CN104342370A - Biomechanical system for three-dimensional perfusion tension and compression culture of cells - Google Patents

Abstract

The invention provides a biomechanical system for three-dimensional perfusion stretching and compression culture of cells, which includes a cell bioscaffold for simulating the real three-dimensional growth environment of human cells; A fixture and a cavity for containing the cell bioscaffold; a peristaltic pump, used to drive the cell culture solution into the bioreactor for perfusion; a load applying device, used to provide a cycle for the bonded fixture in the bioreactor Sexual reciprocating stretching, and then applying stretching or compressing mechanical stimulation to the cells; the control module, connected with the computer, controls the load applying device, and adjusts the sample length, stretching ratio, reciprocating frequency and reciprocating times. The invention imitates the real and complicated mechanical environment in the body, realizes the quantitative research on the effect of the controllable perfusion stretching or compression mechanical environment on cell growth while realizing three-dimensional cell culture, and explores the law of cell mechanics-biology coupling.

Description

Biomechanical system for three-dimensional perfusion stretching and compression culture of cells

technical field

The invention relates to a biomechanical cell culture system, specifically, it is used for the perfusion stretching or compression culture test of cells in a three-dimensional bioelastic support to explore the law of cell mechanics-biology coupling. the

Background technique

Cells, the basic unit of life, have always been in a complex three-dimensional mechanical environment. How mechanical stimulation regulates the cytoskeleton, cell shape, cell proliferation and differentiation is still unclear. Studies have shown that the mechanical factors affecting the proliferation and differentiation of bone marrow mesenchymal stem cells mainly include stretch strain, shear stress, compressive stress, and gravity. Due to the complex environment in real organisms, cell separation, culture and mechanical experiments in vitro are important research methods in the field of biomechanics. In order to investigate the influence of mechanical stimuli on cells, it is necessary to apply mechanical stimuli during the culture process in a three-dimensional environment to study the regulation mechanism of mechanical signals on cells. the

At present, the main loading systems for mechanical stimuli commonly used in biomechanical experiments include four-point bending, axial pulling, vacuum action models, and Flexercell loading systems. The method and device for cell culture and load application of three-dimensional materials are not yet common. The difficulty in device design lies in how to culture cells in a three-dimensional system and perfuse stretch/compression. the

Contents of the invention

The purpose of the present invention is to propose a biomechanical system for three-dimensional perfusion stretching or compression culture in view of the deficiencies of current cell culture and mechanical loading devices, and to observe the effects of perfusion stretching or compression mechanical stimulation on cell growth in a three-dimensional environment. Quantitative research to explore the law of cell mechanics-biology coupling. the

In order to solve the above problems, the present invention provides a biomechanical system for three-dimensional perfusion stretching and compression culture of cells, including:

Cell biological scaffolds, used to simulate the real three-dimensional growth environment of human cells;

The bioreactor is provided with a clamp for clamping the cell bioscaffold and a cavity for accommodating the cell bioscaffold;

A peristaltic pump, used to drive the cell culture solution into the bioreactor for perfusion;

The load applying device is used to provide periodic reciprocating pull to the clamp in the bonded bioreactor, and then apply tensile or compressive mechanical stimulation to the cells;

The control module is connected with the computer, controls the load applying device, and adjusts the length of the sample, the stretching ratio, the reciprocating frequency and the number of reciprocating. the

Further, the cell bioscaffold is an elastic porous structure with uniform pore size and good connectivity; it has good biocompatibility or bioinertness, and is non-toxic and harmless to cells; it has a certain mechanical strength so that it can withstand the action of force. It will not be damaged under the environment and maintain the space environment required by cells; it has certain structural deformation and recovery capabilities. the

Further, the material of the bioreactor is a rigid plastic with good biocompatibility, and is composed of two biological containers and an elastic bracket fixture. There are solution inlets and outlets on the biological container, and the corresponding silicone tube and the cell culture medium are connected. The blue-necked bottles are connected to form a perfusion circulation loop under the action of the peristaltic pump. the

Further, the biological container includes:

