroblasts and evaluation on the secretion capacity of the myocardial extracellular matrix.
Alternatively, the loading time is, for example, 24 h.
Further, the method for acquiring the myocardial cell contraction force and contraction frequency data by taking a part of the whole gel and applying the force-electric coupling type stimulation to the part of the gel in the step S2 includes: the method comprises the steps of simultaneously applying preset tensile stress, extrusion stress and torsional stress to local gel by adopting a force stimulation loading device, continuously loading force for 4-72 hours, simultaneously inserting an inserted microelectrode into the local gel to electrically stimulate cardiac muscle cells, detecting the contraction force and the contraction frequency of the cardiac muscle cells in a force-electricity coupling type loading mode, and recording the data of the contraction force and the contraction frequency of the cardiac muscle cells.
Specifically, a force stimulation loading device is adopted to simultaneously apply preset tensile stress, extrusion stress and torsional stress loading to local gel, so that the myocardial cell membrane generates strain, and the state and function of the myocardial cell can be influenced by a force sensitive ion channel on the cell membrane; the operation of simultaneously loading a schematic of the shear stress F τ in the x-direction and the tensile stress Fp in the y-direction is shown in FIG. 5.
meanwhile, an inserted microelectrode can be inserted into the gel to electrically stimulate the myocardial cells, so that force-electric coupling type stimulation is implemented, and the contraction force and the contraction frequency of the myocardial cells are observed.
further, the method for degrading the gel to obtain the cell suspension in step S3 to collect the parameter data of the cell suspension includes: respectively degrading the remaining parts of the local gel and the whole gel, eluting the cardiac muscle cells from the gel to obtain cell suspension, and detecting parameter data of the cell suspension according to the obtained cell suspension; wherein the parameter data includes: redistribution of various focal adhesion proteins on cell membranes, and expression levels of GATA-4, fi-actin, beta-MHC, NKx2.5, Cx43 and cTnT proteins.
Specifically, after the whole gel is subjected to force stimulation and the local gel is subjected to force-electric coupling type stimulation, relevant data are recorded, the myocardial cells are eluted from the gel through degradation of the gel to obtain a cell suspension, redistribution of various adhesion plaque proteins (such as integrin, talin and vinculin) on a cell membrane is detected according to the obtained cell suspension, and the expression levels of GATA-4, fi-actin, beta-MHC, NKx2.5, Cx43 and cTnT proteins are detected.
Further, the method for testing and regulating the in vitro function of the myocardial cells by adopting the force stimulation mode further comprises the following steps: and step S4, taking a plurality of whole gels, repeating the steps S1-S3 for each whole gel, and applying different preset strains to each whole gel and the local gels obtained from each whole gel.
Specifically, in order to collect sufficient detection data, preset values of strain force applied to the whole gel and preset values of tensile stress, compressive stress and torsional shear stress applied to the local gel are changed, a plurality of whole gels are taken, and the steps S1-S3 are repeated for each whole gel.
further, as shown in fig. 2 to 4, and fig. 6 to 7, the force stimulation loading device includes: the device comprises an accommodating body, a stretching mechanism and a twisting mechanism; wherein the containing body is suitable for containing the gel 2 for wrapping the myocardial cells 21 and adopts a non-rigid material; the stretching mechanism is adapted to stretch or squeeze the containing body from opposite sides thereof to apply a tensile stress, a shear stress or a squeezing stress to the gel 2; and the twisting mechanism is adapted to twist the containment body to apply a torsional shear stress to the gel 2.
Specifically, the force stimulation loading device of the present embodiment can simultaneously apply tensile stress, shear stress, or compressive stress, torsional shear stress to the gel 2 encapsulating the cardiomyocytes 21 through the tensile mechanism and the torsion mechanism.
As a first embodiment of the containing body in the present embodiment:
as shown in fig. 2 and 3, the receiving body includes: the upper cover plate 1 and the lower cover plate 3 are respectively connected through a clamping cover 13 at two sides; the upper cover plate 1 and the lower cover plate 3 are both made of elastic rubber materials; a plurality of first protruding parts 11 are arranged on the inner surface of the upper cover plate 1 at intervals; and a plurality of second protrusions 31 are provided on the inner surface of the lower cover plate 3 at intervals.
