CN101372661B - Adjustable pouring type vascular tissue engineering reactor having rotating cultivation cavity - Google Patents
Adjustable pouring type vascular tissue engineering reactor having rotating cultivation cavity Download PDFInfo
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Abstract
The invention provides a vascular tissue engineering reactor which has the functions of culture cavity rotation, dual perfusion inside and outside vascular tissues and intravascular nutrient solution pulsation. The reactor can simultaneously realize the intravascular perfusion of the culture solution, the extravascular perfusion of the culture solution, the rotation of a culture cavity, the reciprocating tension of blood vessels in culture along the axial direction and any combination thereof. In addition, the reactor can realize multi-blood vessels culture at the same time and handling, recording and/or displaying of the pressure data.
Description
Technical field
The present invention relates to cell cultures, field of tissue engineering technology, more particularly relate to a kind of vascular tissue engineering reactor with culture chamber spinfunction.
Background technology
Up to now, autotransplantation, allogeneic blood vessel, heterogenous blood vessel and synthetic blood vessel all can not become ideal arteries surrogate (the particularly following blood vessel of 6mm bore), but have brought hope with arteries organizational project bioreactor for constructing and research organization's through engineering approaches arteries for this field in recent years.At present, the research contents of arteries used in tissue engineering bio-reactor mainly comprises:
1. vascular tissue is realized and even, the high-density inoculation of control cell in support: the direct filling type bioreactor of discovering in recent years is being better than non-direct filling type bioreactors such as static state, rotation stirring-type aspect cell inoculation density and the homogeneity, this technology has been applied to myocardial cell, vascular tissue and liver cell organizational project
2. improve the mass transfer situation: for vascular tissue, the rotary bio-reactor of wall provides certain low shear-stress by dynamic rotation Laminar Flow for cell, improved mass transfer situation culture effect preferably and be better than rotating-filling rate of the rotary bio-reactor of stirring type bioreactor wall, the problems such as balance of the shearing stress size that pair cell applies, cytotrophy thing and metabolite mass transfer all can exert an influence to culture effect, therefore need be at parameters such as specific organizational controls, adjusting filling rates, with the mass transfer effect that obtains to optimize.
3. in the intravascular tissue engineering cultivation, apply suitable mechanical force, controlling arterial vascular engineering tissue makes up: a large amount of fluid shear stress that studies show that have the influence of highly significant to the growth of vascular endothelial cell, periodically mechanical extension can improve the organizational structure's performance of the smooth muscle cell structure that is planted on the polymeric stent material and the expression of elastin, and radial pulsation stress can improve the structural strength of engineering blood vessel.Therefore, in engineered culturing process, apply mechanical loading, can directly promote cell specific vegetative period to multi-functional differentiation, promote the structure of engineering tissue.
Boris A.Nasseri etc. has designed a kind of engineering blood vessel bio-reactor and has been used for cell inoculation and cultivation (Dynamic Rotational Seeding and CellCulture System for Vascular Tube Formation.TissueEngineering 2003; 9 (2): 291-299.): provide rotary power with hybrid heater for the blood vessel cultivation, produce the wall rotation of nutrient solution around the rotation of hybrid heater central shaft, improve mass transfer with respect to vessel wall by the blood vessel culture chamber; Reactor adopts dabbling mode in the blood vessel.
Ralf Sodian etc. has designed a kind of engineering blood vessel bio-reactor and has been used for the perfusion inoculation of blood vessel and cultivates (Tissue-Engineering Bioreactors:A NewCombined Cell-Seeding and Perfusion System for VascularTissue Engineering.Tissue Engineering 2002; 8 (5): 863-870.): produce pulsating flow with pneumatic mode extruding nutrient solution storage chamber silicone skirt, carry out pulsating flow pouring and cultivate, cell inoculation is combined with the pulsating flow pouring cultivation, dynamically inoculation improves cell distribution; Perfusion pulsating flow in the blood vessel; Reactor adopts dabbling mode in the blood vessel, and culture chamber does not have the wall rotation.
Chrysanthi Williams etc. has designed and a kind ofly provides power by peristaltic pump, engineering blood vessel bio-reactor that can perfusion inside and outside the blood vessel is used for small-caliber vascular and cultivates, outer perfusion improves the cytotrophy situation, interior perfusion provides shear-stress and pulsating flow surrounding, and culture chamber does not have wall rotation (Perfusion Bioreactor for SmallDiameter Tissue-Engineered Arteries.Tissue Engineering2004; 10 (5-6): 930-941.).
Yuji Narita etc. has designed a kind of vascular tissue engineering reactor (Novel Pulse Duplicating Bioreactor System forTissue-Engineered Vascular Construct.Tissue Engineering2004 that simulates physiological pulsating flow; 10 (7-8): 1224-1233.): the to-and-fro movement by sacculus formula chamber promotes the nutrient solution in the conformability chamber, produces pulsating flow, variable valve is set before and after culture chamber and has pulsating waveform under the liquid storage cylinder simulation physiological status of check valve; Reperfusion mode is perfusion in the blood vessel, no wall rotation.
