CN1115407C - composite field three-dimensional cell culture device - Google Patents
composite field three-dimensional cell culture device Download PDFInfo
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- CN1115407C CN1115407C CN98121829A CN98121829A CN1115407C CN 1115407 C CN1115407 C CN 1115407C CN 98121829 A CN98121829 A CN 98121829A CN 98121829 A CN98121829 A CN 98121829A CN 1115407 C CN1115407 C CN 1115407C
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Abstract
The invention relates to a device for constructing a tissue substitute with specific form and function, which is characterized in that tissue cells are planted in a three-dimensional matrix support and are placed in a strain field and an electromagnetic field to enable the cells to grow directionally, improve the metabolism and promote the secretion function of the cells. The design comprises a control part consisting of a microcomputer system, a power amplifier and the like, a mechanical part consisting of a stepping motor and a link mechanism, an electromagnetic part consisting of a direct-current power supply and a Helmholtz coil, and a tissue reactor part consisting of a culture bottle, a three-dimensional matrix bracket planted with cells and the like. Simple structure, convenient use and wide application, and brings good news to patients with tissue defects.
Description
The present invention relates to cell culture experiments equipment, particularly the cell in the three dimensional matrix support carries out the experimental installation of cell cultures with strain field and electromagnetic field compound action.
In China, more and more because of the tissue defect number of patients that wound, disease cause, become modal clinical problem, be the important factor that disables.The tissue defect method for implantation of replacing commonly used is treated.Alternative comprises xenogenesis, allogeneic and autologous tissue and synthetic material.Heteroplasm causes fastish rejection, allograft rejection reaction unavoidably, and tissue-derived limited; The autograft meeting causes the tissue source of damaging and supplying with for the district that limitation is arranged; Traditional synthetic material causes foreign body reaction after implanting, secondary infection and exposed etc., and these all force the scientific worker to seek novel tissue substituent.
The novel tissue substituent of external structure requires the viable cell that the function of high density is relevant to plant in the extracellular matrix of natural or synthetic, form identical with tissue or similar tissue, transplant then in human body " growth on the spot ", reach the purpose that forms the new tissue that function is arranged.And existing cell culture processes can not adapt to the needs of the novel tissue substituent of external structure, as:
Traditional monolayer cell adherent culture is to take out histocyte in body, and physiological condition in the analogue body under aseptic, proper temperature and certain nutritional condition, makes it to survive and grows and keep the method for its 26S Proteasome Structure and Function.Cell is growth and a propagation in two dimensional surface in this method, be subjected to the influence that cells contacting suppresses; Incubation time is long, particularly goes down to posterity repeatedly, is easy to cause cell to morph or anti-differentiating phenomenon (as the chondrocyte); Through digestion process, pair cell has certain damage when going down to posterity.Therefore, low with its cloning efficiency of traditional cell culture method, can not be at external generation engineering three-dimensional tissue structures.
In order to improve cloning efficiency, produced the microcarrier culture method, comprise micro-capsule culture method or tubular fibre culture method.These culture techniques are carrier with micro-capsule or tubular fibre, increased the cell attachment area, reach purpose (Jauregui HO et al Primary cultures of rat hepatocytes inhollow fiber chambers.In Vitro Cell Dev Biol, 1994 of a large amount of culturing cells; 30A:23).Principal feature with the microcarrier cell cultures is the stirring that need suit, to increase the area of cell attachment, but increased the randomness that cell and carrier adhere to and grow, be unfavorable for the external tissue substituent that on purpose produces with specific modality 26S Proteasome Structure and Function.
The cell three-dimensional culture technique of newly-developed, cell and matrix contact area had both been fully taken into account, cell can and be bred in the three-dimensional space growth, it is the one preferred technique (Naughton GK et al.Three-dimensional cell and tissue culture system.Int.PCT.WO90/02796,1990) of present external structure tissue substituent.But it is strong that common cell three-dimensional culture technique still exists cell growth randomness, do not determine direction; And the cell poor growth, organize long limitation of formation cycle, obviously be unfavorable for clinical application.