Specifically, the material of the upper cover plate 1 and the lower cover plate 3 is, for example, but not limited to, polydimethylsiloxane (pdms) or polytetrafluoroethylene; the card cover 13 is also made of, for example, but not limited to, polydimethylsiloxane (pdms) or polytetrafluoroethylene; the first boss 11 is, for example, but not limited to, a rectangular tooth; the second boss 31 is also for example, but not limited to, a rectangular tooth; the gel 2 is clamped between the first protruding part and the second protruding part, the first protruding part and the second protruding part are matched, the gel 2 is convenient to adhere to the upper cover plate and the lower cover plate respectively, the sliding offset of the gel can be greatly reduced, and the force can be uniformly applied to the gel.
as a second embodiment of the containing body in the present embodiment:
As shown in fig. 4, the receiving body includes: the device comprises an upper cover plate 1 and a lower cover plate 3, wherein one side of the upper cover plate 1 is connected with a stretching mechanism through a connecting clamping cover, and one side of the lower cover plate 3 is connected with the stretching mechanism through another connecting clamping cover; the upper cover plate 1 and the lower cover plate 3 are both made of elastic rubber materials; a plurality of first protruding parts 11 are arranged on the inner surface of the upper cover plate 1 at intervals; and a plurality of second protrusions 31 are provided on the inner surface of the lower cover plate 3 at intervals.
further, the stretching mechanism includes: the screw rod mechanisms are respectively and symmetrically arranged on two opposite sides of the accommodating body; the screw mechanism includes: a screw motor (40; 50), a transmission shaft (41; 51), a screw (42; 52) and a nut; wherein the screw rod (42; 52) penetrates through the nut, and one end of the screw rod is connected with the screw rod motor (40; 50) through the transmission shaft (41; 51); the other end of the screw rod (42; 52) is connected with the clamping cover 13; each screw motor (40; 50) is adapted to drive a corresponding screw (42; 52) to move in a direction away from or towards the card cage 13, respectively, to draw or squeeze the gel 2 from opposite sides of the gel 2; and each screw motor (40; 50) is adapted to drive the corresponding screw (42; 52) to move in a direction away from the corresponding attachment clip, stretching the upper and lower cover plates 1 and 3, respectively, from both sides, to apply a shear stress to the gel 2 located in the containing body.
Specifically, the screw rod mechanism adopts a micro screw rod mechanism and is controlled by a control module; the screw motor (40; 50) adopts a micro servo motor to improve the stretching or extruding precision; each screw motor (40; 50) respectively drives the corresponding screw to move in the direction away from or towards the clamping cover 13, so that the gel 2 is stretched or extruded from two opposite sides of the gel 2, and further the stretching stress or the extrusion stress is uniformly applied to the gel 2; each screw motor (40; 50) drives a corresponding screw to move in a direction away from a corresponding connecting clamp cover, and stretches the upper cover plate 1 and the lower cover plate 3 from both sides to apply shear stress to the gel 2 in the containing body.
Further, each nut is located on a respective bracket (43; 53).
As a first embodiment of the torsion mechanism of the present embodiment:
The torsion mechanism is located on the upper cover plate 1 through an upper clamp plate 70, and includes: a torsion motor 84 and a torsion assembly 80; wherein the torsion assembly 80 comprises: a housing, a sun gear 81, a plurality of planetary gears 82 meshed with the sun gear 81, and a peripheral rim 83 meshed with each planetary gear 82; an output shaft of the torsion motor 84 is connected with the central gear 81; the peripheral rim 83 is fixed on the upper clamp plate 70; the gear shaft of the central gear 81 and the gear shafts of the planetary gears 82 are fixed on the shell; the torsion motor 84 is adapted to drive the sun gear 81 to rotate the planetary gears 82 to rotate the peripheral rim 83, so as to rotate the upper cover plate 1 via the upper clamp plate 70, thereby applying a torsional stress to the gel 2.
Specifically, the peripheral rim 83 is fixed on the upper clamping plate 70, so that the upper cover plate 1 is driven to rotate by the rotation of the peripheral rim 83, torsional stress is applied to the gel 2, and the rotating diameter of the embodiment is larger.
as a second embodiment of the torsion mechanism of the present embodiment:
The torsion mechanism is located on the upper cover plate 1 through an upper clamp plate 70, and includes: a torsion motor 84 and a torsion assembly 80; wherein the torsion assembly 80 comprises: a housing, a sun gear 81, a plurality of planetary gears 82 meshed with the sun gear 81, and a peripheral rim 83 meshed with each planetary gear 82; an output shaft of the torsion motor 84 is connected with the central gear 81; the gear shaft of each planetary gear 82 is fixed on the upper clamp plate 70; the gear shaft of the central gear 81 and the peripheral rim 83 are fixed on the shell; the torsion motor 84 is adapted to drive the sun gear 81 to rotate the planetary gears 82, so as to drive the shaft brackets 821 of the planetary gears 82 to rotate, and further drive the upper cover plate 1 to rotate through the upper clamp plate 70, thereby applying a torsional stress to the gel 2.
Specifically, the gear shaft of each planetary gear 82 is fixed on the upper clamp plate 70, so that the upper clamp plate 70 is driven to move by the rotation of the shaft bracket 821 of each planetary gear 82, the upper cover plate 1 is further rotated, torsional stress is applied to the gel 2, the rotation diameter of the embodiment is small, the diameter of the upper clamp plate is smaller than that of the peripheral rim, and the rotation direction is opposite to that of the first embodiment.
