CN110577895B - Dynamic cell culture method and culture device for simulating in-vivo dynamic environment - Google Patents
Dynamic cell culture method and culture device for simulating in-vivo dynamic environment Download PDFInfo
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Abstract
The invention relates to a dynamic cell culture method and a culture device for simulating an in vivo dynamic environment, wherein the culture method comprises the following steps: adhering cells to a flexible base material in vitro for culture, applying a cyclic dynamic load with a certain frequency and a certain direction to the flexible base material in the culture process, wherein the load in each cyclic period is gradually increased along with time until the load reaches a maximum value and then is gradually reduced along with time, and the deformation rate and the load of the flexible base material are synchronously changed, namely are linearly increased and then linearly reduced; the culture device comprises a cell culture chamber, a clamping device for clamping the flexible base material, a pressure head for applying load to the flexible base material and a centering type slider-crank mechanism for driving the pressure head to reciprocate along the direction vertical to the flexible base material, wherein the clamping device and the pressure head are positioned in the cell culture chamber. The culture method can accurately simulate the interaction relationship of the flexible implant-cells in vivo; the culture device is simple and convenient to assemble and convenient to use, and has great popularization value.
Description
Technical Field
The invention belongs to the technical field of cell culture, and relates to a dynamic cell culture method and a dynamic cell culture device for simulating an in-vivo dynamic environment.
Background
In vitro cell culture is of great significance in judging the growth and development of cells and the integration performance of materials and tissues after the materials are implanted into a body. In vitro cell culture refers to the growth, development and migration of cells under in vitro environment by simulating in vivo environment. Compared with animal experiments and in vivo experiments, in vitro cell culture has the advantages of low cost, simple and convenient operation, easy observation and no ethical problem. However, the existing in vitro cell culture still has many defects, and the in vitro cell culture mainly adopts static culture, including a culture bottle method, a culture dish method, a culture plate method and the like, which can not accurately simulate the in vivo environment, particularly the stress condition, and can not achieve the expected effect of experimental design. Taking the hernia repair patch as an example, the patch is made of a flexible material, and the patch material deforms due to contraction or stretching along with the rhythm of the abdominal wall in the breathing process of a person, so that the stress conduction and cell growth environment change, and the change in the body cannot be simulated by static cell culture, and the obtained result is not representative.
Therefore, it is important to realize dynamic in vitro cell culture by simulating in vitro growth environment more truly, particularly under different stress conditions.
Disclosure of Invention
The invention aims to solve the problem that in the prior art, in-vitro cell culture cannot accurately simulate in-vivo environment, particularly dynamic environment under different stress conditions, and provides a dynamic cell culture method and a culture device for simulating in-vivo dynamic environment, particularly provides an in-vitro dynamic cell culture method and a culture device for simulating axial and radial mechanical stimulation of a flexible substrate in vivo. The method and the device have the advantages of high repeatability of the dynamic simulation experiment, simple and convenient operation and reduction of experiment cost.
In order to achieve the purpose, the invention adopts the following scheme:
a dynamic cell culture method for simulating an in vivo dynamic environment is characterized in that cells are adhered to a flexible base material in vitro for culture, and a circulating dynamic load is applied to the flexible base material in the culture process so as to simulate an in vivo cell mechanical environment and realize dynamic cell culture for simulating the in vivo dynamic environment;
the application frequency of the cyclic dynamic load is Fr, and the application direction is Di;
the load in each cycle period is gradually increased along with time, and then gradually decreased along with time when the load reaches the maximum value;
the deformation rate of the flexible base material and the load change synchronously, and the deformation rate is increased from a to b linearly and then decreased from b to a linearly;
b is b1/b0 is 100%, b1 is the maximum displacement of the flexible base material along Di, b0 is the diameter of an equivalent circle when the flexible base material is not subjected to cyclic dynamic load, and the equivalent circle is a circle with the circumference equal to the side length of the flexible base material.
As a preferred technical scheme:
the dynamic cell culture method for simulating the in vivo dynamic environment comprises the steps that the in vivo cell mechanical environment is the cell mechanical environment at the tendon and the ligament during movement of the tendon and the ligament, the value range of Fr is 0.5-2 Hz, Di is the direction parallel to the flexible base material, a is 0%, the value range of b is 4% -10%, the value range of Fr is determined according to the movement frequency of the tendon and the ligament during daily behaviors of a human, and a and b are determined according to the deformation range of the tendon and the ligament daily, so that the values of Fr, a and b can simulate the human body condition in the range, and therefore the cell mechanical environment at the tendon and the ligament during movement of the tendon and the ligament can be better simulated.