The first elastic clamp, the upper end is designed with a part that is bonded with the force applying device, and the lower end has an internal thread;

The second elastic clamp has external threads at both ends, a cavity inside, and a solution inlet and outlet on the side wall;

The first elastic fastener and the second elastic fastener, the first elastic fastener and the second elastic fastener can be spliced into a ring, and the end surface of the ring is provided with a threaded hole;

The third elastic clamp is in the shape of an end cap with a central circular hole and has internal threads;

The first elastic fastener and the second elastic fastener are fixed on one end surface of the second elastic clamp, and the first elastic clamp and the third elastic clamp can be screwed to the first elastic clamp respectively. Two elastic clamping ends. the

Further, the shape of the elastic support clamp is dumbbell-shaped, and the interior is hollow, which is used to place the cell biological support; while the material of the elastic support clamp has good elasticity, it has good biological inertia and is non-toxic to cells; The inner cavity wall of the elastic bracket clamp is solidified together with the cell bio-scaffold. the

Further, the load applying device includes a motor, a motor fixing flange, a coupling, a sliding bearing, a copper slider, a movable head, a smooth guide rail, a lead screw, a guide rail support, and a support plate; The screw rotates, and the movable head moves horizontally on the screw and the smooth guide rail; the load applying device is supported by the motor fixing flange and the guide rail and placed on the support plate, and the middle part of the support plate is cut out. Empty for easy installation of the bioreactor. the

Further, a sliding bearing is provided on the smooth guide rail to ensure the stability of movement in the horizontal direction during the stretching or compression process; during the stretching process, the motor speed is adjustable, which can achieve different stretching frequencies. Stretching test. the

Further, the bioreactor is fixed between the movable head and the rail support by pins. the

Further, the control module runs in the laboratory virtual work platform LabVIEW, first selects the matching serial port name, and quantitatively adjusts the sample length, stretch ratio, round-trip frequency and round-trip times as required; "start", "stop" key control device The start and stop of the peristaltic pump; the peristaltic frequency of the peristaltic pump can control the perfusion flow rate, and the flow rate is measured by a flow meter. the

Compared with the prior art, the present invention has the following technical effects:

Compared with the prior art, the present invention has the advantages of imitating the real complex mechanical environment in the body, realizing quantitative research on the influence of the controllable perfusion tensile or compressive mechanical environment on cell growth while realizing three-dimensional cell culture , to explore the law of cell mechanics-biology coupling. the

Description of drawings

Fig. 1 is a schematic structural diagram of the perfusion stretching or compression device of the present invention. the

Fig. 2 is an exploded view of the bioreactor of the present invention. the

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined arbitrarily with each other. the

Embodiment one:

The present invention provides a biomechanical system for three-dimensional perfusion stretching and compression culture of cells as shown in accompanying drawings 1-2, including a load applying device, a bioreactor, an elastic cell support, and a program control part. the

The cell bio-scaffold (not shown in the figure) is used to simulate the real three-dimensional growth environment of human cells; the cell bio-scaffold is an elastic porous structure with uniform pore size and good penetration; it has good biocompatibility or biological inertness, and is not harmful to cells. It is non-toxic and harmless; it has a certain mechanical strength, so that it can not be damaged under the action of force, and maintains the space environment required by cells; it has a certain structural deformation and recovery ability. It is a common method to prepare porous cell bioscaffold materials by uniformly mixing a pore-forming agent with an organic solvent, solidifying and molding after pouring, and finally removing the pore-forming agent. The method is simple and practical, with high porosity and controllable pore size. In this example, polydimethylsiloxane (PDMS) was combined with hydroxyapatite (HA) to improve the pore-forming agent method, and an elastic porous cell bioscaffold material was prepared. Among them, PDMS, as a biologically inert polymer material, has good elasticity and processability. HA is the main inorganic component of hard tissues in the human body and plays a major role in the strength of bones. The HA component makes the material have good biocompatibility and improves the strength of the material. The three-dimensional elastic cell bioscaffold was prepared by compounding PDMS and HA in a certain ratio, so that the prepared three-dimensional elastic cell bioscaffold material was good for cell adhesion on the inner wall of the hole while maintaining good elasticity. the

