CN111925939A - Dynamic pressurized tissue engineering valve cell inoculator and use method thereof - Google Patents
Dynamic pressurized tissue engineering valve cell inoculator and use method thereof Download PDFInfo
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Abstract
The invention relates to a dynamic pressurization tissue engineering valve cell inoculator and a using method thereof, and the inoculator comprises an upper base, an upper cover, a lower base, a lower cover and a valve clamp, wherein an upper liquid chamber is arranged in the upper base, a lower liquid chamber is arranged in the lower base, an upper air chamber is arranged in the upper cover, a lower air chamber is arranged in the lower cover, the upper cover is connected with the upper base, an upper diaphragm is arranged between the upper air chamber and the upper liquid chamber, the lower cover is connected with the lower base, a lower diaphragm is arranged between the lower air chamber and the lower liquid chamber, the valve clamp is arranged in the upper liquid chamber, and the upper base is connected with the lower base through a buckle. The invention is used for cell inoculation of human heart tissue engineering valves, can realize pressurized dynamic culture of seed cells, enables the seed cells to be evenly inoculated in the stent material and can enter the interior of the valve stent under the action of pressure, realizes the comprehensive cellularization of the stent and avoids the falling of the cells.
Description
The technical field is as follows:
the invention relates to the technical field of biomedical engineering, in particular to a dynamic pressurization tissue engineering valve cell inoculator and a using method thereof.
Background art:
at present, the global heart disease incidence rate is increased year by year, the number of patients is increased continuously, the incidence rate of heart valve diseases is also in an increasing trend, and more than 30 ten thousand heart valve operations are performed every year in the world. The main surgical approach to heart valve surgery is valve replacement, and two valve substitutes are currently used clinically primarily: both mechanical and biological valves have their drawbacks. Mechanical valves require lifelong anticoagulation treatment, with the risk of bleeding and thromboembolism; bioprosthetic valves have a limited useful life of about 10-15 years and many patients are at risk for a secondary valve change. It is currently widely accepted that tissue engineered valves are the most promising ideal valve replacement because of their advantages of lifetime use without anticoagulation.
The tissue engineering valve is constructed by inoculating seed cells on a valve stent for in vitro culture, and implanting the seed cells into a body after forming a new extracellular matrix and a complete cell layer. However, the current tissue engineering valve is not applied to clinical application, and the main problems are the problems of seed cells and culture conditions. In the past, a static inoculation static culture mode is mostly studied, but the mode is simple to operate, but has the problems of nonuniform cell inoculation and low inoculation efficiency. Patent No. ZL200810232069.1 discloses that the collagen and elastin of acellular scaffolds themselves are crosslinked by epichlorohydrin (C3H5Cl), and simultaneously, rgd (YGRGDSP) polypeptide is linked to the collagen and elastin of acellular xenogenic valve/vascular scaffolds as tissue engineering valves/vascular scaffolds, so that the YGRGDSP polypeptide can be uniformly and firmly distributed throughout the acellular valve scaffolds, so as to enhance the adhesion of seed cells and scaffolds and stimulate cell proliferation. However, this method is not only complicated in steps, but also requires chemical treatment of the valve, and is not suitable for cell seeding of human heart valves. Patent No. ZL201720600607.2 is through setting up the cell reactor in the thermostated container, and the thermostated container internal fixation has driving motor, can drive the cell reactor through driving motor and rotate, makes the seeding cell evenly disperse in the cell reactor to disturb nutrient solution and cell in the cell reactor, make the cell evenly distributed in the cell reactor, in order to prevent the emergence of cell contact suppression phenomenon. However, the device can only realize the uniform distribution of cells, the dynamic pressurization of the cells cannot be realized, the tissue engineering valve cultured in vitro is difficult to grow a complete cell layer, the connection between the cells and the stent is loose and is limited to surface connection, once the tissue engineering valve is implanted into a human body, most of the cells can be circularly washed away by body fluid in the human body, and the device cannot play a role.