The dynamic cell culture method for simulating the in-vivo dynamic environment is characterized in that the in-vivo cell mechanical environment is the cell mechanical environment in the abdominal wall during daily behaviors of adults, the Fr value ranges from 0.1Hz to 0.5Hz, the Di value is the direction perpendicular to the flexible base material, and a is 0%;
when the daily behavior of the adult is lying or standing still, the value range of b is 5-30%; when the daily behavior of the adult is slow walking or sitting, the value range of b is 10-30%, the value range of Fr is determined according to the respiratory frequency of the daily behavior of the human, and a and b are determined according to the deformation range of the daily abdominal wall, so that the values of Fr, a and b can better simulate the cell mechanical environment in the abdominal wall during the daily behavior of the adult in the range;
or the in vivo cell mechanics environment is the cell mechanics environment at the cardiac muscle during the movement of the cardiac muscle of the human, the range of Fr is 0.1-0.5 Hz, Di is the direction vertical to the flexible base material, a is 0%, the range of b is 10% -20%, the range of Fr is determined according to the heart beating frequency during the daily behavior of the human, and a and b are determined according to the deformation range during the expansion and contraction of the cardiac muscle, so the values of Fr, a and b can better simulate the cell mechanics environment at the cardiac muscle during the movement of the cardiac muscle of the human;
or the in vivo cell mechanical environment is the cell mechanical environment of osteogenesis in the human osteogenesis process, the range of Fr is 0.5-2 Hz, Di is the direction perpendicular to the flexible base material, a is 0%, the range of b is 5-15%, the range of Fr is determined according to the pressure frequency of osteogenesis tissue during the human daily behavior, a and b are determined according to the tissue deformation range, so that the values of Fr, a and b can better simulate the cell mechanical environment of osteogenesis in the human osteogenesis process in the range, and the range of the application time of the cyclic dynamic load is 1-2 h; after the application of the circulating dynamic load is finished, applying a static load, wherein the direction of the static load is perpendicular to the direction of the flexible base material, the maximum displacement of the flexible base material along the direction of the static load accounts for 0.5 percent of the diameter of the equivalent circle when the flexible base material is not subjected to the static load, and the sum of the application time of the circulating dynamic load and the application time of the static load is 24 hours;
or the in vivo cell mechanical environment is a cell mechanical environment in the bladder during the daily behavior of the human bladder (the simulation process is a process for accelerating and simulating the urine holding), the value range of Fr is 0.02-0.1 Hz, Di is a direction perpendicular to the flexible base material, a is 0%, the value range of b is 0-10%, the value range of Fr is determined according to the pressure frequency of the bladder during the daily behavior of the human and represents a process of gradually filling, a and b are determined according to the bladder deformation range, wherein the upper limit of 10% of b is determined according to the bladder deformation during the urine holding (known as forced urine retention), so that the values of Fr, a and b can better simulate the cell mechanical environment in the bladder during the daily behavior of the human bladder within the range;
or the in vivo cell mechanics environment is a cell mechanics environment in the bladder when the human bladder daily behaviors are performed (the simulation process is an unaccelerated process for simulating the change of the human bladder daily behaviors, the process does not contain held urine, and the frequency accords with the contraction and expansion frequency of a human), Di is a direction perpendicular to the flexible base material, a is 0.5%, the value range of b is 0.5-5.5%, the value range of Fr is determined according to the pressure frequency applied to the bladder when the human bladder daily behaviors are performed, a and b are determined according to the bladder deformation range, namely, the cycle process from no urine to gradual accumulation to discharge, and from normal discharge to no urine to gradual accumulation is performed, so that the cell mechanics environment in the bladder when the human bladder daily behaviors are well simulated by the values of Fr, a and b in the range; before the application of the cyclic dynamic load is started, between two adjacent cyclic periods and after the application of the cyclic dynamic load is finished, applying a static load, wherein the direction of the static load is perpendicular to the direction of the flexible base material, the maximum displacement of the flexible base material in the direction of the static load accounts for 0.5 percent of the equivalent circle diameter of the flexible base material when the flexible base material is not subjected to the static load, the number of the cyclic periods is 4, the application time of the static load before the application of the cyclic dynamic load is 2-2.5 h, the application time of the static load between the two adjacent cyclic periods is 2.5-3 h, the application time of the static load after the application of the cyclic dynamic load is finished is 8h, and the sum of the application time of the cyclic dynamic load and the application time of the static load is 24.
The invention also provides a dynamic cell culture device for simulating the in-vivo dynamic environment by adopting the dynamic cell culture method for simulating the in-vivo dynamic environment, in particular an in-vitro dynamic cell culture device for simulating the in-vivo mechanical stimulation of a flexible substrate, which comprises a cell culture chamber, a clamping device for clamping the flexible substrate, a pressure head for applying load to the flexible substrate and a centering crank-slider mechanism for driving the pressure head to reciprocate along the direction vertical to the flexible substrate, wherein the clamping device and the pressure head are positioned in the cell culture chamber (because the clamping device needs to clamp the material for culturing cells, the pressure head needs to be in contact with the material, and the cell culture environment needs to be carried out in the cell culture chamber, the clamping device and the pressure head are positioned in the cell culture chamber) In contrast, the centering slider-crank mechanism has a simpler structure, so the mechanism is adopted by the invention.
As a preferable scheme:
the centering type slider-crank mechanism mainly comprises a computer, a driving motor, a first connecting rod, a disc, a second connecting rod, a slider, a pressure head rod, a sliding chute, a rack and a spur gear, wherein the pressure head rod is an L-shaped rod and consists of a horizontal rod and a vertical rod, and one end of the vertical rod, which is far away from the horizontal rod, is connected with a pressure head;
the computer is used for controlling the operating frequency of the driving motor to control the displacement of the pressure head, one side disc surface of the disc is connected with an output shaft of the driving motor through a first connecting rod, the other side disc surface of the disc is connected with a second connecting rod through a pin, the second connecting rod is connected with a sliding block, the sliding block is installed in the sliding groove and is connected with the horizontal rod through a T-shaped block, the spur gear is sleeved on the horizontal rod and meshed with the rack, and the sliding groove, the rack and the vertical rod are parallel to each other.