Satish, C.Muluk etc. have designed the vascular reaction device that the enough stretching motors of a kind of energy load tensile stress and load torsional stress with micro-stepping motors, provide pulsating flow by program-controlled valve, adopt dabbling mode in the blood vessel, no wall rotation (Enhancementof tissue factor expression by vein segments exposed tocoronary arterial hemodynamics.Journal of vascularsurgery:official publication, the Society for VascularSurgery[and] International Society for CardiovascularSurgery, North American Chapter 1998; 27 (3): 521-527).
Craig A.Thompson etc. has designed a kind of vascular tissue engineering reactor (A Novel Pulsatile, Laminar Flow Bioreactorfor the Development of Tissue-Engineered VascularStructures.Tissue Engineering 2002 that simulates physiological pulsating flow; 8 (6): 1083-1088.): provide pressurized air flow to promote nutrient solution with ventilator and flow, regulate simulation arteries diastolic pressure by vacuum breaker, with the flow direction of check valve controlled liq in the loop, simulated blood vessel pulsating waveform as pulsation source; Perfusion in the blood vessel, no wall rotation.
In sum, there is bigger defective in existing vascular tissue engineering reactor: the one, do not think over factors such as flow impedance, conformability (being both surge capability of elasticity of vessel wall), resistance, inertial flow, and can't provide the nearly physiology flowing environment that is similar to intra-arterial blood pulses stream mode to the simple analog of vascular flow dynamics environment; The 2nd, can't provide the periodicity axial tension of approximate physiological status, mechanical force such as stretching, distortion load radially simultaneously for vascular tissue; The 3rd, most modes that adopt no wall rotation, at cell evenly, be still waiting improvement aspect high-density inoculation and the mass transfer.Therefore, press for development and can load arteries simultaneously and cultivate required multiple mechanical environment, help cell evenly, the high-density inoculation, help mass transfer, have the bio-reactor of good Modulatory character.
Summary of the invention
The present invention is directed to the body interior mechanics environment of arteries tissue growth, a kind of vascular tissue engineering reactor with culture chamber spinfunction is provided, this reactor has excellent mass transferring performance, can cultivate for arteries nearly physiological pulsating flow surrounding is provided.This reactor can also be as the construction platform with multi-purpose reactor, these functions make it possible to the while loading classes and are similar to the mechanical loadings such as periodic radial stretching that arteries is subjected under the physiological status, and above-mentioned mechanical loading has good Modulatory character; Simultaneously can provide horizontal rotation, make reactor have excellent mass transferring performance, specifically comprise:
1, can drive under the situation of vascular tissue's culture chamber rotation by vascular tissue's culture chamber rotating machine, realize inside and outside while or independent perfusion culture liquid, provide and to have carried out the inside and outside while (or separately) dabbling equipment scheme;
2, can realize pulsing function (require inside and outside perfusion by two independently the nutrient solution loop finish);
The purpose of this invention is to provide a kind of multi-modal arteries tissue engineering reactor, the organizational project arteries that utilizes this reactor can carry out multiple diameter and length is cultivated, also can carry out vascular cell, the biological research of vascular tissue, its characteristics are:
1. adopt new arteries tissue engineering reactor principle of design: simulation arterial blood mobile conformability and resistance to flow on flow circuits produce nearly physiological pulsating flow; Can simulate ripple frequency, pressure and the flow waveform of different artery segments, simulation hypertension, shearing force, hemodynamic situation such as low shear-stress;
2. loading classes is similar to the mechanical loadings such as radially stretching that arteries is subjected under the physiological status simultaneously, and above-mentioned mechanics loads has good Modulatory character;
3. can carry out the rotation of reactor wall with regulatable rotating speed, inside and outside all can the realization of its medium vessels poured into, and makes reactor have excellent mass transferring performance.
According to an aspect of the present invention, provide a kind of vascular tissue engineering reactor, it is characterized in that comprising:
Pour into loop culture chamber inlet ductwork in liquid storage bottle, the blood vessel in perfusion fluid drive unit, the blood vessel by pouring in the continuous successively blood vessel of liquid line;
Vascular tissue's culture chamber, thus the downstream end of described culture chamber inlet ductwork enters into vascular tissue's culture chamber and is used for being connected with the upstream extremity of vascular tissue to be cultivated and is used to make the inside that flows into described vascular tissue from the nutrient solution of perfusion liquid storage bottle in the described blood vessel;
Perfusion loop culture chamber export pipeline in blood vessel, thus its upstream extremity enters into described vascular tissue culture chamber and the nutrient solution of the inside that is used to derive described vascular tissue of being used for being connected with the downstream end of vascular tissue to be cultivated;
An outer filling loop liquid storage bottle;
An outer filling loop liquid driving device, it links to each other by liquid line with described outer filling loop liquid storage bottle;
An outer filling loop culture chamber inlet ductwork, its upstream extremity links to each other with described outer filling liquid driving device by liquid line, and its downstream end enters into described culture chamber inside, thereby is used for the nutrient solution from outer filling loop liquid storage bottle is injected the space outside the described vascular tissue in the culture chamber; And
An outer filling loop culture chamber export pipeline, its upstream extremity is placed in the described culture chamber and its downstream end extends to outside the described culture chamber, is used for the nutrient solution outside the described vascular tissue in the described culture chamber is drawn;
A culture chamber rotating machine;
A culture chamber rotary actuator, the rotating shaft of itself and described culture chamber rotating machine is coupled, and is coupled with described culture chamber, is used for the rotation of described culture chamber rotating machine driven passing to described culture chamber, rotates thereby drive described culture chamber.