The invention has the following patent contents:
technical problem to be solved
The invention aims to provide a dynamic pressurization tissue engineering valve cell inoculator and a using method thereof, which are used for inoculating human heart tissue engineering valve cells and solve the problems that in the prior art, the tissue engineering valve cells cultured in vitro are loosely connected with a stent, and the cells are easily washed away by body fluid in a human body after being implanted into the human body and cannot play a role.
The technical scheme is as follows:
in order to solve the technical problems, the invention adopts the following technical scheme: a dynamic pressurization tissue engineering valve cell inoculator comprises an upper base, an upper cover, a lower base, a lower cover and a valve clamp, wherein an upper liquid chamber is arranged in the upper base, a lower liquid chamber is arranged in the lower base, an upper air chamber is arranged in the upper cover, a lower air chamber is arranged in the lower cover, the upper cover is connected with the upper base, an upper diaphragm is arranged between the upper air chamber and the upper liquid chamber, the lower cover is connected with the lower base, a lower diaphragm is arranged between the lower air chamber and the lower liquid chamber, the valve clamp is arranged in the upper liquid chamber, the upper base is connected with the lower base through a buckle, the upper liquid chamber is communicated with the lower liquid chamber, an upper liquid injection port and an upper air exhaust port which are communicated with the upper liquid chamber are arranged on the upper base, and an upper gas injection port which is communicated with the upper air chamber is arranged on the upper cover, the lower base is provided with a lower liquid pouring port and a lower exhaust port which are communicated with the lower liquid chamber, and the lower cover is provided with a lower gas pouring port which is communicated with the lower gas chamber.
The upper base and the lower base are detachably connected through the buckle, the upper liquid chamber is communicated with the lower liquid chamber, the valve clamp is used for clamping and fixing a sutured three-valve heart valve in the upper liquid chamber, the upper liquid injection port and the lower liquid injection port are respectively used for injecting cell suspensions into the upper liquid chamber and the lower liquid chamber, the upper air exhaust port and the lower air exhaust port are used for exhausting air in the upper liquid chamber and the lower liquid chamber when the cell suspensions are injected, the upper air injection port and the lower air exhaust port are used for inflating and pressurizing the upper air chamber and the lower air chamber through an air pump after the upper liquid chamber and the lower liquid chamber are filled with the cell suspensions, the upper diaphragm and the lower diaphragm deform under the action of air pressure to pressurize the cell suspensions in the upper liquid chamber and the lower liquid chamber, the heart valve seed cells are dynamically cultured in a cell suspension with certain pressure.
Further, valve anchor clamps include seam seat, solid fixed ring and support, gu fixed ring sets up the seam seat with between the support, the support with form the growth chamber between the fixed ring, be equipped with moulding ball in the growth chamber, the seam seat is used for sewing up heart valve's border, and imbeds in the growth chamber, moulding ball is used for pressing heart valve make on the support heart valve with the support laminating, subsequent tissue culture of being convenient for.
Furthermore, a rubber sealing ring is arranged on the joint surface of the upper base and the lower base, and the joint surface of the upper base and the lower base is sealed to prevent the leakage of the cell suspension.
Further, the upper diaphragm and the lower diaphragm are both convex diaphragms and protrude towards the upper liquid chamber and the lower liquid chamber respectively, so that the deformation directions of the upper diaphragm and the lower diaphragm are ensured when the upper air chamber and the lower air chamber are inflated and pressurized.
Furthermore, the upper cover and the lower cover are respectively provided with a pressure gauge communicated with the upper air chamber and the lower air chamber, and the pressure gauges are used for measuring the inflation pressure in the upper air chamber and the lower air chamber, so that the inflation and pressurization pressure can be conveniently controlled.
Further, be equipped with the thread groove in the upper portion liquid chamber, be equipped with the clamping ring in the thread groove, the outside of clamping ring be equipped with thread groove complex external screw thread, valve anchor clamps quilt the clamping ring is fixed in the upper portion liquid chamber, when guaranteeing valve anchor clamps's fixed effect, be convenient for follow take out in the upper portion liquid chamber valve anchor clamps.