According to the dynamic cell culture device for simulating the in-vivo dynamic environment, the driving motor, the first connecting rod and the disc are positioned in the box body, so that the interference of the gas environment caused by the fact that the motor drives the disc to move to an incubator (the incubator is a cell culture chamber and provides gas, temperature and humidity environment for cell culture, the whole device needs to be placed in the incubator to provide proper environment for cell culture) can be reduced, the box body refers to the shell of the dynamic cell culture device, the direct exposure of the internal structure is avoided, the cell culture chamber is a small cabin for applying dynamic culture, cells are cultured in the cell culture chamber, the cell culture chamber is arranged outside the box body, the transmission device is arranged in the box body, and the cell culture chamber and the box body are both arranged in the incubator), and the second connecting rod penetrates through the box body; the sliding groove is arranged on the metal plate; the bottom of the pressure head is provided with a pressure sensor which is connected with a computer; the material of box is iron or steel, and the material of pressure head and pressure head pole is polyethylene or steel, and the material of head rod and second connecting rod is polyethylene, organic glass or steel.
According to the dynamic cell culture device for simulating the in-vivo dynamic environment, the top of the cell culture chamber is provided with the first threaded clamping groove, the cell culture chamber can be rotationally taken down from the whole device to be operated, the top of the first threaded clamping groove is provided with the sealing ring, the sealing ring is sleeved on the pressure head rod, the side wall of the cell culture chamber is provided with the ventilation hole which is communicated with the inside and the outside of the cell culture chamber to connect the inside and the outside gas environment, and the cell culture chamber is made of polyethylene or organic glass. Because the pressure head rod enters the cell culture chamber through the sealing ring, the air flow interference caused by the movement of the pressure head rod in the dynamic culture process can be relieved, and a relatively stable environment is provided.
According to the dynamic cell culture device for simulating the in-vivo dynamic environment, the clamping device is soaked in the culture medium and consists of the second threaded clamping groove and the upper and lower silica gel rings positioned below the second threaded clamping groove. The distance between the upper silica gel ring and the lower silica gel ring can be controlled by rotating the second threaded clamping groove, so that the flexible base material can be clamped.
According to the dynamic cell culture device for simulating the in-vivo dynamic environment, the first threaded clamping groove and the second threaded clamping groove are made of polyethylene, organic glass or steel.
The dynamic cell culture device for simulating the in vivo dynamic environment comprises the following steps:
(1) connecting the box body with a transmission device (a disc, a pin, a second connecting rod, a metal plate, a spur gear, a chute, a slide block, a rack and a pressure head rod) and then placing the box body in an incubator;
(2) planting cells on a flexible substrate, enabling the cells to grow on the flexible substrate, and putting the flexible substrate into a cell culture chamber for clamping;
(3) setting parameters such as the application frequency, the deformation rate, the time and the like of the cyclic dynamic load, carrying out dynamic culture, controlling the pressure head to reciprocate in the vertical direction, causing the flexible base material to generate axial strain, and closing the door of the incubator;
(4) the dynamic culture can be terminated manually or by the end of the culture time, and the cell culture chamber is opened to change the cell liquid or the flexible substrate is taken out to be observed subsequently, so that the dynamic culture is completed.
Has the advantages that:
(1) the culture device prepared by the invention can simulate the mechanical environment of abdominal walls and other parts, culture cells under dynamic conditions, and apply cyclic strain to the material under sterile environment, thereby influencing the behavior of the cells adhered to the material and more accurately simulating the interaction relationship between the flexible implant and the cells in vivo;
(2) the culture device prepared by the invention is simple and convenient to assemble and convenient to use, and axial strain with different action areas can be applied to the flexible base material in the cell culture chamber by replacing the pressurizing chuck;
(3) the culture device prepared by the invention has controllable pressurizing frequency and displacement and can meet the requirements of various materials;
(4) the materials adopted by the culture device prepared by the invention have biocompatibility and cell compatibility;
(5) the culture device prepared by the invention has wide application prospect in the field of in-vitro cell culture;
(6) the culture device prepared by the invention can be used for simulating various in vivo dynamic environments by simple modification.
(7) Compared with static culture, the culture method can simulate the in-vivo environment in the in-vitro culture process, so that cells are cultured under the condition of fitting the in-vivo environment better, and the obtained data is closer to the actual in-vivo situation.
(8) Compared with in vivo experiments, the culture method can complete dynamic culture at lower experiment cost, and has great popularization value.
Drawings
FIG. 1 is a graph of deformation rate versus time in a mechanical environment simulating the cell mechanics of tendons and ligaments during movement of human tendons and ligaments;
FIG. 2 is a graph of deformation rate versus time for a simulation of the mechanical environment of cells within the abdominal wall when an adult is lying flat or standing still;
FIG. 3 is a graph of deformation rate versus time in a mechanical environment of cells in the myocardium during simulated human myocardial motion;
FIG. 4 is a graph of deformation rate versus time for the mechanical environment of cells in the bladder as simulated in example 6 for the daily behavior of the human bladder;
FIG. 5 is a graph of deformation rate versus time for the mechanical environment of cells in the bladder as simulated in example 7 for the daily behavior of the human bladder;
FIG. 6 is a schematic view of the structure of a dynamic cell culture apparatus;
the cell culture device comprises a box body 1, a driving motor 2, a first connecting rod 3, a disc 4, a pin 5, a second connecting rod 6, a metal plate 7, a spur gear 8, a sliding chute 9, a sliding block 10, a rack 11, a pressure head rod 12, a sealing ring 13, a first thread clamping groove 14, a vent hole 15, a pressure head 16, a pressure sensor 17, a cell culture chamber 18, an upper silica gel ring 19, a lower silica gel ring 20 and a second thread clamping groove 21.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
A dynamic cell culture method for simulating an in vivo dynamic environment is characterized in that cells are adhered to a flexible base material in vitro for culture, and a circulating dynamic load is applied to the flexible base material in the culture process so as to simulate the cell mechanical environment at tendons and ligaments when human tendons and ligaments move, thereby realizing the dynamic cell culture for simulating the in vivo dynamic environment;
the application frequency of the circulating dynamic load is Fr, the range of Fr is 0.5-2 Hz, the application direction is Di, and Di is parallel to the flexible base material;
the load in each cycle period is gradually increased along with time, and then gradually decreased along with time when the load reaches the maximum value;
as shown in fig. 1, the deformation rate and the load of the flexible substrate change synchronously, the deformation rate is increased from a to b linearly and then decreased from b to a linearly, a is 0%, and the value range of b is 4-10%;
b is b1/b0 is 100%, b1 is the maximum displacement of the flexible base material along Di, b0 is the diameter of an equivalent circle when the flexible base material is not subjected to cyclic dynamic load, and the equivalent circle is a circle with the circumference equal to the side length of the flexible base material.