Purpose of the present invention provide in order to overcome above-mentioned weak point of the prior art just a kind of with cell seeding in the three dimensional matrix support, place under strain field and the electromagnetic field compound action, make cell oriented growth in the three dimensional matrix support, improve cellular metabolism, promote its secreting function, to construct the device of tissue substituent with specific modality and function.
The control section that the present invention is made up of microsystem, power amplifier etc.; The mechanical part that constitutes by stepper-motor, linkage assembly; The electromagnet portion that constitutes by power supply, Helmholtz coil group and by culturing bottle, plant the three dimensional matrix that is implanted with cell and prop up the tissue reactor that is configured to and partly constitute.The microprocessing systems respectively leading screw on the control step driven by motor linkage assembly is made periodically to-and-fro movement and control direct supply, and the output pulsed current generates an electromagnetic field the Helmholtz coil group on the anchor; Tissue reactor is a garden cylindricality glass culturing bottle, two ends have aperture fixed clamp tool and stretch out for leading screw, plant the three dimensional matrix timbering material that is implanted with cell and place nutrient solution, two ends are fixed by fixture, be provided with the cultivation bottle cap, the three dimensional matrix timbering material that is fixed on the leading screw produces dynamic strain field with guide screw movement, strain field and the cell of electromagnetic field compound action in timbering material, and tissue reactor places CO
2To guarantee the ambient conditions of cell growth, be provided with scatterer in the incubator in the incubator, have special hole to do the pipeline turnover on the incubator.
The present invention compared with prior art has the positively effect of following advantage and generation:
1. adopt three dimensional matrix support folding or that be rolled into,, greatly increased the bond area of cell for the cell growth provides the three-dimensional orientation growing space; The factor that can in support, add various promotion cell adhesions, growth and differentiation simultaneously, the microenvironment of cell growth during simulation is organized in vivo effectively.
2. the cell in the three dimensional matrix support is in dynamic strain field, increased the permeability of cell in the matrix scaffold to a certain extent, improved culture system heat and mass effect, thereby significantly improved the nutritional condition of cell in matrix scaffold, improved and organized formation efficiency.
3. cell is improving the growth conditions of cell in the three dimensional matrix support greatly under the compound action of dynamic strain field and electromagnetic field, thereby accelerated cell growth process, cell culture period is shortened, metabolite content improves, emiocytosis collegen filament amount is big, and presses certain orientation and arrange.
4. the present invention is simple in structure, easy to use, is widely used, and brings glad tidings for the tissue defect patient.
Below be the drawing explanation of accompanying drawing of the present invention:
Fig. 1 is a structural representation of the present invention.
Fig. 2 is that control section of the present invention, mechanical part, electromagnet portion, tissue reactor partly connect block diagram.
Fig. 3 is the schematic circuit diagram of control section of the present invention.
Fig. 4 is the software workflow figure of control section of the present invention.
Fig. 5 is that mechanical part master of the present invention looks synoptic diagram.
Fig. 6 is a mechanical part schematic top plan view of the present invention.
Fig. 7 is that three dimensional matrix of the present invention props up and is configured to synoptic diagram.