Furthermore, the upper air injection port, the upper liquid injection port, the upper air exhaust port, the lower air injection port, the lower liquid injection port and the lower air exhaust port are respectively provided with a detachable threaded plug, so that the plugging is facilitated.
Furthermore, the upper diaphragm and the lower diaphragm are made of high-elasticity silica gel, so that the high-elasticity silica gel diaphragm has high corrosion resistance while ensuring enough elasticity, is not easy to age and is beneficial to long-term use.
The invention also discloses a use method of the dynamic pressurized tissue engineering valve cell inoculator, which mainly comprises the following steps:
firstly, sewing three pieces of heart valves on a valve clamp to form a circular surface, then placing the valve clamp into an upper liquid chamber to fix the valve clamp, and connecting and fixing an upper base and a lower base through a buckle;
injecting a cell suspension with a cell density of 100w/mL from the lower injection port of the lower liquid chamber, simultaneously opening the lower exhaust port of the lower liquid chamber, stopping injection when the liquid level rises to be close to the lower exhaust port of the lower liquid chamber, simultaneously closing the lower injection port and the lower exhaust port of the lower liquid chamber, injecting a cell suspension with a cell density of 100w/mL from the upper injection port of the upper liquid chamber, simultaneously opening the upper exhaust port of the upper liquid chamber, stopping injection when the liquid level rises to be close to the upper exhaust port of the upper liquid chamber, and simultaneously closing the upper injection port and the upper exhaust port of the upper liquid chamber;
adjusting the position of the cell inoculator, injecting liquid and exhausting until the air in the upper liquid chamber and the lower liquid chamber is completely exhausted, connecting the upper air injection port of the upper air chamber with an air pump, pressurizing the upper air chamber through the air pump, connecting the lower air injection port of the lower air chamber with the air pump, pressurizing the lower air chamber through the air pump, closing the upper air injection port and the lower air injection port after pressurization is finished, fixing the cell inoculator on a vertical rotary shaking table, putting the shaking table into a cell culture box for dynamic pressurization inoculation, taking out a valve after inoculation is finished, and putting the valve into a dynamic bioreactor for continuous dynamic culture.
(III) the beneficial effects are as follows:
compared with the prior art, the invention has the beneficial effects that: the upper liquid chamber and the lower liquid chamber are respectively separated from the upper air chamber and the lower air chamber through the upper diaphragm and the lower diaphragm, the valve clamp is arranged in the upper liquid chamber, the upper liquid chamber and the lower liquid chamber which are filled with cell suspension can be pressurized through inflating the upper air chamber and the lower air chamber, heart valve seed cells on the valve clamp are dynamically cultured in the cell suspension with certain pressure, the close fusion of the seed cells and the support is promoted, the comprehensive cellularization of the support is realized, and the inoculation effect of tissue engineering valve cells is ensured.
Description of the drawings:
in order to more clearly illustrate the technical solution in the patent embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below.