Example 2
A dynamic cell culture method for simulating an in vivo dynamic environment is characterized in that cells are adhered to a flexible base material in vitro for culture, and a circulating dynamic load is applied to the flexible base material in the culture process to simulate the mechanical environment of cells in the abdominal wall when an adult lies flat or stands still, so that dynamic cell culture for simulating the in vivo dynamic environment is realized;
the application frequency of the circulating dynamic load is Fr, the range of Fr is 0.1-0.5 Hz, the application direction is Di, and Di is perpendicular to the flexible base material;
the load in each cycle period is gradually increased along with time, and then gradually decreased along with time when the load reaches the maximum value;
as shown in fig. 2, the deformation rate and the load of the flexible substrate change synchronously, and the deformation rate and the load are increased from a to b linearly and then decreased from b to a linearly, wherein a is 0% and b is 5-30%;
b is b1/b0 is 100%, b1 is the maximum displacement of the flexible base material along Di, b0 is the diameter of an equivalent circle when the flexible base material is not subjected to cyclic dynamic load, and the equivalent circle is a circle with the circumference equal to the side length of the flexible base material.
Example 3
A dynamic cell culture method for simulating an in vivo dynamic environment comprises the steps of adhering cells on a flexible base material in vitro for culture, and applying a circulating dynamic load to the flexible base material in the culture process to simulate the mechanical environment of cells in an abdominal wall when an adult walks slowly or sits up, so as to realize dynamic cell culture for simulating the in vivo dynamic environment;
the application frequency of the circulating dynamic load is Fr, the range of Fr is 0.1-0.5 Hz, the application direction is Di, and Di is perpendicular to the flexible base material;
the load in each cycle period is gradually increased along with time, and then gradually decreased along with time when the load reaches the maximum value;
the deformation rate and the load of the flexible base material change synchronously, the deformation rate is increased from a to b linearly and then decreased from b to a linearly, a is 0%, and the value range of b is 10-30%;
b is b1/b0 is 100%, b1 is the maximum displacement of the flexible base material along Di, b0 is the diameter of an equivalent circle when the flexible base material is not subjected to cyclic dynamic load, and the equivalent circle is a circle with the circumference equal to the side length of the flexible base material.
Example 4
A dynamic cell culture method for simulating an in vivo dynamic environment is characterized in that cells are adhered to a flexible base material in vitro for culture, and a circulating dynamic load is applied to the flexible base material in the culture process so as to simulate a cell mechanical environment at cardiac muscle when human cardiac muscle moves and realize dynamic cell culture for simulating the in vivo dynamic environment;
the application frequency of the circulating dynamic load is Fr, the range of Fr is 0.1-0.5 Hz, the application direction is Di, and Di is perpendicular to the flexible base material;
the load in each cycle period is gradually increased along with time, and then gradually decreased along with time when the load reaches the maximum value;
as shown in fig. 3, the deformation rate and the load of the flexible substrate change synchronously, and the deformation rate and the load are increased from a to b linearly and then decreased from b to a linearly, wherein a is 0% and b ranges from 10% to 20%;
b is b1/b0 is 100%, b1 is the maximum displacement of the flexible base material along Di, b0 is the diameter of an equivalent circle when the flexible base material is not subjected to cyclic dynamic load, and the equivalent circle is a circle with the circumference equal to the side length of the flexible base material.
Example 5
A dynamic cell culture method for simulating an in vivo dynamic environment is characterized in that cells are adhered to a flexible base material in vitro for culture, and a circulating dynamic load is applied to the flexible base material in the culture process so as to simulate the mechanical environment of osteoblasts in the human osteogenesis process and realize dynamic cell culture for simulating the in vivo dynamic environment;
the application frequency of the circulating dynamic load is Fr, the range of Fr is 0.5-2 Hz, the application direction is Di, and Di is the direction vertical to the flexible base material;
the load in each cycle period is gradually increased along with time, and then gradually decreased along with time when the load reaches the maximum value;
the deformation rate and the load of the flexible base material change synchronously, the deformation rate is increased from a to b linearly and then decreased from b to a linearly, a is 0%, and the value range of b is 5-15%;
b is b1/b0 is 100%, b1 is the maximum displacement of the flexible base material along Di, b0 is the diameter of an equivalent circle when the flexible base material is not subjected to cyclic dynamic load, and the equivalent circle is a circle with the circumference equal to the side length of the flexible base material;
the value range of the application time of the cyclic dynamic load is 1-2 h; and after the application of the cyclic dynamic load is finished, applying a static load, wherein the direction of the static load is the direction vertical to the flexible base material, the maximum displacement of the flexible base material along the direction of the static load accounts for 0.5 percent of the equivalent circle diameter of the flexible base material when the static load is not applied, and the sum of the application time of the cyclic dynamic load and the application time of the static load is 24 h.
