CN103966095A - Bioreactor for three-dimensional cell perfusion culture - Google Patents
Bioreactor for three-dimensional cell perfusion culture Download PDFInfo
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- CN103966095A CN103966095A CN201410218150.XA CN201410218150A CN103966095A CN 103966095 A CN103966095 A CN 103966095A CN 201410218150 A CN201410218150 A CN 201410218150A CN 103966095 A CN103966095 A CN 103966095A
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- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate
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Abstract
The invention relates to a bioreactor for three-dimensional tissue cell perfusion culture. A reaction chamber, a liquid storage tank and a peristaltic pump are sequentially connected through transfusion pipes, wherein the reaction chamber comprises a box cover and a box body comprising lateral walls, a bottom wall, a cut-off device, an overflow insert plate, screen frames, a static pressure channel, a sluice channel and a draining channel; the cut-off device is a gate or a weir flow dam, and a plurality of uniformly arranged water holes are formed in the same plane along the longitudinal section of the cut-off device; an overflowing opening matched with the draining channel is formed in the lateral wall of the static pressure channel; and the screen frames are fixed on two lateral walls of the box body. The invention also relates to a bioreactor with a laminated structure, which can be used for solving the problem of cell death easily caused by non-uniform flow field and non-uniform nutritional transfer in the existing perfusion culture, is suitable for three-dimensional culture of tissue cells, can be used for improving the quality of tissue engineering products, and has the characteristics of good expansibility, strong universality and wide application range.
Description
Technical field
The invention belongs to cell culture apparatus field, relate to a kind of bio-reactor that is applicable to three-dimensional tissue's cell perfusion culture, the bio-reactor that especially Various Tissues organ is cultivated altogether.
Background technology
The engineering tissue (or claiming three-dimensional construction) having built under normal circumstances need to carry out the cultivation of for some time in vitro, make could transplant after its maturation or for research, and the most frequently used training mode is the static cultivation method that adopts culture plate regularly to change liquid at present, first remove old nutrient solution and add again new nutrient solution, then put into incubator and left standstill cultivation, its advantage is the cultivation that is applicable to short run, changes the liquid cycle and can take corresponding strategy with the difference of tissue, cell.But its shortcoming is apparent: the one, and the consumption of nutritive medium is the decline of carrying out property, and meta-bolites constantly increases, and can cause some physiological damages to cell; The 2nd, rely on disperse mode that nutrition is provided, there is the limited remarkable shortcoming of nutrition supply, even cause the cell of engineering tissue inside to can not get nutrition and death; The 3rd, periodically change liquid and make nutritive ingredient be cyclic fluctuation, be unfavorable for the even growth of cell.
In vivo, abundant capillary network framework plays an effective mass transfer channel, for cell provides sufficient oxygen and nutrition.Therefore when vitro culture in order to ensure nutraceutical supply, must set up the method for nutrient solution Efficient Cycle, there is at present following strategy: roller bottle cultivation, rotating and culturing and filling type are cultivated, the advantage that wherein filling type is cultivated is particularly remarkable, on the one hand, because flow through continuously three-dimensional construction of nutrient solution is inner or around, can overcome nutritive substance and transmit limited shortcoming, even if exceeding 500 μ m, construction thickness also can obtain reliable nutrition supply; On the other hand, liquid stream when perfusion culture can produce certain shearing force, and suitable mechanical stimulus is provided, and is conducive to growth and the maturation of construction.(the Kalyanaraman BM such as Kalyanaraman, Dorothy MS, Steven TB.Medium Flow Rate Regulates Viability and Barrier Function of Engineered Skin Substitutes in Perfusion Culture. Tis Eng:Part A. 2008, 14 (5): 583-93) designed a kind of biological respinse chamber, enter, water outlet pipe is to access respectively culturing room from the both sides, bottom surface of culturing room, by being set, the height of drain pipe controls the height of culturing room's liquid level, be culturing room's liquid level higher than rising pipe after will overflow inlet pipe and flowing out of liquid, by the organization engineering skin building is carried out to slaking cultivation, result shows that at rate of flooding be under 5ml/min or 15ml/min culture condition, histological structure is better than static cultivation, wherein, show by cell viability detected result, cell viability under the rate of flooding of 5ml/min is significantly better than the cell viability under static cultivation, adopt the BrdU method of mixing to detect the hyperplasia situation of cell and also show, the hyperplasia situation in the time of rate of flooding 5ml/min or 15ml/min is significantly better than the hyperplasia situation under static cultivation.Contrast only shows while also finding rate of flooding 50ml/min that a small amount of hyperplasia, experimental study have proved that low flow velocity can increase cell viability and the barrier that maintains epidermis, is suitable for transplanting, and high flow rate produces the degeneration necrosis that high shearing force can cause cell.