As shown in Figure 2, the bioreactor is provided with a clamp for clamping the cell bio-scaffold and a cavity for accommodating the cell bio-scaffold; the material of the bioreactor is a rigid plastic with good biocompatibility, consisting of two bio-containers 10 and an elastic support The fixture 11 is composed of a solution inlet and outlet on the biological container 10, which is connected with a blue bottle containing a cell culture solution through a corresponding silicone tube, and forms a perfusion circulation loop under the action of a peristaltic pump. the

The biocontainer 10 comprises: a first elastic clamp 13, the upper end is designed with a part 19 bonded with the force applying device, the bioreactor is fixed between the movable head 9 and the guide rail support 12 by a pin, and the lower end has an internal thread; Elastic clamping 14, both ends have external threads, the inside is a cavity, the side wall has a solution inlet and outlet 18, and the lower end has a groove to connect with the elastic support clamp 11; the first elastic fastener 15 and the second elastic fastener 16, The first elastic fastener 15 and the second elastic fastener 16 can be spliced into a ring, and the end surface of the ring is provided with a threaded hole for fixing the elastic bracket clamp 11; the third elastic clamp 17 has a central circular hole The shape of the end cap has an internal thread; the first elastic clamp 13 and the third elastic clamp 17 can be screwed to the two ends of the second elastic clamp 14 respectively, and the first elastic fastener 15 and the second elastic clamp The firmware 16 is fixed between the third elastic clamp 17 and the second elastic clamp 14 to ensure the sealing of the cavity. the

The shape of the elastic support fixture 11 is dumbbell-shaped and hollow inside, which is used to place the cell biological support; while the material of the elastic support fixture 11 has good elasticity, it has good biological inertia and is non-toxic to cells; the inner cavity wall of the elastic support fixture 11 and The cell bio-scaffold is solidified and bonded together. The elastic holder clamp 11 and the biocontainer 10 are fixed, and then bonded with a stretching or compressing device. the

The peristaltic pump is used to drive the cell culture solution into the bioreactor for perfusion. the

The load-applying device is used to provide periodic reciprocating pull to the fixture in the bonded bioreactor, and then apply tensile or compressive mechanical stimulation to the cells; the load-applying device includes a motor 2, a motor fixing flange 1, and a coupling 3. Sliding bearing 7, copper slider 8, movable head 9, smooth guide rail 4, lead screw 5, guide rail support 12, support plate 6; motor 2 drives screw 5 to rotate through shaft coupling 3, movable head 9 is on the lead screw 5 and the smooth guide rail 4 to move horizontally; the load-applying device is placed on the support plate 6 by the motor fixing flange 1 and the guide rail support 12, and the middle part of the support plate 6 is hollowed out to facilitate the installation of the bioreactor. A sliding bearing 7 is arranged on the smooth guide rail 4, which is used to ensure the movement stability in the horizontal direction during the stretching or compression process; during the stretching process, the rotation speed of the motor 2 is adjustable, which can realize stretching at different stretching frequencies. Stretch test. the

The control module is connected with the computer, controls the load applying device, and adjusts the length of the sample, the stretch ratio, the reciprocating frequency and the reciprocating times. the

The control module runs in the laboratory virtual work platform LabVIEW, first select the matching serial port name, and quantitatively adjust the sample length, stretch ratio, round-trip frequency and round-trip times as required; "Start" and "Stop" keys control the start and stop of the device ; The peristaltic frequency of the peristaltic pump can control the perfusion flow rate, and the flow rate is measured with a flow meter. the

The cell culture solution in the biological container 10 is connected to the solution inlet and outlet 18 of the bioreactor through a silicone tube, and can be perfused; under the action of a peristaltic pump, the solution enters the bioreactor from the inlet for a solution perfusion experiment, and flows out from the outlet to form A circulation loop provides nutrients for cell growth, and the peristaltic frequency of the peristaltic pump controls the flow rate of the solution during perfusion. At the same time, the experimental parameters are set through the software, the computer controls the load-applying device, and the elastic support fixture 11 is pulled by the movable head 9 and the lead screw 5 to drive the stretching movement of the support material and cells. the