Example 6
A dynamic cell culture method for simulating an in vivo dynamic environment is characterized in that cells are adhered to a flexible base material in vitro for culture, and a circulating dynamic load is applied to the flexible base material in the culture process so as to simulate the mechanical environment of cells in a bladder during the daily behavior of the human bladder and realize the dynamic cell culture for simulating the in vivo dynamic environment;
the application frequency of the circulating dynamic load is Fr, the range of Fr is 0.02-0.1 Hz, the application direction is Di, and Di is perpendicular to the flexible base material;
the load in each cycle period is gradually increased along with time, and then gradually decreased along with time when the load reaches the maximum value;
as shown in fig. 4, the deformation rate and the load of the flexible substrate change synchronously, and the deformation rate and the load are increased from a to b linearly and then decreased from b to a linearly, wherein a is 0% and b ranges from 0 to 10%;
b is b1/b0 is 100%, b1 is the maximum displacement of the flexible base material along Di, b0 is the diameter of an equivalent circle when the flexible base material is not subjected to cyclic dynamic load, and the equivalent circle is a circle with the circumference equal to the side length of the flexible base material.
Example 7
A dynamic cell culture method for simulating an in vivo dynamic environment is characterized in that cells are adhered to a flexible base material in vitro for culture, and a circulating dynamic load is applied to the flexible base material in the culture process so as to simulate the mechanical environment of cells in a bladder during the daily behavior of the human bladder and realize the dynamic cell culture for simulating the in vivo dynamic environment;
the application frequency of the cyclic dynamic load is Fr, the application direction is Di, and Di is the direction vertical to the flexible base material;
the load in each cycle period is gradually increased along with time, and then gradually decreased along with time when the load reaches the maximum value;
as shown in fig. 5, the deformation rate and the load of the flexible substrate change synchronously, the deformation rate is increased from a to b linearly, and then the deformation rate and the load are decreased from b to a linearly, wherein a is 0.5%, and the value range of b is 0.5-5.5%;
b is b1/b0 is 100%, b1 is the maximum displacement of the flexible base material along Di, b0 is the diameter of an equivalent circle when the flexible base material is not subjected to cyclic dynamic load, and the equivalent circle is a circle with the circumference equal to the side length of the flexible base material;
before the application of the cyclic dynamic load is started, between two adjacent cyclic periods and after the application of the cyclic dynamic load is finished, applying a static load, wherein the direction of the static load is perpendicular to the direction of the flexible base material, the maximum displacement of the flexible base material in the direction of the static load accounts for 0.5 percent of the equivalent circle diameter of the flexible base material when the flexible base material is not subjected to the static load, the number of the cyclic periods is 4, the application time of the static load before the application of the cyclic dynamic load is 2-2.5 h, the application time of the static load between the two adjacent cyclic periods is 2.5-3 h, the application time of the static load after the application of the cyclic dynamic load is finished is 8h, and the sum of the application time of the cyclic dynamic load and the application time of the static load is 24.
Example 8
A dynamic cell culture apparatus for simulating an in vivo dynamic environment, which employs the dynamic cell culture method for simulating an in vivo dynamic environment according to any one of embodiments 2 to 7, as shown in fig. 6, includes a cell culture chamber 18, a holding device for holding a flexible substrate, a pressure head 16 for applying a load to the flexible substrate, and a centering slider-crank 10 mechanism for driving the pressure head 16 to reciprocate in a direction perpendicular to the flexible substrate, the holding device and the pressure head 16 being located in the cell culture chamber 18;
the centering type crank slider 10 mechanism mainly comprises a computer, a driving motor 2, a first connecting rod 3, a disc 4, a second connecting rod 6, a slider 10, a pressure head rod 12, a chute 9, a rack 11 and a spur gear 8, wherein the pressure head rod 12 is an L-shaped rod and consists of a horizontal rod and a vertical rod, and one end of the vertical rod, which is far away from the horizontal rod, is connected with a pressure head 16;
the computer is used for controlling the working frequency of the driving motor 2 so as to control the displacement of the pressure head 16, one side disc surface of the disc 4 is connected with an output shaft of the driving motor 2 through a first connecting rod 3, the other side disc surface is connected with a second connecting rod 6 through a pin 5, the second connecting rod 6 is connected with a sliding block 10, the sliding block 10 is installed in a sliding groove 9 and is connected with a horizontal rod through a T-shaped block, a spur gear 8 is sleeved on the horizontal rod and meshed with a rack 11, and the sliding groove 9, the rack 11 and a vertical rod are parallel to each other;
the driving motor 2, the first connecting rod 3 and the disc 4 are positioned in the box body 1, so that the interference of the motor driving the disc 4 to the gas environment in the incubator when the motor drives the disc 4 to move can be reduced, and the second connecting rod 6 penetrates through the box body 1; the chute 9 is arranged on the metal plate 7; the bottom of the pressure head 16 is provided with a pressure sensor 17, and the pressure sensor 17 is connected with a computer; the box body 1 is made of iron or steel, the pressure head 16 and the pressure head rod 12 are made of polyethylene or steel, and the first connecting rod 3 and the second connecting rod 6 are made of polyethylene, organic glass or steel;
a first thread clamping groove 14 is formed in the top of the cell culture chamber 18, a sealing ring 13 is arranged on the top of the first thread clamping groove 14, the sealing ring 13 is sleeved on the pressure head rod 12, a vent hole 15 is formed in the side wall of the cell culture chamber 18, and the cell culture chamber 18 is made of polyethylene or organic glass;
the clamping device consists of a second thread clamping groove 21, an upper silica gel circular ring 19 and a lower silica gel circular ring 20 which are positioned below the second thread clamping groove;
the first thread clamping groove 14 and the second thread clamping groove 21 are made of polyethylene, organic glass or steel.