Although existing filling type culture apparatus has been realized the circulation of liquid simply, but do not consider the overall condition in flow field, on the one hand, because of nutrient solution flow velocity in working cycle unstable, change frequent, cannot ensure at the uniform flow field in low flow velocity in the time cultivating tissue engineering product of flowing through, unfavorable to hyperplasia; On the other hand, the nutrient solution in edge is difficult for flowing, culture apparatus flow field heterogeneity, and nutrition is transmitted uneven, easily causes Growth of Cells inhomogeneous; Moreover the shearing force that liquid applies cell in the situation that of flow velocity inequality varies in size, when flow velocity is fast shearing force excessive and make cell be vulnerable to damage, when flow velocity too low (approaching static), be unfavorable for again hyperplasia.The inventor discloses a kind of filling type bioreactor in the 35th the 7th phase of volume of Third Military Medical University's journal " design of novel skin organizational project filling type biological respinse chamber and Preliminary Simulation based on quasistatic Plane Fluid Field ", comprises medium bottle, peristaltic pump, aseptic silica gel pipe, dashpot, overfall dike, culturing room and earial drainage ditch.This bio-reactor is that nutrient solution is realized quasistatic Plane Fluid Field by the mode of overflow, nutrient solution enters dashpot, enter culturing room through high overfall dike overflow, thereby in cultivation, histocyte is supported, then nutrient solution is spilled over to earial drainage ditch through low overfall dike, and last nutrient solution enters into lower one deck reactor dashpot.Utilize this bio-reactor, because nutrient solution exists surface tension, flow into the pressure head that the flow velocity of dashpot is non-constant and inhomogeneous caused unstable, in addition the earial drainage inclined-plane of high overfall dike can not be in definitely smooth, so nutrient solution is flowed through after dashpot savings potential energy when the earial drainage inclined-plane of high overfall dike, form one or several strands of inhomogeneous flow liquids, What is more, under lower flow velocity, some region on earial drainage inclined-plane has formed cutout so that substratum and cultivates section and occurred inhomogeneous flow field when flow field entering, cause the cell in some region to can not get or obtain little nutritive substance and can not get rid of meta-bolites in time and occur damage or dead, and existing screen cloth is fixed on the diapire of reaction chamber mostly, must need certain support to be used for fixing screen cloth, will cause thus in flow field direction, the pillar rear side of stationary screen rack can not form uniform flow field and also can affect to partly cultivating region.
As can be seen here, in filling type is cultivated, make to flow through that it is even to wait to cultivate histiocytic liquid flow field, flow velocity remains at low flow velocity, to improving tissue engineering product quality, shorten the cultivation time, improve that to cultivate efficiency most important.
Summary of the invention
For the problems referred to above, the technical problem to be solved in the present invention is to provide a kind of bio-reactor that is applicable to three-dimensional tissue's cell cultures, and the nutrient solution flow velocity that this bio-reactor can be realized Plane Fluid Field is suitable, and even flow field is stable; The present invention also provides a kind of compact construction, favorable expandability, more optimal stacked bio-reactor.