Specific implementation: The preparation of the three-dimensional porous cell elastic scaffold is as follows:

Preparation of PDMS/HAP slurry: Ethyl acetate is mixed with PDMS, PDMS: ethyl acetate = 3: 1 (mass ratio). After adding a certain amount of HAP, ball milling and mixing for 5 hours;

Porous material preparation: pour the sieved NaCl and PDMS/HAP slurry into the mold, apply a certain pressure at 80°C, and solidify after 2 hours. The solidified material is taken out from the mold, soaked in ultrapure water, ultrasonically treated, and the water is changed every two hours. After the NaCl is completely dissolved (1-2 days), the required elastic porous material is obtained; the obtained sample is A cylinder with a diameter of 6mm and a length of 10mm. the

The prepared elastic support is coated with curing solution (PDMS and its curing agent Dow Corning 184 are mixed according to the mass ratio of 10:1), put into the inner cavity of the fixture, and cured at 80 degrees Celsius for 2 hours; the material of the elastic support fixture is medical grade silicone rubber, which is soft , elastic, dumbbell-shaped, hollow inside, cavity diameter 6mm, upper and lower base circle diameter 18mm, height 10mm. the

The material of the biological container 10 is a rigid plastic with good biocompatibility, and the inner cavity can hold 5ml-10ml liquid. The inner diameter of the solution inlet and outlet is 2mm, and the outer diameter is 4mm, which is the same material as the reactor and has threads. The screw is fixed, the diameter of the circular groove at the lower end is 18mm, and it is connected with the cavity through a circular channel with a diameter of 6mm. the

Sterilize the above materials, blue-necked bottles, peristaltic silicone tubes, bioreactors, etc. under high temperature and high pressure, and slowly drop the suspension containing rat bone marrow mesenchymal stem cells (rBMSCs) into the scaffold from the top of the scaffold material in a super clean bench. material, and cultivated in the _CO2 incubator for 2h; after planting, place the two ends of the elastic clamp in the groove of the second elastic clamp 14, pass the first elastic fastener 15, the second elastic fastener 16 and 6 The screws are fixed, and the first elastic clamp 13 and the third elastic clamp 17 at both ends are screwed tightly to form a sealed channel. The third elastic clamp 17 is a silicone pad to prevent local seepage; the solution inlet and outlet 18 are connected to the silicone tube , and fixed with an iron wire, the inlet end of the silicone tube is passed into the blue-mouth bottle through the peristaltic pump, and the outlet end is directly connected into the blue-mouth bottle to form a perfusion circuit. The peristaltic frequency of the peristaltic pump adjusts the perfusion rate, which is measured by a flowmeter; The load applying device is bonded by pins, one end is fixed on the guide rail support 12, and the other end is fixed on the movable head 9. The motor 2 drives the screw 5 and the movable head 9 to move horizontally on the smooth guide rail 4 through the coupling 3, thereby pulling The elastic bracket fixture 11 drives the three-dimensional porous elastic cell bio-scaffold attached to the wall to move horizontally, and applies stretching force stimulation to the cells; the load applying device is connected to the computer through the control module, and the corresponding serial port name is selected in the stretching control software, and the parameters are set. . In this example, the sample length is 10mm, the stretching ratio is 10 (10%), the reciprocating frequency is 1HZ, the reciprocating times are 21600, and the peristaltic frequency is 5HZ; the power is turned on, and the three-dimensional perfusion stretching culture of cells begins.