The present invention is described in further detail below with reference to drawings and Examples:
Embodiment 1: a kind of composite field three dimentional cell cultivation equipment (seeing accompanying drawing 1-7): make periodically to-and-fro movements and control direct supply 5 by the leading screw 4 in the microprocessor system 1 difference control step motor 2 drivening rod mechanisms 3, the output pulsed current makes the Helmholtz coil group 7,8,9,10 on the anchor 6 produce pulse electromagnetic fields; Tissue reactor 11 is garden cylindricality glass culturing bottles, two ends have aperture fixed clamp tool 12 and stretch out for leading screw, plant the three dimensional matrix timbering material 14 that is implanted with cell 13 and place nutrient solution 15, two ends are fixed by fixture 12 and 16,17 for cultivating bottle cap, the three dimensional matrix timbering material that is fixed on the leading screw produces dynamic strain field with guide screw movement, strain field and the cell of electromagnetic field compound action in timbering material, and tissue reactor places CO
2To guarantee the ambient conditions of cell growth, be provided with scatterer 19 in the incubator in the incubator 18, special hole 20 arranged for the pipeline turnover on the incubator, its concrete control section, mechanical part, electromagnet portion and tissue reactor part division are as follows:
(1) control section is made up of hardware components and software section:
(1.1) hardware components is by 8 chip microprocessors of a slice reduce power consumption, a LCD liquid-crystal display, a keyboard monitoring interface, the photosignal testing circuit of a zero passage detection position, each latch of segmentation value mutually of stepper-motor, power amplifier; And the pulse signal driving circuit, the VMOS switching tube, direct supply and Helmholtz coil are formed,
(1.2) the software work principle is as follows: initialize routine is carried out in the start back; on LCD, show the start original state subsequently; wait for keyboard input machine operation pre-set parameter and electromagnetic field pre-set parameter; after this starting microprocessor sends starting impulse driving stepper-motor and triggers the VMOS switching tube; motor is rotated by preset rotation speed and make direct supply output pulsed current; after the machine operation time finishes; send position detection pulse and desired location comparison by the zero passage detection photoswitch circuit; motor is stopped to predeterminated position; finish stop time; motor is started working again; after net cycle time and the end of electromagnetic field effect time; EP (end of program), its workflow diagram is seen Figure of description 4.
(2) mechanical part is to make the actuating arm 24 of connection and connecting rod 25 drive the catch bar 27 periodically to-and-fro movements of do that have motion bearings 26 by the chute 23 that the stepper-motor 2 that is positioned at annular seal space 21 drives on the rotary disk 22, the timbering material that is fixed on the leading screw 4 on the catch bar and connects 14 moves with catch bar, be provided with scale 28 indication collapsing lengths, on the dish of rotation garden eccentric orfice 29 is arranged, regulate eccentricity by eccentricity adjusting knob 30, the fulcrum 31 of chute is positioned on the actuating arm, each parts is fixing by trip bolt 32,33 is slide shaft, 34 is mat insulation, 35 is support, inlet pipe 36 has two, vapor pipe 37 has one, advances, vapor pipe together with the lead of control step motor from CO
2The special use of incubator is portalled and 20 is drawn,
(2.1) linkage assembly work reciprocating amplitude of periodicity and frequency are by the eccentricity and the rotating speed decision of motor-driven disk, 0,2,4,6,8,10,15,20mm the eccentricity of electric motor driving garden dish:, totally 8 grades, rotating speed of motor is 0～300 rev/min, strain field intensity: 0～100%, frequency 0～5Hz, strain field intensity and frequency independent variation
(3) electromagnet portion is a microprocessor output start pulse signal, the control electric power outputting current drives the Helmholtz coil group on the anchor, to generate an electromagnetic field, the intensity of electromagnetic field is directly proportional with the electric current that flows through Helmholtz coil, regulate electromagnetic intensity by regulating direct supply outward current size, regulate electromagnetic field frequency by the frequency of regulating start pulse signal
(3.1) power supply is a direct supply, and the relation of the electromagnetic intensity B that its outward current I and Helmholtz coil produce is:
R, n are respectively the Helmholtz coil radius and the number of turn, R=40mm, nI=0～10000 ampere-turns, μ
0=0.4 π * 10
-6Newton/meter
2,
Electromagnetic intensity: 0～0.1 tesla (T), frequency: 0～1kHz, electromagnetic intensity and frequency no dependence,