In order to solve the problems of the technologies described above, be applicable to the bio-reactor of three-dimensional tissue's cell cultures, comprise that container for storing liquid, peristaltic pump and at least one comprise the rectangular parallelepiped box body of open top and the reaction chamber of lid composition; A cut-off equipment is fixed in box body one end, forms static pressure groove with box body, and the other end is fixed an overflow plate, forms dredging flow groove with box body, is fixed with and cut-off equipment and all discontiguous screen bed bases of overflow plate between static pressure groove and dredging flow groove; Screen bed base is fixed on box body, and screen cloth is fixed in the middle of screen bed base; Between cut-off equipment and overflow plate, form conversion zone; On reaction chamber, arrange one and be communicated with the inlet opening that also liquid directly can be injected to static pressure groove with static pressure groove, on the wall of box body, arrange one and be communicated with the fluid hole that also liquid directly can be discharged to dredging flow groove with dredging flow groove; Cut-off equipment longitudinally offers the some evenly distributed permeable holes that the liquid in static pressure groove at the uniform velocity evenly can be introduced to conversion zone in the same level of cross section; On the sidewall of static pressure groove, be provided with overflow port, on the sidewall of box body, be provided with the unnecessary liquid that static pressure groove can be overflowed through overflow port and enter by inflow port the drainage trough of dredging flow groove; Cut-off equipment is equal to or less than the degree of depth of box body, screen bed base is lower than the height of cut-off equipment, overflow plate is greater than or equal to the height of screen bed base upper screen cloth, the lower edge of overflow port is lower than the height of the upper surface of cut-off equipment and higher than the height of overflow plate upper surface, inflow port and permeable hole upper limb, to form a stair-stepping poor position liquid level in static pressure groove, conversion zone and dredging flow groove; Tubing is connected to container for storing liquid, peristaltic pump between fluid hole and inlet opening in turn.
In the present invention, screen bed base is fixed on the two side of box body, cut-off equipment, overflow plate and screen bed base pass through engagement connection with box body respectively, on the face that cut-off equipment, overflow plate contact with box body and the face that contacts with box side wall of screen bed base be provided with step-like clamp, the sidewall of box body with on diapire, be provided with the groove that engages fastening with clamp.Facilitate dismounting and the cleaning of reaction chamber by engagement connection, and compared with screen bed base is fixed on was fixed on diapire with former screen bed base on the sidewall of box body, reduced screen bed base support to the stopping of nutrient solution in conversion zone, formed uniform flow field.
Overflow port in preferred the present invention comprise U-shaped groove and in static pressure groove U-shaped groove other arrange can by locating shaft rotates cover U-shaped groove also can be by regulating U-shaped groove lower edge height to carry out the catch of liquid level in regulating static groove.By overflow port is set, the height of the interior liquid level of regulating static groove flows through the permeable hole of cut-off equipment equably to control nutrient solution easily.
In the present invention, permeable hole is several evenly distributed identical bar hole, circular port, semi-circular hole or profile holes.By evenly distributed permeable hole is set, can realizes when substratum enters conversion zone and form uniform runner, to realize uniform flow field.
In the present invention, be provided with the specification mould contour with the support of screen bed base on the net plane of screen cloth, specification mould can be square, circular, trilateral or polymorphic structure.
In the present invention, to exceed screen bed base upper screen cloth height be 0～4mm to preferred overflow plate.By the overflow plate a little more than screen cloth height is set, can make nutrient solution form good contacting with the culture on screen cloth.
In improvement project of the present invention, cut-off equipment is the gate with permeable hole, and permeable hole is positioned at the middle and lower part of gate.
In another improvement project of the present invention, cut-off equipment is the weir flow dam with earial drainage inclined-plane, weir flow dam comprises dam body and lower dam body, the side that lower dam body is adjacent with screen bed base has earial drainage inclined-plane, permeable hole is positioned at the upper dam body on this weir flow dam, this earial drainage inclined-plane and box bottom are 15 °～25 ° angles, and the height on earial drainage inclined-plane is 1/3～1/2 of weir flow height of dam degree.
By above-mentioned two kinds of improved cut-off equipments, compared with existing cut-off equipment, nutrient solution not only can produce flow field at the uniform velocity uniformly during by permeable hole, and dismounting, clean convenient.