Example 9
The dynamic cell culture using the dynamic cell culture apparatus for simulating an in vivo dynamic environment of example 8 was carried out as follows:
(1) sterilizing the culture device, and placing the sterile box body and a transmission device (a disc, a pin, a second connecting rod, a metal plate, a spur gear, a chute, a slide block, a rack and a pressure head rod) connected inside and outside the box body into the culture box;
(2) cutting the flexible substrate-electrostatic spinning polycaprolactone fiber membrane material to 3 multiplied by 3cm2Inoculating human fibroblasts onto a flexible substrate in a clean bench;
(3) placing the electrostatic spinning polycaprolactone fiber membrane material under a microscope to observe whether cells grow on the material, placing the flexible base material into a cell culture chamber of a culture device in an ultra-clean workbench, and screwing the second thread clamping groove to enable the flexible base material to be clamped between the two silica gel circular rings;
(4) installing a cell culture chamber at a first thread clamping groove outside a box body and screwing, setting dynamic culture time to be 24 hours by using a computer, setting the application frequency Fr of a circulating dynamic load to be 0.2Hz, a to be 0 percent and b to be 10 percent, setting a contrast group, namely culturing an electrostatic spinning polycaprolactone fiber membrane material with the same size for 24 hours under a static condition, and closing the culture box for cell culture;
(5) taking down the cell culture chamber after 24 hours, putting the cell culture chamber into a super clean workbench, loosening the second threaded clamping groove, and taking out the flexible base material;
(6) the flexible substrate is treated, the cell morphology on the material is observed by using a scanning electron microscope, the cell proliferation rate is measured by treating the cell by using CCK8, the distribution condition of actin stress fibers is observed by staining the cell by using phalloidin, and the collagen yield is measured by using a kit.
After the dynamic culture is carried out on the cells growing on the electrospun polycaprolactone fiber membrane material through the steps, the experimental result shows that the arrangement rule of the fibroblasts is realized through the static culture, the growth of the fibroblasts is more disordered through the dynamic culture, and the actin stress fibers are disordered and arranged. The proliferation rate of the dynamically cultured fibroblasts was increased by 33% compared to the statically cultured cells, and the collagen production was increased by 150%.
Example 10
The dynamic cell culture using the dynamic cell culture apparatus for simulating an in vivo dynamic environment of example 8 was carried out as follows:
(1) sterilizing the culture device, and placing the sterile box body and a transmission device (a disc, a pin, a second connecting rod, a metal plate, a spur gear, a chute, a slide block, a rack and a pressure head rod) connected inside and outside the box body into the culture box;
(2) electrostatic deposition of flexible substratesCutting the spinning polycaprolactone fiber membrane material to 3 multiplied by 3cm2Inoculating human fibroblasts onto a flexible substrate in a clean bench;
(3) placing the electrostatic spinning polycaprolactone fiber membrane material under a microscope to observe whether cells grow on the material, placing the flexible base material into a cell culture chamber of a culture device in an ultra-clean workbench, and screwing the second thread clamping groove to enable the flexible base material to be clamped between the two silica gel circular rings;
(4) installing the cell culture chamber at a first thread clamping groove outside the box body, screwing, setting dynamic culture time to be 24 hours by using a computer, setting the application frequency Fr of a circulating dynamic load to be 0.2Hz, setting a to be 0 percent and setting b to be 5 percent, 10 percent and 20 percent, and closing the culture box for cell culture;
(5) taking down the cell culture chamber after 24 hours, putting the cell culture chamber into a super clean workbench, loosening the second threaded clamping groove, and taking out the flexible base material;
(6) the flexible substrate is treated, the cell morphology on the material is observed by using a scanning electron microscope, the cell proliferation rate is measured by treating the cell by using CCK8, the distribution condition of actin stress fibers is observed by staining the cell by using phalloidin, and the collagen yield is measured by using a kit.
After the dynamic culture is carried out on the cells growing on the electrostatic spinning polycaprolactone fiber membrane material through the steps, the experimental result shows that the growth disorder degree of the fibroblasts is increased and the disorder arrangement degree of actin stress fibers is increased along with the increase of the dynamic culture pressure. The cell proliferation rate of b 20% is 30% lower than that of b 5%, indicating that excessive pressure inhibits cell proliferation, and the collagen production of fibroblasts with b 20% is 41% lower than that of b 5%, indicating that excessive pressure inhibits cell collagen production.