The invention discloses a kind of a kind of infinite embodiment of vascular tissue engineering reactor, this embodiment has following feature:
Reactor has perfusion and two perfusions of blood vessel outer filling loop in the blood vessel, and mechanical loading loads and proofing unit is positioned on the loop, and the observing and controlling part is made up of concentrator, small signal amplifier, driving mechanism, treater and indicating meter.
One concrete but among the infinite embodiment, liquid storage bottle is provided with the sterile air interchanger, the pH meter interface is arranged; The material of liquid storage bottle for can the withstand high temperatures disinfectant, the glass that does not have bio-toxicity, stainless steel, plastics, polycarbonate; The capacity of liquid storage bottle is 0.2L-2L;
As concrete but infinite embodiment, liquid driving device is peristaltic pump, linear electric motors and/or pulsation source, can realize flowing environment such as steady state flow, simple pulsating flow, nearly physiological pulsating flow by industrial computer controlling flow moving curve.
One concrete but among the infinite embodiment, peristaltic pump provides steady state flow, flow range is 0-1000ml/ minute, pressure is 0-250mmHg; In another specific embodiment, linear electric motors provide simple pulsating flow, and ripple frequency is 0-200 time/minute, and flow range is 0-1000ml/ minute, and pressure is 0-250mmHg; Pulsation source provides nearly physiological pulsating flow, and pulsation source is made of pulsation chamber, the linear electric motors that two ends are equipped with unidirectional non-return valve.
The pulsation chamber is the volume fixed closed chamber of full of liquid, the below of closed chamber links to each other with linear electric motors by Air-tight piston, closed chamber links to each other with the reactor pipeline of upstream and downstream by its central elastic hose, in the upstream and downstream exit of flexible pipe a unidirectional non-return valve is arranged respectively; The material of elastic hose is resilient organic materials.
One concrete but in the infinite embodiment, respectively there is a liquid dispenser at vascular tissue culture chamber two ends, connect perfusion upstream and downstream pipeline and vascular tissue's culture in the blood vessel with removably; Also respectively there is an outlet at vascular tissue culture chamber two ends, link to each other with blood vessel outer filling pipeline with removably respectively.
As concrete but infinite embodiment, the material of vascular tissue's culture chamber for can the withstand high temperatures disinfectant, the glass that does not have bio-toxicity, stainless steel, plastics, polycarbonate, be shaped as right cylinder or symmetric polyhedron, length is for example 10-50cm, and capacity is for example 50-500ml; Vascular tissue's culture chamber keeps cultivating cavity airtight by gasket and bolt, but the material of gasket and bolt is the organic and inorganic material of withstand high temperatures disinfectant.
As concrete but infinite embodiment, the volume of priming line is for example 100-500ml in the reactor blood vessel, and the volume of outer filling pipeline is for example 100-800ml.
As concrete but infinite embodiment, liquid dispenser is positioned at vascular tissue's culture chamber, the container that outlet is all arranged for two ends, its end that links to each other with perfusion upstream and downstream pipeline in the reactor blood vessel has an outlet, the end that links to each other with the vascular tissue culture has for example 1-6 outlet, liquid in the reactor blood vessel in the priming line is entered by the upstream extremity of upstream liquid divider, flowing out dispersion by downstream end enters in each vascular tissue's culture, flow out by the upstream extremity inflow of downstream divider and from its downstream end again, compile priming line in the intravasation.
As concrete but infinite embodiment, one of liquid dispenser is fixing vertically, and another can move back and forth in vascular tissue's culture chamber vertically.
As concrete but infinite embodiment, the liquid dispenser material for can the withstand high temperatures disinfectant, the glass that does not have bio-toxicity, stainless steel, plastics, polycarbonate, the diameter of its outlet is 1-12mm.
As concrete but infinite embodiment, stepper-motor drives vascular tissue's culture chamber rotary transmission gear.
As concrete but infinite embodiment, reactor is used for the arterial vascular cultivation of organizational project, and analog approximation in the overall waveform of physiological pulsating flow, secondaries, amplitude with the time mutually, haemodynamics environment such as the senior executive internal pressure of analogy under pathological state, shearing force, low shear-stress;
As concrete but infinite embodiment, the linear electric motors to-and-fro movement is penetrated the process that blood is gone into aorta to the overflow mould pseudocardia that the pulsation chamber produces, by regulating linear electric motors, conformability setter and dragulator, adjust pulse waveform, pressure and flow range and the frequency of beating within the specific limits, ripple frequency, pressure and flow waveform to the different artery segments of blood vessel simulation cultivated, simulation hypertension, shearing force, hemodynamic situation such as low shear-stress.
As concrete but infinite embodiment, stepper-motor, vascular tissue's culture chamber rotary transmission gear constitute swivel arrangement; Wherein vascular tissue's culture chamber rotary transmission gear group is positioned at vascular tissue's culture chamber one end, is coupled with the vascular tissue culture chamber;
As concrete but infinite embodiment, vascular tissue engineering reactor of the present invention is used for the arterial vascular cultivation of organizational project, arteries tubing comprises through taking off the animal blood vessels that cell is handled, tubing that constitutes by natural materialss such as collagen, silk fiber, wool fibers and the tubing that constitutes by superpolymer such as PLGA, PLA, PLG, sodium alginate, tetrafluoroethylene, blood vessel diameter is for example between the 1-12mm, length of vessel is for example between the 10-30cm, and the cell on the blood vessel tubing comprises vascular endothelial cell, vascular smooth muscle cell and adventitia inoblast.