FIG. 1 is a schematic diagram of the overall structure of a dynamic pressurized tissue engineering valve cell inoculator according to an embodiment of the present invention;
FIG. 2 is a cross-sectional view of a dynamic pressurized tissue engineering valve cell inoculator according to a patented embodiment of the invention;
FIG. 3 is a schematic structural view of the upper base of a dynamic pressurized tissue engineering valve cell inoculator according to an embodiment of the present invention;
FIG. 4 is a schematic structural view of the lower base of a dynamic pressurized tissue engineering valve cell inoculator according to an embodiment of the present invention;
FIG. 5 is a schematic diagram of a valve clamp of a dynamic pressurized tissue engineering valve cell inoculator according to an embodiment of the present invention;
FIG. 6 is a schematic view of the compression ring of the dynamic pressurized tissue engineering valve cell inoculator according to the patented embodiment of the present invention;
in the figure: 1. an upper base; 2. an upper cover; 3. a lower base; 4. a lower cover; 5. a valve clamp; 51. a sewing seat; 52. a fixing ring; 53. a support; 54. a growth chamber; 55. shaping balls; 6. an upper liquid chamber; 7. a lower liquid chamber; 8. an upper air chamber; 9. a lower air chamber; 10. an upper diaphragm; 11. a lower diaphragm; 12. buckling; 13. an upper liquid injection port; 14. an upper vent; 15. an upper gas injection port; 16. a lower liquid pouring port; 17. a lower exhaust port; 18. a lower air inlet; 19. a rubber seal ring; 20. a manometer; 21. a thread groove; 22. pressing a ring; 23. an external thread; 24. threaded plug
The specific implementation mode is as follows:
the technical solution in the patent embodiment of the present invention will be clearly and completely described below with reference to the accompanying drawings in the patent embodiment of the present invention.
As shown in fig. 1, 2, 3, 4, 5 and 6, the dynamic pressurized tissue engineering valve cell inoculator comprises an upper base 1, an upper cover 2, a lower base 3, a lower cover 4 and a valve clamp 5, wherein an upper liquid chamber 6 is arranged in the upper base 1, a lower liquid chamber 7 is arranged in the lower base 3, an upper air chamber 8 is arranged in the upper cover 2, a lower air chamber 9 is arranged in the lower cover 4, the upper cover 2 is connected with the upper base 1, an upper diaphragm 10 is arranged between the upper air chamber 8 and the upper liquid chamber 6, the lower cover 4 is connected with the lower base 3, a lower diaphragm 11 is arranged between the lower air chamber 9 and the lower liquid chamber 7, the valve clamp 5 is arranged in the upper liquid chamber 6, the upper base 1 and the lower base 3 are connected through a buckle 12, the upper liquid chamber 6 is communicated with the lower liquid chamber 7, an upper liquid injection port 13 and an upper exhaust port 14 which are communicated with the upper liquid chamber 6 are arranged on the upper base 1, an upper gas injection port 15, the lower base 3 is provided with a lower injection port 16 and a lower exhaust port 17 which are communicated with the lower liquid chamber 7, and the lower cover 4 is provided with a lower injection port 18 which is communicated with the lower air chamber 9.
Preferably, the valve clamp 5 comprises a sewing seat 51, a fixing ring 52 and a support 53, the fixing ring 52 is arranged between the sewing seat 51 and the support 53, a growth cavity 54 is formed between the support 53 and the fixing ring 52, a shaping ball 55 is arranged in the growth cavity 54, the sewing seat 51 is used for sewing the edge of the heart valve and placing the heart valve into the growth cavity 54, and the shaping ball 55 is used for pressing the heart valve on the support 53 to attach the heart valve to the support 53, so as to facilitate subsequent tissue culture.
Preferably, a rubber seal 19 is provided on the joint surface of the upper base 1 and the lower base 3 to seal the joint surface of the upper base 1 and the lower base 3 and prevent the cell suspension from leaking.
Preferably, the upper diaphragm 10 and the lower diaphragm 11 are both convex diaphragms and protrude in the direction of the upper liquid chamber 6 and the lower liquid chamber 7, respectively, so as to ensure the deformation direction of the upper diaphragm 10 and the lower diaphragm 11 when the upper air chamber 8 and the lower air chamber 9 are inflated and pressurized.
Preferably, the upper cover 2 and the lower cover 4 are respectively provided with a pressure gauge 20 communicated with the upper air chamber 8 and the lower air chamber 9, and the pressure gauges 20 are used for measuring the inflation pressure in the upper air chamber 8 and the lower air chamber 9 so as to control the inflation pressurization pressure.