Another kind of improvement project of the present invention has been to provide a kind of stacked box body that can produce in batches, and this stacked box body is to consist of the mutual reverse imbedding of box body together.
In the present invention, the outside of the upper surface of the sidewall of box body is provided with step-like flange, and the surrounding of the diapire of box body is provided with the object gomphosis groove with the flange tabling of sidewall upper surface.Multiple box bodys consist of the stacked box body of box body chimeric docking, cover cap is combined on the top box body of stacked box body, and the box body on lid and top forms reaction chamber, a reaction chamber of the common composition of the diapire of the box body on top and the box body of adjacent bottom; The fluid hole of stacked box body middle and upper part box body is connected with the inlet opening of bottom box body by tubing, and the fluid hole of the box body of stacked box body bottom is connected with the inlet opening of the reaction chamber at top by tubing, container for storing liquid, peristaltic pump; Stacked box body and lid, tubing, container for storing liquid, peristaltic pump form stacked bio-reactor jointly.
The fluid hole of the box body on the top in preferred above-mentioned improvement project in stacked box body is arranged on the diapire of dredging flow groove, and the static pressure groove that is adjacent the box body of chimeric bottom is connected, form the inlet opening of bottom box body, so that liquid flows directly in the middle of the static pressure groove of bottom box body from the top box body of stacked box body.By being opened in the fluid hole on diapire, simplify the structure of stacked bio-reactor.
The number of the box body in preferred stacked box body is 2 to 20;
Above-mentioned stacked bio-reactor can also be provided with in the outside of stacked box body by casing and case lid and form and with the aseptic reaction box that can make the duct that tubing passes through matching with top box body static pressure groove and bottom box body fluid hole, laminated cartridge body is arranged in casing, and container for storing liquid and peristaltic pump are positioned at outside reaction box.By reaction box is set, can make stacked bio-reactor react under sterile closed environment.
Said external can also be arranged with cyclic water jacket outward at reaction box with the bio-reactor of reaction box, and container for storing liquid and peristaltic pump are positioned at outside cyclic water jacket.So that bio-reactor inside is in temperature constant state.
In technique scheme, when inject nutrient solution in static pressure groove, nutrient solution is put aside in static pressure groove, regulate the height of overflow port, unnecessary liquid overflows from overflow port, ensures after formation steady pressure, to flow into lentamente screen bed base bottom with the permeable hole on cut-off equipment in static pressure groove, and in flow field, there is no stopping of screen bed base support, make to react box body middle part and form uniform stable state Plane Fluid Field, carry out uniform nutrition diffusion; Naturally soak and flow into screen bed base bottom because nutrient solution forms steady pressure in static pressure groove, therefore the nutrient solution of inflow screen bed base bottom remains at lower uniform and stable flow velocity, this flow velocity can provide for organ or tissue of the three-dimensional tissue engineering product that the plan on screen bed base is cultivated or produced the shearing force of uniformity, is conducive to hyperplasia and tissue development.
The present invention is a kind of bio-reactor that is applicable to three-dimensional tissue's organ, cell or tissue engineering product perfusion culture, is particularly useful for organization engineering skin or other membranaceous tissues to comprise common cultivation as cornea, heart valve and Various Tissues organ, cell carry out perfusion culture.Its principle of work is: first, by intending on the material solution filling sieve rack of organ or tissue of three-dimensional tissue engineering product of cultivation or production, put into specification mould on screen bed base making the specification of specified shape, take out specification mould, cover lid, then in static pressure groove, inject nutrient solution, nutrient solution is put aside in static pressure groove, unnecessary liquid overflows from overflow port, in guarantee static pressure groove, after formation steady pressure, the permeable hole on cut-off equipment flows to screen bed base, make to react box body middle part and form uniform stable state Plane Fluid Field, the nutritive medium submergence that organ or tissue of the three-dimensional tissue engineering product that plan on screen bed base is cultivated or produced is flowed just, also can suitably reduce liquid level makes three-dimensional tissue to be cultivated on screen bed base all the time in liquid-gas interface cultivation conditions, for organ or tissue of the three-dimensional tissue engineering product of intending research or produce provides sufficient nutrient and suitable shearing force, when the liquid level retaining height at reaction box body middle part is during higher than overflow plate height, nutrient solution flows into dredging flow groove through overflow plate top, and then flow in container for storing liquid from fluid hole, by peristaltic pump again by indent in meditation liquor pump, realize enclosed Cyclic culture.