As concrete but infinite embodiment, the whole priming line of vascular tissue engineering reactor of the present invention is kept 37 ℃, 5-15%CO by putting into the animal cell culture case
2, the culture condition of 95% relative humidity.
Reactor assembly of the present invention is installed, convenient disassembly; Whole reactor, comprise that perfusion channel pipeline and joint are detachable, can sterilize that exemplary conditions for sterilization is for for example: 130 ℃, 3 normal atmosphere, 1 hour time.
Vascular tissue engineering reactor of the present invention, compared following beneficial effect with pre-existing reactors:
1. vascular tissue engineering reactor of the present invention concrete but infinite embodiment, overcome the arteries tissue engineering reactor of using always and can't simulate the impedance characteristics such as conformability, inertial flow and resistance to flow of arteries blood flow, the shortcoming that is similar to intra-arterial blood pulses stream environment can't be provided, can on flow circuits, simulate impedance characteristics such as arterial blood mobile conformability, inertial flow and resistance to flow, can on flow circuits, simulate the pressure and the flow pulse wave of nearly physiological pulsating flow, produce nearly physiological pulsating flow; Can adjust pulse waveform, pressure and flow range within the specific limits, reach ripple frequency, simulate ripple frequency, pressure and the flow waveform of different artery segments, simulation hypertension, shearing force, hemodynamic situation such as low shear-stress;
2. in conduct of vascular tissue engineering reactor of the present invention but among the infinite embodiment, can on the blood vessel culture simultaneously or separately loading classes be similar to that arteries is subjected to mechanical loadings such as radially stretching under the physiological status, above-mentioned mechanical loading has good Modulatory character;
3. concrete but among the infinite embodiment of vascular tissue engineering reactor of the present invention, reactor culturing room partly is spirotheca-direct fill-up mode, can carry out the rotation of reactor wall with regulatable rotating speed, inside and outside all can the realization of blood vessel poured into, and makes reactor have excellent mass transferring performance;
4. vascular tissue engineering reactor of the present invention a kind of concrete but infinite embodiment, both can be used for cultivating the arteries tissue, also can be used as research vascular cell, the biology of tissue and the instrument of mechanical property, have bigger popularizing application prospect and bigger potential economic results in society.
Description of drawings
Fig. 1 has shown an embodiment with vascular tissue engineering reactor of culture chamber spinfunction of the present invention.
Fig. 2 has been used to show the structure and the work of a kind of embodiment of pulsation source of the present invention.
Fig. 3 has shown a further embodiment of the present invention, wherein uses pulsation source as perfusion fluid drive unit in the blood vessel.
Embodiment
As shown in Figure 1, have in the embodiment of vascular tissue engineering reactor of culture chamber spinfunction in the present invention, perfusion liquid storage bottle 101 links to each other with perfusion fluid drive unit 104 in the blood vessel by pipeline in the blood vessel.Perfusion fluid drive unit 104 can adopt peristaltic pump in the blood vessel, also can adopt pulsation source or other proper device, and its different concrete formation can make it have function corresponding and/or produce corresponding effects, as hereinafter will specifically discussing.
Perfusion fluid drive unit 104 links to each other by the upstream extremity of pipeline with interior perfusion loop culture chamber inlet ductwork 107 in the blood vessel.The downstream end of interior perfusion loop culture chamber inlet ductwork 107 is passed upstream seal plug 109 and is entered into vascular tissue's culture chamber 110, and the upstream extremity of vascular tissue 111 to be cultivated is enclosed within on the downstream end of inlet ductwork 107.Upstream seal plug 109 engages with culture chamber in the mode of rotatable (rotation of culture chamber 110) sealing, thereby realizes culture chamber sealing to external world.
Perfusion upstream, loop support in label 108 is represented one, perfusion loop inlet ductwork 107 in being used for fixing and/or keeping.
In being enclosed within, the downstream end of vascular tissue 111 to be cultivated pours on the upstream extremity of loop culture chamber export pipeline 115.The upstream extremity of export pipeline 115 is placed in the culture chamber 110, and the downstream end of export pipeline 115 is passed downstream sealing plug 112 and is reached outside the culture chamber 110, and by pipeline be connected to blood vessel in the pipeline of perfusion liquid storage bottle 101 link to each other, flow back to the road thereby form the interior perfusion fluid of a complete blood vessel.
Downstream sealing plug 112 engages with culture chamber in the mode of rotatable (rotation of culture chamber 110) sealing, thereby realizes culture chamber sealing to external world.
Perfusion downstream, loop support in label 116 is represented one, perfusion loop export pipeline 115 in being used for fixing and/or keeping.