In practical applications, the appropriate torsion mechanism is selected based on the size of the biological sample and the amount of force required to load.
In particular, the torsion mechanism is also controlled by the control module; the gel 2 is twisted around the rotation center 12, and the torsion motor 84 employs a micro-servo motor to improve the accuracy of the torsion.
specifically, in this embodiment, the shear stress is achieved by respectively pulling the upper cover plate and the lower cover plate through the two lead screws, so that the upper portion and the lower portion of the gel are respectively pulled in opposite directions; and the torsional stress is realized by the matching action of the torsional motor, the torsional component and the upper clamping plate.
Specifically, in the present embodiment, the applied torsional stress is different from the shear stress, the range of action of the torsional stress is a circular region, and the achieved torsional strain is a continuous strain along a ring shape.
Further, the upper plates 70 of different diameters or the upper plates 70 of different shapes may be selected according to the size of the rotation circular area.
Further, the torque motor 84 is located on a support assembly; the support assembly includes: a cross bar 92 and support bars (91; 93) respectively located at both ends of the cross bar 92.
further, a lower clamping plate 60 is arranged below the lower cover plate 3.
In summary, the method for testing and regulating the in vitro function of the cardiomyocyte by adopting the force stimulation mode applies stress to the whole gel through the force stimulation loading device, and simultaneously applies tensile stress, extrusion stress, shear stress and coupled electrical stimulation to the local gel according to the response condition of a cell population, so that the cardiomyocyte can sense static and dynamic mechanical stimulation in a cell microenvironment through a force sensitive ion channel (such as TRPV4, BK) on the cell membrane, and the electrophysiology on the cell membrane and the biochemical response in the cell are activated, which plays an important role in controlling the structure and the function of the cardiomyocyte; in addition, the force stimulation loading device can simultaneously apply tensile stress or extrusion stress and torsional shear stress to the gel wrapping the myocardial cells through the stretching mechanism and the twisting mechanism, namely simultaneously apply the tensile stress or the extrusion stress and the torsional shear stress to the myocardial cells; this application still through the cooperation of the first bellying on the upper cover plate and the second bellying on the apron down, the gel of being convenient for respectively with the bonding of upper and lower apron, and can reduce the gel greatly and slide the skew, ensure that power can evenly be exerted on the gel, evenly exert on the cardiomyocyte promptly.
in light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (11)
1. a method for testing and regulating the in vitro function of myocardial cells by adopting a force stimulation mode is characterized by comprising the following steps:
step S1, applying force stimulation to the whole gel wrapping the myocardial cells to acquire extension and migration data of the myocardial cells;
Step S2, taking local part of the whole gel, and applying force-electric coupling type stimulation to the local gel to collect the myocardial cell contractility and contraction frequency data; and
And step S3, degrading the gel to obtain cell suspension, so as to collect parameter data of the cell suspension.
2. the method of claim 1, wherein the force stimulation is used to perform in vitro functional testing and control of cardiomyocytes,
The method for acquiring extension and migration data of the myocardial cells by applying force stimulation to the whole gel wrapping the myocardial cells in the step S1 comprises the following steps:
And applying preset strain force loading including one or more of tensile stress, extrusion stress, shear stress or torsional shear stress to the whole gel by adopting a force stimulation loading device, continuously loading the force for 4-72 h, detecting the stretching and migration conditions of the myocardial cells by a timing interval microscopic camera shooting technology, and recording stretching and migration data of the myocardial cells.
3. the method of claim 1, wherein the force stimulation is used to perform in vitro functional testing and control of cardiomyocytes,
The method for taking part of the whole gel and applying force-electricity coupling type stimulation to the part gel to collect the myocardial cell contraction force and contraction frequency data in the step S2 comprises the following steps:
The method comprises the steps of simultaneously applying preset tensile stress, extrusion stress and torsion force loading on local gel by adopting a force stimulation loading device, continuously loading the force for 4-72 hours, simultaneously inserting an inserted microelectrode into the local gel to electrically stimulate cardiac muscle cells, detecting the contraction force and the contraction frequency of the cardiac muscle cells in a force-electricity coupling type loading mode, and recording the data of the contraction force and the contraction frequency of the cardiac muscle cells.
4. the method of claim 1, wherein the force stimulation is used to perform in vitro functional testing and control of cardiomyocytes,
the method for degrading the gel to obtain the cell suspension in the step S3 to collect the parameter data of the cell suspension comprises the following steps:
Respectively degrading the remaining parts of the local gel and the whole gel, eluting the cardiac muscle cells from the gel to obtain cell suspension, and detecting parameter data of the cell suspension according to the obtained cell suspension;
Wherein the parameter data includes: redistribution of various focal adhesion proteins on cell membranes, and expression levels of GATA-4, fi-actin, beta-MHC, NKx2.5, Cx43 and cTnT proteins.