Combining the data in the experiment and comparing with the static culture control group, analyze the influence of the three-dimensional dynamic fluid shear environment and stretching or compression stimulation on the life activities of cells, and at the same time, stretching or compression at different frequencies and different stretching ratios can also be carried out Stimulus quantitative experiments and separate perfusion experiments reveal the law of mechanics-biology coupling. the

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention. the

Claims (9)

1. A biomechanical system for three-dimensional perfusion stretching and compression culture of cells, characterized in that it comprises: Cell biological scaffolds, used to simulate the real three-dimensional growth environment of human cells; A bioreactor is provided with a clamp for clamping the cell bioscaffold and a cavity for accommodating the cell bioscaffold; A peristaltic pump, used to drive the cell culture solution into the bioreactor for perfusion; a load applying device, which is used to provide periodic reciprocating pull to the bonded fixture in the bioreactor, and then apply tensile or compressive mechanical stimulation to the cells; The control module is connected with the computer, controls the load applying device, and adjusts the length of the sample, the stretching ratio, the reciprocating frequency and the number of reciprocating. 2. A biomechanical system for three-dimensional perfusion stretch-compression culture of cells as claimed in claim 1, characterized in that: The cell bioscaffold is an elastic porous structure with uniform pore size and good connectivity; it has good biocompatibility or bioinertness, and is non-toxic and harmless to cells; it has a certain mechanical strength so that it can not As for damage, it maintains the space environment required by cells; it has certain structural deformation and recovery capabilities. 3. A biomechanical system for three-dimensional perfusion stretching and compression culture of cells as claimed in claim 2, characterized in that: The material of the bioreactor is rigid plastic with good biocompatibility, and it is composed of two biological containers and elastic bracket fixtures. The bottles are connected to form a perfusion circulation loop under the action of the peristaltic pump. 4. A biomechanical system for three-dimensional perfusion tension compression culture of cells as claimed in claim 3, wherein the biological container comprises: The first elastic clamping, the upper end is designed with a component that is bonded to the force applying device, and the lower end has an internal thread; The second elastic clamp has external threads at both ends, a cavity inside, and a solution inlet and outlet on the side wall; The first elastic fastener and the second elastic fastener, the first elastic fastener and the second elastic fastener can be spliced into a ring, and the end surface of the ring is provided with a threaded hole; The third elastic clamp is in the shape of an end cap with a central circular hole and has internal threads; The first elastic fastener and the second elastic fastener are fixed on one end surface of the second elastic clamp, and the first elastic clamp and the third elastic clamp can be screwed to the first elastic clamp respectively. Two elastic clamping ends. 5. A biomechanical system for three-dimensional perfusion stretching and compression culture of cells as claimed in claim 4, characterized in that: The shape of the elastic support fixture is dumbbell-shaped, and the interior is hollow, which is used to place the cell biological support; while the material of the elastic support fixture has good elasticity, it has good biological inertia and is non-toxic to cells; the elastic support The internal cavity wall of the fixture is solidified with the cell bio-scaffold. 6. A biomechanical system for three-dimensional perfusion stretch-compression culture of cells as claimed in claim 5, characterized in that: The load applying device includes a motor, a motor fixing flange, a coupling, a sliding bearing, a copper slider, a movable head, a smooth guide rail, a lead screw, a guide rail support, and a support plate; the motor drives the wire through the coupling. The rod rotates, and the movable head moves horizontally on the lead screw and smooth guide rail; the load applying device is supported by the motor fixing flange and guide rail and placed on the support plate, and the middle part of the support plate is hollowed out. Easy to install bioreactor. 7. A biomechanical system for three-dimensional perfusion stretching and compression culture of cells as claimed in claim 6, characterized in that: A sliding bearing is provided on the smooth guide rail to ensure the stability of movement in the horizontal direction during the stretching or compression process; during the stretching process, the motor speed can be adjusted to achieve stretching at different stretching frequencies test. 8. A biomechanical system for three-dimensional perfusion stretch-compression culture of cells as claimed in claim 7, characterized in that: The bioreactor is fixed between the movable head and rail support by pins. 9. A biomechanical system for three-dimensional perfusion stretch-compression culture of cells as claimed in claim 8, characterized in that: The control module runs in the laboratory virtual work platform LabVIEW, first selects the matching serial port name, and quantitatively adjusts the sample length, stretch ratio, round-trip frequency and number of round-trips as required; "start", "stop" keys control the device and stop; the peristaltic frequency of the peristaltic pump can control the perfusion flow rate, and the flow rate is measured by a flow meter.