Embodiment shown in Figure 1 also comprises the blood vessel outer filling loop of a nutrient solution.As shown in Figure 1, this blood vessel outer filling loop comprises: outer filling loop liquid storage bottle 102; Outer filling liquid driving device 103, it links to each other through liquid line with liquid storage bottle 102; Outer filling loop culture chamber inlet ductwork 105, its upstream extremity links to each other with drive unit 103 by pipeline, and its downstream end is passed upstream seal plug 109 and is entered into culture chamber 110 inside, is used for the nutrient solution from outer filling loop liquid storage bottle 102 is injected in the culture chamber 110; And, outer filling loop culture chamber export pipeline 114, its upstream extremity is placed in the culture chamber 110, and its downstream end is passed downstream sealing plug 112 and extended to outside the culture chamber 110, be used for nutrient solution is drawn from culture chamber 110, and link to each other with outer filling loop liquid storage bottle 102 by liquid line.
The embodiment of Fig. 1 also comprises a kind of embodiment of the part of the rotation that realizes culture chamber, and these parts comprise: culture chamber rotary drive motor 118; The culture chamber rotary transmission gear group 113 that is coupled with the rotating shaft 119 of motor 118.A follower gear in the gear unit 113 is fixedly mounted on (in the neck portion that is mounted in culture chamber 110 that shows among Fig. 1) on the culture chamber 110, thereby the rotating drive power of motor 118 is passed to culture chamber 110, makes culture chamber 110 rotations.
By embodiment shown in Figure 1, can realize simultaneously: the rotation of nutrient solution perfusion in the blood vessel, the outer nutrient solution perfusion of blood vessel, culture chamber, and their any combination.
The structure and the work of a kind of embodiment of pulsation source of the present invention have been shown among Fig. 2.This pulsation source of the present invention can be used as interior perfusion fluid drive unit 104 of blood vessel and/or the blood vessel outer filling liquid driving device 103 among Fig. 1.Among Fig. 2 with Fig. 1 in identical label represent identical or suitable part, and these identical or suitable parts no longer are repeated in this description.
As shown in Figure 2, as the embodiment of an indefiniteness of the present invention, in blood vessel, between perfusion liquid storage bottle 101 and the vascular tissue's culture chamber 110, be connected with pulsation source 201, first dragulator 204, the first conformability setter 205 in turn.
As shown in Figure 2, pulsation source 201 of the present invention comprises pulsation chamber 202, passes the elastic hose 203 in pulsation chamber 202, the unidirectional non-return valve 208 in upstream that is arranged on the upstream port place of elastic hose, the unidirectional non-return valve 209 in downstream, the packed-piston 210 of downstream port that are arranged on elastic hose and the linear electric motors 211 that are used to drive packed-piston 210.Pulsation chamber 202 is volume fixed closed chamber that are used for full of liquid.And elastic hose 203 has constituted the part that pulsation chamber 202 is passed in (or outer) perfusion loop in the blood vessel.The to-and-fro movement of linear electric motors 211 by driven plunger 210, and produces squeezing action to the liquid that pulsation is full of in the chamber 202, this extruding and then act on the nutrient solution in the elastic hose 203, thus in blood vessel, produce corresponding pulsating flow in the perfusion loop.
As a kind of preferred but infinite embodiment, can make this pulsating flow simulation cardiac ejection go into the fluctuation of aorta, and can regulate ripple frequency, flow and the pressure of pulsating flow within the specific limits.Unidirectional non-return valve 208,209 is used to guarantee be unidirectional pulsating flow from the effusive nutrient solution stream of pulsation source.
One first dragulator of label 204 expressions.Dragulator is the mechanic adjustment unit that is connected on the pipeline, variable valve for example, and it is used for by regulating the flow of tube chamber size adjustment loop liquid, and is accompanied by the adjusting of perfusion pressure.
One first conformability setter of label 205 expressions.The conformability setter is the setting device that is connected on the pipeline, is used to regulate because the variation of the liquid capacity that pressure change caused.
One second conformability setter of label 206 expressions, one second dragulator of label 207 expressions.
First and second dragulators 204,207 respectively are used for regulating the perfusion pressure of nutrient solution of culture chamber 110 and waveform, the wave amplitude of nutrient solution perfusion pressure change.The first and second conformability setters 205,206 respectively are used for regulating the inertial flow of the nutrient solution of culture chamber.First and second dragulators 204,207 and the first and second conformability setters 205,206 be used for regulating jointly respectively with the overall waveform that obtains to be similar to physiological pulsating flow, secondaries, amplitude with the time mutually, acquisition is similar to hypertensive senior executive's internal pressure, haemodynamics environment such as shearing force, or the hemodynamic situation of simulation arteries when hanging down shear-stress.
It should be understood that pulsation source of the present invention not only can be used as the liquid driving device 104 in the perfusion loop in the blood vessel, also can be used as the liquid driving device 103 in the blood vessel outer filling loop.
By embodiment shown in Figure 1, can realize the interior perfusion of nutritive medium of the vascular tissue in the interior cultivation of culture chamber and the rotation of outer filling and culture chamber simultaneously, thereby make the vascular tissue in the cultivation obtain even, effective, abundant, nutrient solution supply dynamically.
It should be understood that the just corresponding example that rotates the rotation transmission device that transmits of realization of above-mentioned gear unit 113; Also can replace gear unit 113, these other transmission mechanism such as chain gearing, belt transmission, transmission rod or the like with other transmission mechanism.