5. The method of claim 1, wherein the force stimulation is used to perform in vitro functional testing and control of cardiomyocytes,
the method for testing and regulating the in-vitro function of the myocardial cells by adopting the force stimulation mode further comprises the following steps:
and step S4, taking a plurality of whole gels, repeating the steps S1-S3 for each whole gel, and applying different preset strain forces to each whole gel and the local gels obtained from each whole gel.
6. The method for in vitro functional testing and regulation of cardiomyocytes according to claim 2 or 3,
the force stimulation loading device comprises:
the device comprises an accommodating body, a stretching mechanism and a twisting mechanism; wherein
The containing body is suitable for containing gel for wrapping the myocardial cells and is made of non-rigid materials;
The stretching mechanism is suitable for stretching or extruding the accommodating body from two opposite sides of the accommodating body so as to apply stretching stress, shearing stress or extrusion stress to the gel; and
The twisting mechanism is adapted to twist the containment body to apply a torsional shear stress to the gel.
7. the method of claim 6, wherein the force stimulation is used to perform in vitro functional testing and control of cardiomyocytes,
the containing body includes: the upper cover plate and the lower cover plate are connected through a clamping cover respectively at two sides;
The upper cover plate and the lower cover plate are both made of elastic rubber materials;
A plurality of first protruding parts are arranged on the inner surface of the upper cover plate at intervals; and
a plurality of second protruding parts are arranged on the inner surface of the lower cover plate at intervals.
8. The method of claim 6, wherein the force stimulation is used to perform in vitro functional testing and control of cardiomyocytes,
The containing body includes: the device comprises an upper cover plate and a lower cover plate, wherein one side of the upper cover plate is connected with a stretching mechanism through a connecting clamping cover, and one side of the lower cover plate is connected with the stretching mechanism through another connecting clamping cover;
the upper cover plate and the lower cover plate are both made of elastic rubber materials;
a plurality of first protruding parts are arranged on the inner surface of the upper cover plate at intervals; and
A plurality of second protruding parts are arranged on the inner surface of the lower cover plate at intervals.
9. the method for in vitro functional testing and regulation of cardiomyocytes according to claim 7 or 8,
the stretching mechanism includes: the screw rod mechanisms are respectively and symmetrically arranged on two opposite sides of the accommodating body;
The screw mechanism includes: the screw rod motor, the transmission shaft, the screw rod and the nut; wherein
The screw rod penetrates through the nut, and one end of the screw rod is connected with the screw rod motor through the transmission shaft;
The other end of the screw rod is connected with the clamping cover;
Each screw motor is suitable for respectively driving the corresponding screw to move in the direction away from or towards the clamping cover so as to stretch or extrude the gel from two opposite sides of the gel; and
Each lead screw motor is suitable for driving corresponding lead screw respectively and moves in the direction of keeping away from corresponding connection card cover, stretches upper cover plate and lower cover plate respectively from both sides to exert shear stress to the gel that is located in the container body.
10. the method of claim 9, wherein the force stimulation is used to perform in vitro functional testing and control of cardiomyocytes,
The torsion mechanism is located on the upper cover plate through an upper clamping plate and comprises: a torsion motor and a torsion assembly; wherein
The torsion assembly includes: the device comprises a shell, a central gear, a plurality of planetary gears meshed with the central gear, and peripheral rims meshed with the planetary gears;
an output shaft of the torsion motor is connected with the central gear;
The peripheral rim is fixed on the upper clamping plate;
The gear shaft of the sun gear and the gear shaft of each planetary gear are fixed on the shell;
The torsion motor is suitable for driving the central gear to drive the planet wheels to rotate so as to drive the peripheral rim to rotate, so that the upper cover plate is driven to rotate through the upper clamping plate, and torsion stress is applied to the gel.
11. The method of claim 10, wherein the force stimulation is used to perform in vitro functional testing and control of cardiomyocytes,
The torsion mechanism is located on the upper cover plate through an upper clamping plate and comprises: a torsion motor and a torsion assembly; wherein
The torsion assembly includes: the device comprises a shell, a central gear, a plurality of planetary gears meshed with the central gear, and peripheral rims meshed with the planetary gears;
an output shaft of the torsion motor is connected with the central gear;
The gear shaft of each planetary gear is fixed on the upper clamping plate;
The gear shaft and the peripheral rim of the central gear are fixed on the shell;
the torsion motor is suitable for driving the central gear to drive each planetary gear to revolve to drive the shaft bracket of each planetary gear to move, and then the upper cover plate is driven to rotate through the upper clamping plate, so that torsion stress is applied to the gel; and
The diameter of the upper clamping plate is smaller than that of the peripheral rim.
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