Due to above structure, the present invention has the following advantages: 1. by overflow port being set and with the cut-off equipment that the liquid in static pressure groove at the uniform velocity evenly can be introduced to the permeable hole of conversion zone, when nutrient solution flows through permeable hole, the tension force of easily breaking through self forms uniform runner, make nutrient solution in the time flowing through screen bed base, remain lower stable even velocity of flow, the Plane Fluid Field that forms stable and uniform in reaction box precursor reactant region, is conducive to hyperplasia; 2, bio-reactor of the present invention can singlely independently use, also can multiple series connection or stacked use, there is good extendability, can maintain the biological characteristics of three-dimensional tissue's organ, the quality that has ensured three-dimensional tissue's organ or product, has improved again the efficiency of fundamental research or production; 3. cut-off equipment of the present invention, specification mould, screen bed base and overflow plate can retract replacing according to actual needs, not only be applicable to the cultivation of organization engineering skin, also be applicable to film material as cornea, heart valve, nerve, blood vessel, mesentery, or the cultivation of other organizer official rank, and can cultivate dissimilar histoorgan simultaneously, remove the seed cell that also can be used for cultivating adherent growth after screen bed base, there is highly versatile, feature applied widely.
Brief description of the drawings
Fig. 1 is the schematic perspective view of reaction chamber of the present invention;
Fig. 2 is the schematic perspective view of box body body wall of the present invention;
Fig. 3 is the structural representation of bio-reactor of the present invention;
Fig. 4 is the schematic perspective view of gate in the present invention;
Fig. 5 is the schematic perspective view on weir flow dam in the present invention;
Fig. 6 is the schematic perspective view of screen cloth and screen bed base in the present invention;
Fig. 7 is the structural representation of the stacked bio-reactor of the present invention;
Fig. 8 is the structural representation of the embodiment of the present invention three;
Fig. 9 is the schematic diagram in flow field of the present invention;
In figure: 1, box body; 2, lid; 3, cut-off equipment; 4, gate; 5, weir flow dam; 6, permeable hole; 7, static pressure groove; 8, overflow plate; 9, dredging flow groove; 10, upper dam body; 11, lower dam body; 12, earial drainage inclined-plane; 13, overflow port; 14, locating shaft; 15, catch; 16, drainage trough; 17, inflow port; 18 inlet openings; 19, fluid hole; 20, screen bed base; 21, screen cloth; 22, specification mould; 23 clamps; 24, engaging groove; 25, flange; 26, gomphosis groove; 27, tubing; 28, container for storing liquid; 29, peristaltic pump; 30, stacked box body; 31, casing; 32, case lid; 33, cyclic water jacket; 34, duct.
Embodiment
Below in conjunction with accompanying drawing, the present invention will be further described.