Should also be understood that owing under two dabbling situations in managing and outside the pipe, manage interior pulsation corresponding to perfusion loop and the fluctuation of managing the pressure difference of liquid in the outer filling loop in the pipe.So,, also can be arranged on pulsation source on the pipe outer filling loop as a concrete but infinite embodiment of the present invention; Perhaps, a further concrete but infinite embodiment of the present invention can pour into the pulsation source that is provided with simultaneously on the loop separately in blood vessel outer filling loop and blood vessel.These distortion all belong to scope of the present invention.
Fig. 3 has shown a further embodiment of the present invention.
Compare with the embodiment of Fig. 1, in embodiment shown in Figure 3, use pulsation source 201 as perfusion fluid drive unit 104 in the blood vessel.
In addition, as a further concrete but not determinate embodiment of the present invention, in Fig. 3, further comprised the downstream nutritive medium divider 304 that the upstream nutritive medium divider 303 that links to each other with the downstream end of interior perfusion loop culture chamber inlet ductwork 107 links to each other with upstream extremity with interior perfusion loop culture chamber export pipeline 115.Upstream and downstream liquid dispenser 303,304 is used for being provided with the vascular tissue of many cultivations between them, thereby cultivates when having realized many vascular tissues.
In embodiment shown in Figure 3, further comprised following optional part: outer filling loop culture chamber inlet pressure sensor 301 is used for detecting the liquid pressure in the outer filling loop of culture chamber ingress; Outer filling loop culture chamber outlet pressure sensor 306 is used for detecting the liquid pressure in the outer filling loop in culture chamber exit; In perfusion loop culture chamber inlet pressure sensor 302, be used for detecting the liquid pressure in the interior perfusion loop of culture chamber ingress; In perfusion loop culture chamber outlet pressure sensor 305, be used for detecting the liquid pressure in the interior perfusion loop in culture chamber exit.
The output of transmitter 301,302,305,306 is sent to concentrator 307, to be amplified the back by amplifier 308 by treater 310 with indicating meter 311 is handled, write down and/or demonstration etc.Driving mechanism of label 309 expressions.
Among Fig. 3, in the small circle of each transmitter place band arrow and numeral, represent that the data of this transmitter are sent to the concentrator joint at the small circle place of concentrator 307 place's band respective digital.
By embodiment shown in Figure 3, can realize simultaneously: nutrient solution perfusion in the blood vessel, the outer nutrient solution perfusion of blood vessel, the rotation of culture chamber, the blood vessel reciprocal stretching vertically in the cultivation, and their any combination; In addition, it can also realize that many blood vessels cultivate simultaneously, and can realize processing, the record of pressure data and/or show.
Specific embodiments of the invention are below described.
Outer filling in embodiment 1 inner rotary
1. according to the inside and outside perfusion of connection blood vessel shown in Figure 1 loop, the inside and outside perfusion fluid drive unit of its medium vessels is peristaltic pump (Cole-Parmer company, a Masterflex series);
2. reactor is carried out disinfection, conditions for sterilization is: 130 ℃, and 3 normal atmosphere, 1 hour time;
3. engineering blood vessel that will be to be cultivated under sterile state is installed in vascular tissue's culture chamber medium vessels on the upstream and downstream pipeline in perfusion loop, engineering blood vessel internal diameter vessel diameter 6mm wherein, and length is 10cm, material is an acellular matrix;
4. according to connection shown in Figure 1 vascular tissue culture chamber swivel arrangement, wherein vascular tissue's culture chamber rotating machine is selected Hayden's 57000 serial stepper-motors for use;
5. require the preparation substratum according to cultivating, aseptic culture medium is injected perfusion liquid storage bottle and blood vessel outer filling liquid storage bottle in the blood vessel;
6. open the power supply preheating of each equipment;
7. irrigation flow is 0-1.6ml/s in the setting blood vessel, and intake pressure is 100-120mmHg, and top hole pressure is 75-115mmHg; Blood vessel outer filling flow is 0-1.2ml/s, and intake pressure is 100-120mmHg, and top hole pressure is 80-110mmHg;
8. set turning to and rotating speed of vascular tissue's culture chamber rotating machine: be rotated counterclockwise 15rpm;
10. operation reactor;
Pulsating flow pouring outer filling in embodiment 2 inner rotary
1. according to the inside and outside perfusion of connection blood vessel shown in Figure 3 loop, the perfusion fluid drive unit is a pulsation source in its medium vessels, and blood vessel outer filling liquid driving device is peristaltic pump (Cole-Parmer company, a Masterflex series);
2. reactor is carried out disinfection, conditions for sterilization is: 130 ℃, and 3 normal atmosphere, 1 hour time;
3. engineering blood vessel that will be to be cultivated under sterile state is installed on the liquid dispenser of vascular tissue's culture chamber upstream and downstream, engineering blood vessel internal diameter vessel diameter 6mm wherein, and length is 20cm, PLGA;
4. according to connection shown in Figure 3 vascular tissue culture chamber swivel arrangement, wherein vascular tissue's culture chamber rotating machine is selected Hayden's 57000 serial stepper-motors for use;
5. the pressure transmitter of importing and exporting according to medium vessels tissue culture chamber, the inside and outside perfusion of connection blood vessel shown in Figure 3 loop, and signal supervisory instrument;
6. require the preparation substratum according to cultivating, aseptic culture medium is injected perfusion liquid storage bottle and blood vessel outer filling liquid storage bottle in the blood vessel;
7. open the power supply preheating of each equipment;
10. the perfusion ripple frequency is 70 times/minute in the setting blood vessel, and the motor gain is 1-5%, sets starting position; Setting the interior irrigation flow of blood vessel is 0-1.6ml/s, and intake pressure is 100-140mmHg, and top hole pressure is 75-115mmHg; Blood vessel outer filling flow is 0-1.0ml/s, and intake pressure is 110-140mmHg, and top hole pressure is 85-110mmHg;
11. set turning to and rotating speed of vascular tissue's culture chamber rotating machine: turn clockwise 20rpm;
12. operation reactor;
13. regulate dragulator and conformability setter, pressure, the flow waveform in controlling flow intravascular tissue culture chamber, the arterial pulse wave waveform of analogy under physiological status.