Preferably, a thread groove 21 is formed in the upper liquid chamber 6, a pressing ring 22 is formed in the thread groove 21, an external thread 23 matched with the thread groove 21 is formed on the outer side of the pressing ring 22, and the valve clamp 5 is fixed in the upper liquid chamber 6 through the pressing ring 22, so that the valve clamp 5 can be conveniently taken out of the upper liquid chamber 6 while the fixing effect of the valve clamp 5 is guaranteed.
Preferably, the upper air injection port 15, the upper liquid injection port 13, the upper air exhaust port 14, the lower air injection port 18, the lower liquid injection port 16 and the lower air exhaust port 17 are respectively provided with a detachable threaded plug 24, so that plugging is facilitated.
Preferably, the upper diaphragm 10 and the lower diaphragm 11 are made of high-elasticity silica gel, so that the high-elasticity silica gel has sufficient elasticity, good corrosion resistance, is not easy to age, and is beneficial to long-term use.
The use method of the dynamic pressurized tissue engineering valve cell inoculator comprises the following steps:
sewing three heart valves on a sewing seat 51 of a valve clamp 5 into a circular surface, and placing the circular surface into a growth cavity 54 to ensure that the heart valves are pressed and fixed on a support 53 by a shaping ball 55; the valve clamp 5 is put into the thread groove 21 of the upper liquid chamber 6, and the pressing ring 22 is screwed into the thread groove 21, so that the valve clamp 5 is fixed; connecting an upper base 1 and a lower base 3 through a buckle 12, arranging a rubber sealing ring 19 on the joint surface of the upper base 1 and the lower base 3, and sealing the joint surface of the upper base 1 and the lower base 3; injecting a cell suspension with a cell density of 100w/mL from the lower injection port 16 of the lower liquid chamber 7 while opening the lower vent 17 of the lower liquid chamber 7, stopping injection when the liquid level rises to be close to the lower vent 17 of the lower liquid chamber 7, and simultaneously closing the lower injection port 16 and the lower vent 17 of the lower liquid chamber 7; injecting a cell suspension with a cell density of 100w/mL from the upper injection port 13 of the upper liquid chamber 6, simultaneously opening the upper vent 14 of the upper liquid chamber 6, stopping injection when the liquid level rises to be close to the upper vent 14 of the upper liquid chamber 6, and simultaneously closing the upper injection port 13 and the upper vent 14 of the upper liquid chamber 6; adjusting the position of the cell inoculator, and discharging air while injecting liquid until the air in the upper liquid chamber 6 and the lower liquid chamber 7 is completely discharged; connecting an upper gas injection port 15 of the upper gas chamber 8 with an air pump, pressurizing the upper gas chamber 8 through the air pump, measuring the pressure through a pressure gauge 20, and closing the upper gas injection port 15 when the pressure value reaches a required pressure value (80-120 mmHg); connecting a lower air inlet 18 of the lower air chamber 9 with an air pump, pressurizing the lower air chamber 9 by the air pump, measuring the pressure by a pressure gauge 20, and closing the lower air inlet 18 when the pressure value reaches a required pressure value (80-120 mmHg); fixing a cell inoculator on a vertical rotary shaking table, setting the rotation speed of the shaking table to be 1rpm, putting the shaking table into a cell incubator, and dynamically pressurizing and inoculating for 12-24 hours; after inoculation is finished, the upper air injection port 15 and the lower air injection port 18 are opened to deflate and reduce pressure, cell suspensions in the upper liquid chamber 6 and the lower liquid chamber 7 are sucked out, the buckle 12 is loosened, the valve is taken out, and the valve is put on the dynamic bioreactor to continue dynamic culture.
In conclusion, the dynamic pressurization tissue engineering valve cell inoculator and the use method thereof provided by the invention are used for inoculating human heart tissue engineering valve cells, and solve the problems that in the prior art, the tissue engineering valve cells cultured in vitro are loosely connected with a stent, and the cells are easily washed away by body fluid in a human body after being implanted into the human body, so that the cells cannot play a role.
The invention patent is described above by way of example, but the invention patent is not limited to the above-described specific embodiments, and any changes or modifications made based on the invention patent are within the scope of the invention patent claims.