Embodiment mono-:
Referring to Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 6, Fig. 9, the present invention is a kind of is applicable to the bio-reactor that histocyte dimensional perfusion is cultivated, and comprises that container for storing liquid 28, peristaltic pump 29 and at least one comprise the rectangular parallelepiped box body 1 of open top and the reaction chamber that lid 2 forms; A cut-off equipment 3 is fixed in box body 1 one end, forms static pressure groove 7 with box body 1, and the other end is fixed an overflow plate 8, forms dredging flow groove 9 with box body 1, is fixed with and cut-off equipment 3 and all discontiguous screen bed bases 20 of overflow plate 8 between static pressure groove 7 and dredging flow groove 9; Screen bed base 20 blocks and on the two side of box body 1, screen cloth 21 is fixed in the middle of screen bed base 20; Cut-off equipment 3 longitudinally offers the some evenly distributed permeable holes 6 that the liquid in static pressure groove 7 at the uniform velocity evenly can be introduced to conversion zone in the same level of cross section, and permeable hole 6 is several evenly distributed bar holes, circular port, semi-circular hole or profile hole; On static pressure groove 7 and the shared sidewall of box body 1, be provided with overflow port 13, overflow port 13 comprises U-shaped groove and being rotated and be covered U-shaped groove and can regulate U-shaped groove height and big or small catch 15 by locating shaft 14 of static pressure groove 7 interior settings, is provided with the unnecessary liquid that static pressure groove 7 can be overflowed through overflow port 13 and enters by inflow port 17 drainage trough 16 of dredging flow groove 9 on the sidewall of box body 1; On reaction chamber, arrange one and be communicated with the inlet opening 18 that also liquid directly can be injected to static pressure groove 7 with static pressure groove 7, on the wall of box body 1, arrange one and be communicated with the fluid hole 19 that also liquid directly can be discharged to dredging flow groove 9 with dredging flow groove 9; Cut-off equipment 3 is equal to or less than the degree of depth of box body 1, screen bed base 20 is lower than the height of cut-off equipment 3, overflow plate 8 is greater than or equal to the height of screen bed base 20 upper screen cloths 21, and the lower edge of overflow port 13 is lower than the height of the upper surface of cut-off equipment 3 and higher than the height of overflow plate 8 upper surfaces, inflow port 17 and permeable hole 6 upper limbs; Tubing 27 is connected to container for storing liquid 28, peristaltic pump 29 between fluid hole 19 and inlet opening 18 in turn.
As Fig. 2, shown in Fig. 3, cut-off equipment 3 in the present embodiment is gate 4, peristaltic pump 29 by the cultivation liquid pump in container for storing liquid 28 in the process in static pressure groove 7, inject the nutrient solution higher than permeable hole 6 height, unnecessary liquid overflows from overflow port 13, pass through overflow groove, inflow port 17 flows in dredging flow groove 9, nutrient solution is by the permeable hole 6 on gate 4, and flow into lentamente screen bed base 20 bottoms, make box body 1 middle part form uniform Plane Fluid Field (referring to Fig. 9), carry out uniform nutrition diffusion, and make plan organization of production engineering product on screen cloth 21 be subject to the shearing force of uniformity, avoid because of the inhomogeneous or too high necrocytosis causing of shearing force of nutrient solution nutrition diffusion.
In order to make, the scope of application of the present invention is wider, dismounting cleaning is more convenient, on box body 1 sidewall and diapire, there is engaging groove 24, on gate 4, screen bed base 20 and overflow plate 8, have and can insert the clamp 23 being fixed in described engaging groove 24, gate 4, screen bed base 20 and overflow plate 8 are separable to be fixed on box body 1.Overflow plate 8 is 0～4mm higher than screen bed base 20 upper screen cloths 21, can regulate the liquid level at box body 1 middle part by changing overflow plate 8, not only can provide liquid-gas interface culture environment for organization engineering skin, and can be film material as cornea, heart valve, neural, blood vessel, mesentery etc. provide submerged culture environment, by changing gate 4, screen bed base 20, overflow plate 8 and the other catch 15 of slip overflow port 13, shearing force size and the liquid level in adjustable flow field, be used for other three-dimensional tissue's organ as liver, kidney, lung, bone, the tissues such as cartilage and cell are as stem cell, the cultivation of cancer cells, and in the time removing screen bed base 20, also can be used for cultivating the seed cell of adherent growth.
Described screen bed base 20 can be an entirety, also can be multiple separate screen bed bases 20, certain arranged with interval of being separated by.Described specification mould 22 is fastened on the screen cloth 21 of screen bed base 20 and is contour with the support of screen bed base 20, and specification mould 22 can be designed as square, circular, trilateral or polymorphic structure according to range of product demand.