Claims (6)
1. vascular tissue engineering reactor is characterized in that comprising:
Pour into loop culture chamber inlet ductwork (107) in liquid storage bottle (101), the blood vessel in perfusion fluid drive unit (104), the blood vessel by pouring in the continuous successively blood vessel of liquid line;
Vascular tissue's culture chamber (110), the downstream end of described culture chamber inlet ductwork (107) enter into vascular tissue's culture chamber (110) and be used for vascular tissue to be cultivated (111) thus upstream extremity be connected and be used to make the inside that flows into described vascular tissue (111) from the nutrient solution that pours into liquid storage bottle (101) in the described blood vessel;
Perfusion loop culture chamber export pipeline (115) in blood vessel, its upstream extremity enter into described vascular tissue culture chamber (110) and be used for vascular tissue to be cultivated (111) thus the be connected nutrient solution of the inside that is used to derive described vascular tissue (111) of downstream end;
An outer filling loop liquid storage bottle (102);
An outer filling loop liquid driving device (103), it links to each other by liquid line with described outer filling loop liquid storage bottle (102);
An outer filling loop culture chamber inlet ductwork (105), its upstream extremity links to each other with described outer filling liquid driving device (103) by liquid line, and its downstream end enters into described culture chamber (110) inside, thereby is used for the nutrient solution from outer filling loop liquid storage bottle (102) is injected the space outside the described vascular tissue (111) in the culture chamber (110); And
An outer filling loop culture chamber export pipeline (114), its upstream extremity are placed in the described culture chamber (110) and its downstream end extends to outside the described culture chamber, are used for the nutrient solution outside the described vascular tissue (111) in the described culture chamber is drawn;
A culture chamber rotating machine (118);
A culture chamber rotary actuator (113), the rotating shaft (119) of itself and described culture chamber rotating machine (118) is coupled, and be coupled with described culture chamber (110), be used for the rotation of described culture chamber rotating machine driven and pass to described culture chamber, rotate thereby drive described culture chamber;
Perfusion fluid drive unit (104) is a pulsation source (201) in the described blood vessel, and described pulsation source comprises:
Pulsation chamber (202) is used for full of liquid;
Pass the elastic hose (203) in described pulsation chamber, this elastic hose has constituted from pouring into the part that liquid storage bottle (101) passes described pulsation chamber to the interior nutrient solution path that pours into loop culture chamber inlet ductwork (107) of described blood vessel in the described blood vessel;
Be arranged on the unidirectional non-return valve in upstream (208) at the upstream port place of elastic hose;
Be arranged on the unidirectional non-return valve in downstream (209) of the downstream port of described elastic hose;
Can act on the packed-piston (210) on the liquid in the described pulsation chamber;
Linear electric motors (211), it is reciprocating to be used to drive described packed-piston, thereby the liquid that is full of in the pulsation chamber (202) is produced squeezing action by described packed-piston, this squeezing action and then be delivered on the nutrient solution in the described elastic hose, thus produce from pouring into the nutrient solution pulsating flow of liquid storage bottle (101) in the blood vessel to the inside of described vascular tissue (111);
Described vascular tissue engineering reactor further comprises:
Be arranged on one first dragulator (204) in described pulsation source (201) downstream, be used for regulating the perfusion pressure of perfusion culture liquid in the blood vessel of culture chamber (110) and waveform, the wave amplitude of nutrient solution perfusion pressure change;
Be arranged on one the first conformability setter (205) between described first dragulator (204) and the described culture chamber, be used for regulating culture chamber because the variation of the liquid capacity that pressure change caused;
Be arranged on one the second conformability setter (206) in described culture chamber downstream, be used for regulating culture chamber because the variation of the liquid capacity that pressure change caused;
Be arranged on one second dragulator (207) in described second conformability setter (206) downstream, be used to regulate the perfusion pressure of dabbling nutrient solution in the described culture chamber medium vessels and waveform, the wave amplitude of nutrient solution perfusion pressure change.
2. vascular tissue engineering reactor as claimed in claim 1 is characterized in that further comprising:
A upstream seal plug (109), its mode with rotatable sealing engages with described culture chamber, and is used to make interior perfusion culture chamber inlet ductwork (107) of described blood vessel and described outer filling loop culture chamber inlet ductwork (105) to enter described culture chamber (110) in the mode that seals;
An outer filling pipeline downstream sealing plug (117), its mode with rotatable sealing engages with described culture chamber, and is used to make interior perfusion culture chamber export pipeline (115) of described blood vessel and described outer filling loop culture chamber export pipeline (114) to extend to outside the described culture chamber (110) in described culture chamber (110) in the mode that seals.