Example 11
The dynamic cell culture using the dynamic cell culture apparatus for simulating an in vivo dynamic environment of example 8 was carried out as follows:
(1) sterilizing the culture device, and placing the sterile box body and a transmission device (a disc, a pin, a second connecting rod, a metal plate, a spur gear, a chute, a slide block, a rack and a pressure head rod) connected inside and outside the box body into the culture box;
(2) cutting the expanded polytetrafluoroethylene film material formed by flexible substrate-hot drawing to 3 x 3cm2Inoculating human chondrocytes onto a flexible substrate in a clean bench;
(3) placing the polytetrafluoroethylene material under a microscope to observe whether cells grow on the material, placing the flexible base material into a cell culture chamber of a culture device in an ultra-clean workbench, and screwing the second threaded clamping groove to enable the flexible base material to be clamped between the two silica gel circular rings;
(4) installing a cell culture chamber at a first thread clamping groove outside a box body and screwing, setting dynamic culture time to be 24 hours by using a computer, setting the application frequency Fr of a circulating dynamic load to be 1Hz, setting a to be 0 percent and setting b to be 20 percent, setting a control group, namely culturing polytetrafluoroethylene materials with the same size for 24 hours under a static condition, and closing the culture box to culture cells;
(5) suspending dynamic culture after 24 hours, taking the cell culture chamber down, putting the cell culture chamber into a clean bench, loosening the second threaded clamping groove, taking out the flexible base material, sucking out the culture medium in the cell culture chamber for liquid replacement, putting the material into the cell culture chamber again after liquid replacement, and screwing the second threaded clamping groove to clamp the flexible base material between the two silica gel rings;
(6) installing the cell culture chamber at an interface outside the box body, screwing, setting the same dynamic culture time and the application frequencies Fr, a and b of the circulating dynamic load by using a computer, starting circulating pressurization, closing the incubator and continuously culturing for 24 hours;
(7) after the dynamic culture is finished, taking down the cell culture chamber, putting the cell culture chamber into a super clean workbench, unscrewing the second threaded clamping groove, and taking out the flexible base material;
(8) the flexible substrate was treated, the morphology of the cells on the material was observed using a scanning electron microscope, the proliferation rate of the cells was measured by treating the cells with CCK8, and the collagen production was measured using the kit.
After the dynamic culture of the cells growing on the expanded polytetrafluoroethylene membrane material formed by hot drawing through the steps, experimental results show that the arrangement rule of the cartilage fibroblasts is realized by static culture. The proliferation rate of dynamically cultured chondrocytes was increased by 23% compared to statically cultured cells, and the collagen production was increased by 140%.
Example 12
The dynamic cell culture using the dynamic cell culture apparatus for simulating an in vivo dynamic environment of example 8 was carried out as follows:
(1) sterilizing the culture device, and placing the sterile box body and a transmission device (a disc, a pin, a second connecting rod, a metal plate, a spur gear, a chute, a slide block, a rack and a pressure head rod) connected inside and outside the box body into the culture box;
(2) cutting the expanded polytetrafluoroethylene film material formed by flexible substrate-hot drawing to 3 x 3cm2Inoculating human chondrocytes onto a flexible substrate in a clean bench;
(3) placing the polytetrafluoroethylene material under a microscope to observe whether cells grow on the material, placing the flexible base material into a cell culture chamber of a culture device in an ultra-clean workbench, and screwing the second threaded clamping groove to enable the flexible base material to be clamped between the two silica gel circular rings;
(4) installing the cell culture chamber at a first thread clamping groove outside the box body, screwing, setting dynamic culture time to be 24 hours by using a computer, setting the application frequency Fr of a circulating dynamic load to be 1Hz, setting a to be 0 percent and setting b to be 5 percent, 10 percent and 20 percent, and closing the culture box for cell culture;
(5) suspending dynamic culture after 24 hours, taking the cell culture chamber down, putting the cell culture chamber into a clean bench, loosening the second threaded clamping groove, taking out the flexible base material, sucking out the culture medium in the cell culture chamber for liquid replacement, putting the material into the cell culture chamber again after liquid replacement, and screwing the second threaded clamping groove to clamp the flexible base material between the two silica gel rings;
(6) installing the cell culture chamber at an interface outside the box body, screwing, setting the same dynamic culture time and the application frequencies Fr, a and b of the circulating dynamic load by using a computer, starting circulating pressurization, closing the incubator and continuously culturing for 24 hours;
(7) after the dynamic culture is finished, taking down the cell culture chamber, putting the cell culture chamber into a super clean workbench, unscrewing the second threaded clamping groove, and taking out the flexible base material;
(8) the flexible substrate was treated, the morphology of the cells on the material was observed using a scanning electron microscope, the proliferation rate of the cells was measured by treating the cells with CCK8, and the collagen production was measured using the kit.
After the dynamic culture of the cells growing on the expanded polytetrafluoroethylene membrane material formed by hot drawing through the steps, the experimental result shows that the growth disorder degree of the human chondrocytes is increased along with the increase of the dynamic culture pressure. The cell proliferation rate of b 20% is 28% lower than that of b 5%, indicating that excessive pressure inhibits cell proliferation, and the collagen production of human chondrocytes with b 20% is 60% lower than that of b 5%, indicating that excessive pressure inhibits cell collagen production.