(1) by intending on the material solution filling sieve rack 20 of the three-dimensional construction of research or plan organization of production engineering product, put into specification mould 22 on screen cloth 21 making the specification of specified shape, take out specification mould 22, cover lid 2; Then with the HT speed of 400ml/min to the interior perfusion culture liquid of static pressure groove 7, in the time that the liquid level of nutrient solution is put aside to a certain extent, the catch 15 of slip overflow port 13 is to regulate overflow port 13 height and size, make nutrient solution have a small amount of liquid just will overflow from overflow port 13, produce stable static pressure, nutrient solution flows to screen bed base 20 from 4 permeable holes 6 of gate; Experiment contrast shows, under the low flow conditions of this 400ml/min, pours into every day 2 times, each half an hour, cultivates 10 days, and hyperplasia is 2.37 times of identical number of days hyperplasia under static culture conditions, can shorten the organization engineering skin 5 days time of cultivation.
(2) by intending on the material solution filling sieve rack 20 of the three-dimensional construction of research or plan organization of production engineering product, put into specification mould 22 on screen cloth 21 making the specification of specified shape, take out specification mould 22, cover lid 2; Then with the medium rate of flooding of 200ml/min to the interior perfusion culture liquid of static pressure groove 7, in the time that the liquid level of nutrient solution is put aside to a certain extent, the catch 15 of slip overflow port 13 is to regulate overflow port 13 height and size, make nutrient solution have a small amount of liquid just will overflow from overflow port 13, produce stable static pressure, nutrient solution flows to screen bed base 20 from 4 permeable holes 6 of gate; Experiment contrast shows, under the low flow conditions of this 200ml/min, pours into every day 2 times, each half an hour, cultivates 10 days, and hyperplasia is 2.48 times of identical number of days hyperplasia under static culture conditions, can shorten the organization engineering skin 5 days time of cultivation.
(3) by intending on the material solution filling sieve rack 20 of the three-dimensional construction of research or plan organization of production engineering product, put into specification mould 22 on screen cloth 21 making the specification of specified shape, take out specification mould 22, cover lid 2; Then with the low rate of flooding of 100ml/min to the interior perfusion culture liquid of static pressure groove 7, in the time that the liquid level of nutrient solution is put aside to a certain extent, the catch 15 of slip overflow port 13 is to regulate overflow port 13 height and size, make nutrient solution have a small amount of liquid just will overflow from overflow port 13, produce stable static pressure, nutrient solution flows to screen bed base 20 from 4 permeable holes 6 of gate; Experiment contrast shows, under the low flow conditions of this 100ml/min, pours into every day 2 times, each half an hour, cultivates 10 days, and hyperplasia is 2.75 times of identical number of days hyperplasia under static culture conditions, shortens the tissue engineering product 5 days time of cultivation.
Embodiment bis-:
As shown in Figure 1, Figure 2, shown in Fig. 3, Fig. 5, Fig. 6, Fig. 9, in above-described embodiment one, the function of gate 4 also can realize by weir flow dam 5, described weir flow dam 5 comprises dam body 10 and lower dam body 11, the side that lower dam body 11 is adjacent with screen bed base 20 has earial drainage inclined-plane 12, permeable hole is positioned at the upper dam body 10 on this weir flow dam, and this earial drainage inclined-plane 12 is 15 °～25 ° angles with box bottom, and the height on earial drainage inclined-plane 12 is 1/3～1/2 of weir flow height of dam degree.Because earial drainage inclined-plane 12 has certain altitude, for the nutrient solution on the earial drainage inclined-plane 12 of avoiding flowing through caused large impact to screen bed base 20, the height control on capable of regulating earial drainage inclined-plane 12 flows to the flow rate of liquid of screen bed base 20 in low flow rate regime.