(4) tissue reactor is a garden cylindricality culturing bottle, cultivating bottle cap is placed on the culturing bottle, the culturing bottle two ends have aperture fixed clamp tool 12 and stretch out for leading screw 4, dynamically sealing is done with sealing film in the slit of leading screw and aperture, the three dimensional matrix that kind is implanted with cell props up and is placed in the nutrient solution, two ends are by fixture 12 and 16 fixing, and the three dimensional matrix support is to be that to be woven into mesh be 4～1000 μ m for the polymer fiber of 5～100 μ m by diameter
2Fibrous reticulum constitute, tissue reactor places CO
2Be suitable for the CO of cell growth in the incubator with maintenance
2Concentration and the air ambient that humidity is arranged, electromagnetic field and the cell of strain field compound action in the timbering material of tissue reactor,
(4.1) culture condition: CO
2Concentration 5～10%, 35～37 ℃ of temperature, relative humidity is more than 90%,
The three dimensional matrix timbering material is a polymkeric substance, comprises degradable polymer, non-degradable polymkeric substance and with degradable polymer embedding non-degradable polymkeric substance:
(1) degradable polymer has poly(lactic acid), polyglycolic acid, poly(lactic acid) and polyglycolic acid in 90～10: the random copolymers of 10～90 ratio copolymerization; The non-degradable polymkeric substance has polyvinyl alcohol, polycaprolactam, urethane, hemacol,
(2) have with degradable polymer embedding non-degradable polymkeric substance: poly(lactic acid) embedding polyvinyl alcohol, polyglycolic acid embedding urethane, poly(lactic acid) and polyglycolic acid multipolymer embedding polycaprolactam,
Polymeric web can be rolled into rectangular parallelepiped support, Cylinder support, prism support, ellipse Cylinder support or other form.
In the tissue reactor and the Helmholtz coil group that are parallel with on the catch bar of linkage assembly more than two covers, to increase work efficiency.
The change of the parameter setting in above-mentioned (2.1), (3), (3.1), (4), three dimensional matrix timbering material (1), (2) is to select according to institute's cultured cells characteristics difference.
2. 1 kinds of composite field three dimentional cell cultivation equipments of embodiment (seeing accompanying drawing 1-7): the structure of its device is with embodiment 1, and the three dimensional matrix timbering material is to be that to be woven into mesh be 4 μ m for the degradable polymer acid fiber by polylactic of 5 μ m by diameter
2Fibrous reticulum be rolled into the rectangular parallelepiped support, in support, behind the plantation chondrocyte, under the situation of no strain field (intensity is 0%) and non-electromagnetic field (field intensity is 0 tesla), carry out the cultivation of static three-dimensional cell.
3. 1 kinds of composite field three dimentional cell cultivation equipments of embodiment (seeing accompanying drawing 1-7): the structure of its device is with embodiment 1, the three dimensional matrix timbering material uses degradable polymer, as poly(lactic acid) and polyglycolic acid random copolymers in ratio copolymerization in 10: 90, polymeric web can be rolled into the hexagonal prism support, in support behind the plantation chondrocyte, strain field that be 1% in intensity, frequency is 0.5Hz and field intensity are that 0.05 tesla, frequency are to carry out three-dimensional cell under the electromagnetic field compound action of 500Hz to cultivate.
4. 1 kinds of composite field three dimentional cell cultivation equipments of embodiment (seeing accompanying drawing 1-7): the structure of its device is with embodiment 1, the three dimensional matrix timbering material is the biodegradable polymer fiber of 20 μ m by diameter, and becoming mesh as poly(lactic acid) and polyglycolic acid in the random copolymers fibrage of ratio copolymerization in 90: 10 is 20 μ m
2Fibrous reticulum be rolled into the Cylinder support, in support behind the plantation Tenocyte cell, strain field that be 5% in intensity, frequency is 5Hz and field intensity are that 0.01 tesla, frequency are to carry out three-dimensional cell under the electromagnetic field compound action of 1kHz to cultivate.
5. 1 kinds of composite field three dimentional cell cultivation equipments of embodiment (seeing accompanying drawing 1-7): the structure of its device is with embodiment 1, the three dimensional matrix timbering material is the biodegradable polymer fiber of 40 μ m by diameter, and becoming mesh as poly(lactic acid) and polyglycolic acid in the random copolymers fibrage of ratio copolymerization in 50: 50 is 50 μ m
2Fibrous reticulum be rolled into ellipse Cylinder support, in support behind the plantation sarcoplast, strain field that be 100% in intensity, frequency is 2Hz and field intensity are that 0.1 tesla, frequency are to carry out three-dimensional cell under the electromagnetic field compound action of 50Hz to cultivate.