3. vascular tissue engineering reactor as claimed in claim 1 is characterized in that further comprising:
A upstream nutritive medium divider (303), it links to each other with the end of described interior perfusion loop culture chamber inlet ductwork (107) in culture chamber (110); And
A downstream nutritive medium divider (304), it links to each other with the end of described interior perfusion loop culture chamber export pipeline (115) in culture chamber (110),
Described upstream and downstream liquid dispenser (303,304) is used for being provided with the vascular tissue of many cultivations between them, thereby cultivates when having realized many vascular tissues.
4. vascular tissue engineering reactor as claimed in claim 1, its culture chamber rotary actuator (113) comprise any in gear unit, chain gearing, the belt transmission.
5. vascular tissue engineering reactor as claimed in claim 1 is characterized in that further comprising:
Perfusion upstream, loop support (108) in one is used for fixing and/or keeps described interior perfusion loop inlet ductwork (107);
Perfusion downstream, loop support (116) in one is used for fixing and/or keeps described interior perfusion loop export pipeline (115);
Perfusion loop culture chamber inlet pressure sensor (302) in one is used for detecting the liquid pressure in the interior perfusion loop of culture chamber ingress;
Perfusion loop culture chamber outlet pressure sensor (305) in one is used for detecting the liquid pressure in the interior perfusion loop in culture chamber exit;
An outer filling loop culture chamber inlet pressure sensor (301) is used for detecting the liquid pressure in the outer filling loop of culture chamber ingress;
An outer filling loop culture chamber outlet pressure sensor (306) is used for detecting the liquid pressure in the outer filling loop in culture chamber exit;
A concentrator (307) is used to receive the output of the sensor (301,302,305,306);
An amplifier (308) is used to amplify the transmitter output that concentrator (307) is received;
A treater (310) is used for the data of amplifying through amplifier (308) are handled;
An indicating meter (311) is used for video-stream processor (310) data processed.
6. vascular tissue engineering reactor as claimed in claim 1 is characterized in that perfusion fluid drive unit (103) is a pulsation source (201) in the described blood vessel, and described pulsation source comprises:
Pulsation chamber (202) is used for full of liquid;
Pass the elastic hose (203) in described pulsation chamber, this elastic hose has constituted the part that the nutrient solution path from described blood vessel outer filling liquid storage bottle (102) to described blood vessel outer filling loop culture chamber inlet ductwork (105) passes described pulsation chamber;
Be arranged on the unidirectional non-return valve in upstream (208) at the upstream port place of elastic hose;
Be arranged on the unidirectional non-return valve in downstream (209) of the downstream port of described elastic hose;
Can act on the packed-piston (210) on the liquid in the described pulsation chamber; Linear electric motors (211), it is reciprocating to be used to drive described packed-piston, thereby the liquid that is full of in the pulsation chamber (202) is produced squeezing action by described packed-piston, this squeezing action and then be delivered on the nutrient solution in the described elastic hose, thus the nutrient solution pulsating flow of inside produced from blood vessel outer filling liquid storage bottle to described culture chamber.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101021438A CN101372661B (en) | 2008-03-18 | 2008-03-18 | Adjustable pouring type vascular tissue engineering reactor having rotating cultivation cavity |
| US12/344,586 US20090181448A1 (en) | 2007-12-28 | 2008-12-28 | Perfusion type vascular tissue bioreactor with rotary and stretching functions |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2008101021438A CN101372661B (en) | 2008-03-18 | 2008-03-18 | Adjustable pouring type vascular tissue engineering reactor having rotating cultivation cavity |
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| CN101930685B (en) * | 2010-08-04 | 2011-11-16 | 上海交通大学 | Tuning fork carotid bifurcate flow cavity and circulating system device |
| CN102127507B (en) * | 2010-12-15 | 2013-06-05 | 中国人民解放军第四军医大学 | Biological tissue engineering vasculogenesis system |
| CN104388311B (en) * | 2014-11-20 | 2016-09-21 | 杭州电子科技大学 | Organizational project stem cell inducing cardiomyocytes is cultivated and measures bioreactor |
| EP3249039A1 (en) * | 2016-05-25 | 2017-11-29 | Université Catholique De Louvain | Perfusion-bioreactor and method for processing vascularized composite tissue |
| EP3275992A1 (en) * | 2016-07-29 | 2018-01-31 | Bayer Aktiengesellschaft | Adapter for cell-culture vessel |
| CN106591121B (en) * | 2016-11-30 | 2019-04-05 | 广州博敏科技有限公司 | Rotating and culturing room and rotary pulse culture systems |
| CN111426821B (en) * | 2020-03-24 | 2021-07-06 | 大连理工大学 | Micro-fluidic chip-level extracorporeal circulation system for vascular endothelial cell mechanics biology research |
| CN115747065A (en) * | 2022-11-15 | 2023-03-07 | 南京中医药大学 | Experimental device for in vitro circulation dosing simulation is to vascular endothelium effect |
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| CN2647864Y (en) * | 2003-08-28 | 2004-10-13 | 中国人民解放军第三军医大学 | Reactor for in vitro blood vessel culture |
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| CN2647864Y (en) * | 2003-08-28 | 2004-10-13 | 中国人民解放军第三军医大学 | Reactor for in vitro blood vessel culture |
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