Claims (6)
1. A dynamic cell culture method for simulating an in vivo dynamic environment is characterized in that: adhering cells to a flexible base material in vitro for culture, and applying a circulating dynamic load to the flexible base material in the culture process to simulate an in vivo cell mechanical environment so as to realize dynamic cell culture simulating the in vivo dynamic environment;
the application frequency of the cyclic dynamic load is Fr, and the application direction is Di;
the load in each cycle period is gradually increased along with time, and then gradually decreased along with time when the load reaches the maximum value;
the deformation rate of the flexible base material and the load change synchronously, and the deformation rate is increased from a to b linearly and then decreased from b to a linearly;
b = b1/b0 × 100%, b1 is the maximum displacement of the flexible base material along Di, b0 is the diameter of an equivalent circle when the flexible base material is not subjected to cyclic dynamic load, and the equivalent circle is a circle with the circumference equal to the side length of the flexible base material;
the in-vivo cell mechanical environment is the cell mechanical environment in the abdominal wall in daily behaviors of adults, the value range of Fr is 0.1-0.5 Hz, Di is the direction vertical to the flexible base material, and a is 0%;
when the daily behavior of the adult is lying or standing still, the value range of b is 5% -30%; when the daily behavior of the adult is slow walking or sitting, the value range of b is 10% -30%;
or the in-vivo cell mechanics environment is a cell mechanics environment at the cardiac muscle during the motion of the cardiac muscle of the human, the Fr has a value range of 0.1-0.5 Hz, the Di is a direction vertical to the flexible base material, a is 0%, and b has a value range of 10-20%;
or the in-vivo cell mechanical environment is the cell mechanical environment of an osteogenesis part in a human osteogenesis process, the Fr has a value range of 0.5-2 Hz, the Di is the direction perpendicular to the flexible base material, a is 0%, b has a value range of 5-15%, and the application time of the cyclic dynamic load has a value range of 1-2 h; after the application of the circulating dynamic load is finished, applying a static load, wherein the direction of the static load is perpendicular to the direction of the flexible base material, the maximum displacement of the flexible base material along the direction of the static load accounts for 0.5 percent of the diameter of the equivalent circle when the flexible base material is not subjected to the static load, and the sum of the application time of the circulating dynamic load and the application time of the static load is 24 hours;
or the in-vivo cell mechanical environment is the cell mechanical environment in the bladder during the daily behavior of the human bladder, the Fr value ranges from 0.02 Hz to 0.1Hz, the Di value ranges from 0% to 0% in the direction perpendicular to the flexible base material, and the b value ranges from 0% to 10%;
or the in-vivo cell mechanical environment is the cell mechanical environment in the bladder during the daily behavior of the human bladder, Di is the direction vertical to the flexible base material, a is 0.5%, and the value range of b is 0.5-5.5%; before the application of the cyclic dynamic load is started, between two adjacent cyclic periods and after the application of the cyclic dynamic load is finished, applying a static load, wherein the direction of the static load is perpendicular to the direction of the flexible base material, the maximum displacement of the flexible base material in the direction of the static load accounts for 0.5 percent of the equivalent circle diameter of the flexible base material when the flexible base material is not subjected to the static load, the number of the cyclic periods is 4, the application time of the static load before the application of the cyclic dynamic load is 2-2.5 h, the application time of the static load between the two adjacent cyclic periods is 2.5-3 h, the application time of the static load after the application of the cyclic dynamic load is finished is 8h, and the sum of the application time of the cyclic dynamic load and the application time of the static load is 24;
the culture method adopts a dynamic cell culture device for simulating an in vivo dynamic environment, and comprises a cell culture chamber, a clamping device for clamping a flexible substrate, a pressure head for applying a load to the flexible substrate and positioned above the flexible substrate, and a centering crank slide block mechanism for driving the pressure head to reciprocate along a direction vertical to the flexible substrate, wherein the clamping device and the pressure head are positioned in the cell culture chamber.
2. A dynamic cell culture apparatus for simulating an in vivo dynamic environment for carrying out a dynamic cell culture method simulating an in vivo dynamic environment according to claim 1, characterized in that: the device comprises a cell culture chamber, a clamping device for clamping a flexible substrate, a pressure head which is used for applying load to the flexible substrate and is positioned above the flexible substrate, and an alignment crank slide block mechanism which is used for driving the pressure head to do reciprocating motion along the direction vertical to the flexible substrate, wherein the clamping device and the pressure head are positioned in the cell culture chamber; the centering type crank sliding block mechanism mainly comprises a computer, a driving motor, a first connecting rod, a disc, a second connecting rod, a sliding block, a pressure head rod, a sliding chute, a rack and a spur gear, wherein the pressure head rod is an L-shaped rod and consists of a horizontal rod and a vertical rod, and one end of the vertical rod, which is far away from the horizontal rod, is connected with a pressure head; the computer is used for controlling the working frequency of the driving motor, one side disc surface of the disc is connected with an output shaft of the driving motor through a first connecting rod, the other side disc surface of the disc is connected with a second connecting rod through a pin, the second connecting rod is connected with a sliding block, the sliding block is installed in the sliding groove and is connected with the horizontal rod through a T-shaped block, the spur gear is sleeved on the horizontal rod and is meshed with the rack, and the sliding groove, the rack and the vertical rod are parallel to each other.
3. The dynamic cell culture device for simulating an in vivo dynamic environment according to claim 2, wherein the driving motor, the first connecting rod and the disc are located in the case, and the second connecting rod passes through the case; the sliding groove is arranged on the metal plate; the bottom of the pressure head is provided with a pressure sensor which is connected with a computer; the material of box is iron or steel, and the material of pressure head and pressure head pole is polyethylene or steel, and the material of head rod and second connecting rod is polyethylene, organic glass or steel.
4. The dynamic cell culture device for simulating in vivo dynamic environment of claim 3, wherein the top of the cell culture chamber is provided with a first threaded groove, the top of the first threaded groove is provided with a sealing ring, the sealing ring is sleeved on the pressure head rod, the side wall of the cell culture chamber is provided with a vent hole, and the cell culture chamber is made of polyethylene or organic glass.
5. The dynamic cell culture device for simulating in vivo dynamic environment according to claim 4, wherein the holding device is composed of a second threaded slot and two upper and lower silicone rings located below the second threaded slot.
6. The dynamic cell culture device for simulating an in vivo dynamic environment according to claim 5, wherein the first threaded locking groove and the second threaded locking groove are made of polyethylene, plexiglass or steel.
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