6. 1 kinds of composite field three dimentional cell cultivation equipments of embodiment (seeing accompanying drawing 1-7): the structure of its device is with embodiment 1, the three dimensional matrix timbering material is the non-degradable polymer fiber of 80 μ m by diameter, and to become mesh be 1000 μ m in fibrage as hemacol
2Fibrous reticulum be rolled into the tri-prismoid support, in support behind the plantation scleroblast, strain field that be 0.5% in intensity, frequency is 0.2Hz and field intensity are that 0.08 tesla, frequency are to carry out three-dimensional cell under the electromagnetic field compound action of 200Hz to cultivate.
7. 1 kinds of composite field three dimentional cell cultivation equipments of embodiment (seeing accompanying drawing 1-7): the structure of its device is with embodiment 1, the three dimensional matrix timbering material is the fiber of 100 μ m by the diameter that degradable polymer embedding non-degradable polymkeric substance constitutes, as: it is 1000 μ m that poly(lactic acid) and polyglycolic acid multipolymer embedding capron(e) are woven into mesh
2Fibrous reticulum be rolled into the rectangular parallelepiped support, in support behind the plantation alveolar cell, strain field that be 80% in intensity, frequency is 1.5Hz and field intensity are that 0.06 tesla, frequency are to carry out three-dimensional cell under the electromagnetic field compound action of 150Hz to cultivate.
Claims (4)
1. composite field three dimentional cell cultivation equipment, it is characterized in that: make periodically to-and-fro movement and control direct supply (5) by the leading screw (4) in microprocessor system (1) difference control step motor (2) the drivening rod mechanism (3), the output pulsed current makes Helmholtz coil group (7), (8), (9), (10) on the anchor (6) produce pulse electromagnetic field; Tissue reactor (11) is a garden cylindricality glass culturing bottle, two ends have aperture fixed clamp tool (12) and stretch out for leading screw, plant the three dimensional matrix timbering material (14) that is implanted with cell (13) and place nutrient solution (15), two ends are fixing by fixture (12) and (16), (17) for cultivating bottle cap, the three dimensional matrix timbering material that is fixed on the leading screw produces dynamic strain field with guide screw movement, strain field and the cell of electromagnetic field compound action in timbering material, and tissue reactor places CO
2Ambient conditions to guarantee that cell is grown in the incubator (18), be provided with scatterer (19) in the incubator, special hole (20) is arranged for the pipeline turnover on the incubator, the control section that it is concrete, mechanical part, electromagnet portion and tissue reactor part division are as follows: the hardware of (1.1) control section is by 8 chip microprocessors of a slice reduce power consumption, a LCD liquid-crystal display, a keyboard monitoring interface, the photosignal testing circuit of a zero passage detection position, each latch of segmentation value mutually of stepper-motor, power amplifier, and start pulse signal driving circuit, the VMOS switching tube, direct supply and Helmholtz coil are formed, (1.2) mechanical part is to make the actuating arm (24) of connection and connecting rod (25) drive the periodically to-and-fro movement of catch bar (27) do that has motion bearings (26) by the chute (23) that the stepper-motor that is positioned at annular seal space (21) (2) drives on the rotary disk (22), the timbering material (14) that is fixed on the leading screw (4) on the catch bar and connects moves with catch bar, be provided with scale (28) indication material collapsing length, on the dish of rotation garden eccentric orfice (29) is arranged, regulate eccentricity by eccentricity adjusting knob (30), the fulcrum of chute (31) is positioned on the actuating arm, each parts is fixing by trip bolt (32), (33) be slide shaft, be located at actuating arm (24) one sides, connecting rod (25), actuating arm (24) from top to bottom is connected by screw (32) with slide shaft (33), can it be horizontally slipped on slide shaft, slide shaft is embedded in the table top of support (35), (34) be mat insulation, be attached to the wall inboard, chamber of the annular seal space (21) under the support (35), (35) be support, inlet pipe (36) has two, vapor pipe (37) has one, advances, vapor pipe together with the lead of control step motor from CO