Claims (9)
1. A dynamic pressure tissue engineering valve cell inoculator, characterized in that: the valve clamp is arranged in the upper liquid chamber, the upper base is connected with the lower base through a buckle, the upper liquid chamber is communicated with the lower liquid chamber, an upper liquid injection port and an upper gas injection port which are communicated with the upper liquid chamber are arranged on the upper base, and a lower liquid injection port and a lower gas injection port which are communicated with the lower liquid chamber are arranged on the lower base, and a lower air inlet communicated with the lower air chamber is arranged on the lower cover.
2. The dynamic pressurized tissue engineered valve cell inoculator of claim 1, wherein: the valve clamp comprises a sewing seat, a fixing ring and a support, wherein the fixing ring is arranged between the sewing seat and the support, a growth cavity is formed between the support and the fixing ring, and a shaping ball is arranged in the growth cavity.
3. The dynamic pressurized tissue engineered valve cell inoculator of claim 1, wherein: and a rubber sealing ring is arranged on the joint surface of the upper base and the lower base.
4. The dynamic pressurized tissue engineered valve cell inoculator of claim 1, wherein: the upper diaphragm and the lower diaphragm are both convex diaphragms that project in the direction of the upper liquid chamber and the lower liquid chamber, respectively.
5. The dynamic pressurized tissue engineered valve cell inoculator of claim 1, wherein: and the upper cover and the lower cover are respectively provided with a pressure gauge communicated with the upper air chamber and the lower air chamber.
6. The dynamic pressurized tissue engineered valve cell inoculator of claim 1, wherein: be equipped with the thread groove in the upper portion liquid chamber, be equipped with the clamping ring in the thread groove, the outside of clamping ring be equipped with thread groove complex external screw thread, the valve anchor clamps quilt the clamping ring is fixed in the upper portion liquid chamber.
7. The dynamic pressurized tissue engineered valve cell inoculator of claim 1, wherein: the upper air injection port, the upper liquid injection port, the upper air exhaust port, the lower air injection port, the lower liquid injection port and the lower air exhaust port are all provided with detachable threaded plugs.
8. The dynamic pressurized tissue engineered valve cell inoculator of claim 1, wherein: the upper diaphragm and the lower diaphragm are made of high-elasticity silica gel.
9. Use of a dynamic pressurized tissue engineering valve cell inoculator according to any of claims 1-8, comprising the steps of:
firstly, sewing three pieces of heart valves on a valve clamp to form a circular surface, then placing the valve clamp into an upper liquid chamber to fix the valve clamp, and connecting and fixing an upper base and a lower base through a buckle;
injecting a cell suspension with a cell density of 100w/mL from the lower injection port of the lower liquid chamber, simultaneously opening the lower exhaust port of the lower liquid chamber, stopping injection when the liquid level rises to be close to the lower exhaust port of the lower liquid chamber, simultaneously closing the lower injection port and the lower exhaust port of the lower liquid chamber, injecting a cell suspension with a cell density of 100w/mL from the upper injection port of the upper liquid chamber, simultaneously opening the upper exhaust port of the upper liquid chamber, stopping injection when the liquid level rises to be close to the upper exhaust port of the upper liquid chamber, and simultaneously closing the upper injection port and the upper exhaust port of the upper liquid chamber;
adjusting the position of the cell inoculator, injecting liquid and exhausting until the air in the upper liquid chamber and the lower liquid chamber is completely exhausted, connecting the upper air injection port of the upper air chamber with an air pump, pressurizing the upper air chamber through the air pump, connecting the lower air injection port of the lower air chamber with the air pump, pressurizing the lower air chamber through the air pump, closing the upper air injection port and the lower air injection port after pressurization is finished, fixing the cell inoculator on a vertical rotary shaking table, putting the shaking table into a cell culture box for dynamic pressurization inoculation, taking out a valve after inoculation is finished, and putting the valve into a dynamic bioreactor for continuous dynamic culture.
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