2The special use of incubator portal (20) draw, (1.2.1) linkage assembly work reciprocating amplitude of periodicity and frequency are by the eccentricity and the rotating speed decision of motor-driven disk, the eccentricity of electric motor driving garden dish: 0,2,4,6,8,10,15,20mm, totally 8 grades, rotating speed of motor is 0～300 rev/min, strain field intensity: 0～100%, frequency 0～5Hz, strain field intensity and frequency independent variation, (1.3) electromagnet portion is by microprocessor output start pulse signal, the control electric power outputting current drives the Helmholtz coil group on the anchor, to generate an electromagnetic field, the intensity of electromagnetic field is directly proportional with the electric current that flows through Helmholtz coil, regulate electromagnetic intensity by regulating direct supply outward current size, regulate electromagnetic field frequency by the frequency of regulating start pulse signal, (1.4) tissue reactor is a garden cylindricality culturing bottle, cultivating bottle cap is placed on the culturing bottle, the culturing bottle two ends have aperture fixed clamp tool (12) and stretch out for leading screw (4), dynamically sealing is done with sealing film in the slit of leading screw and aperture, the three dimensional matrix that kind is implanted with cell props up and is placed in the nutrient solution, two ends are fixing by fixture (12) and (16), and the three dimensional matrix support is to be that to be woven into mesh be 4～1000 μ m for the polymer fiber of 5～100 μ m by diameter
2Fibrous reticulum constitute, tissue reactor places CO
2Be suitable for the CO of cell growth in the incubator with maintenance
2Concentration and the air ambient that humidity is arranged, electromagnetic field and the cell of strain field compound action in the timbering material of tissue reactor, (1.4.1) culture condition: CO
2Concentration 5～10%, 35～37 ℃ of temperature, relative humidity is more than 90%.
2. composite field three dimentional cell cultivation equipment according to claim 1, it is characterized in that: the three dimensional matrix timbering material is a polymkeric substance, comprise degradable polymer, non-degradable polymkeric substance and with degradable polymer embedding non-degradable polymkeric substance: (2.1) degradable polymer has poly(lactic acid), polyglycolic acid, poly(lactic acid) and polyglycolic acid are in 90～10: the random copolymers of 10～90 ratio copolymerization, (2.2) the non-degradable polymkeric substance has polyvinyl alcohol, polycaprolactam, urethane, hemacol, (2.3) have with degradable polymer embedding non-degradable polymkeric substance: poly(lactic acid) embedding polyvinyl alcohol, polyglycolic acid embedding urethane, poly(lactic acid) and polyglycolic acid multipolymer embedding polycaprolactam.
3. composite field three dimentional cell cultivation equipment according to claim 1 is characterized in that: polymeric web can be rolled into rectangular parallelepiped support, Cylinder support, prism support, ellipse Cylinder support.
4. composite field three dimentional cell cultivation equipment according to claim 1 is characterized in that: in the tissue reactor and the Helmholtz coil group that are parallel with on the catch bar of linkage assembly more than two covers.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98121829A CN1115407C (en) | 1998-11-05 | 1998-11-05 | composite field three-dimensional cell culture device |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98121829A CN1115407C (en) | 1998-11-05 | 1998-11-05 | composite field three-dimensional cell culture device |
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| CN1259568A CN1259568A (en) | 2000-07-12 |
| CN1115407C true CN1115407C (en) | 2003-07-23 |
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| CN98121829A Expired - Fee Related CN1115407C (en) | 1998-11-05 | 1998-11-05 | composite field three-dimensional cell culture device |
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Also Published As
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| CN1259568A (en) | 2000-07-12 |
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