Embodiment tri-:
Referring to Fig. 1, Fig. 3, Fig. 7, the outside of the upper surface of the sidewall of the box body 1 of the bio-reactor in first embodiment or second embodiment is provided with step-like flange 25, and the surrounding of the diapire of box body 1 is provided with the object gomphosis groove 26 with flange 25 tablings of sidewall upper surface.Multiple box bodys 1 are docked and are formed stacked box body 30 by reverse imbedding, and lid 2 covers on the box body 1 at top; The diapire of box body 1 and the box body of bottom 1 on adjacent stacked box body 30 tops form reaction chamber, and box body 1 and the lid 2 at top form reaction chamber; Wherein the fluid hole 19 of box body 1 is positioned on the diapire of box body 1, the fluid hole 19 being opened on box body 1 diapire has formed the inlet opening 18 of bottom box body 1 and the liquid in the dredging flow groove of top box body 19 directly can have been inputted in the middle of the static pressure groove 7 of bottom box body 1, the inlet opening 18 of top box body 1 is positioned on its reaction chamber, and the liquid of box body 1 fluid hole 19 in bottom being discharged by peristaltic pump 29 is directly pumped in the middle of the static pressure groove 7 of top box body 1; The number of the box body 1 in stacked box body 30 can be 2 to 20, stacked box body 30 and lid 2, tubing 27, container for storing liquid 28, the folded bio-reactor of the common constituting layer of peristaltic pump 29.
When use, nutrient solution is from the static pressure groove 7 of top box body 1 flow through cut-off equipment 3, screen bed base 20, the rear inflow dredging flow groove 9 of overflow plate 8, unnecessary liquid flows into dredging flow groove 9 from overflow port 13 through drainage trough 16, then directly inject the static pressure groove 7 of lower floor's box body 1 by fluid hole 19, flow into container for storing liquid 28 from the fluid hole 19 of the box body 1 of bottom again, through peristaltic pump 29, the nutrient solution in container for storing liquid 28 is pumped in the static pressure groove 7 of top box body 1 again, realizes the enclosed circulation of nutrient solution.Wherein, the quantity of box body 1 that is arranged on middle part can increase as required or reduce.So circulate, the flow rate of liquid at the middle part of all box bodys 1 of flowing through is all the time in low flow rate regime, the three-dimensional tissue's organ that makes the plan organization of production engineering product on screen bed base 20 or intend cultivating can obtain sufficient nutrient and suitable shearing force, has ensured quality product.
Box body 1 in stacked box body 30 in present embodiment also can use as the box body 1 in the middle of Fig. 1, Fig. 3, and when use, the fluid hole 19 of the box body 1 on stacked box body 30 tops and the inlet opening 18 of adjacent bottom box body 1 also can be connected by tubing 27.
Embodiment tetra-:
Accompanying drawing 8 is that the stacked box body 30 in embodiment tri-is put into aseptic reaction box, reaction box is made up of casing 31 and case lid 32, on reaction box, offer the duct 34 that can make tubing 27 pass through of matching with the inlet opening 18 of stacked box body 30 top box bodys 1 and the fluid hole 19 of bottom box body 1, stacked box body 30 is arranged in casing 31, and container for storing liquid 28 and peristaltic pump 29 are positioned at outside casing 31.
Can also establish a cyclic water jacket 33 in reaction box periphery, container for storing liquid 28 and peristaltic pump 29 are positioned at outside cyclic water jacket 33.When use, reaction box can make stacked box body 30 under gnotobasis, cyclic water jacket can make laminated cartridge body in temperature constant state, nutrient solution flows at reaction box inner-outer circulation, flow through the flow rate of liquid at middle part of box body 1 all the time in low flow rate regime, for the plan organization of production engineering product in reaction box or three-dimensional tissue's organ of intending cultivating provide sufficient nutrient and suitable shearing force.The cyclic water jacket 33 arranging in reaction box outside, has ensured the response behaviour in constant temperature in reaction box.
The present invention is by arranging gate 4 or weir flow dam 5, match with overflow port 13, no matter under which kind of rate of flooding, can make nutrient solution at the stable pressure of the interior formation of static pressure groove 7, lower flow velocity remains while flowing through screen bed base 20, box body 1 middle part forms uniform stable state Plane Fluid Field, has overcome the flow field heterogeneity, the nutrition that in existing filling type cultivation, exist and has transmitted the inhomogeneous problem that easily causes necrocytosis, has improved quality product, has shortened the cultivation time; Also can be used for common culture studies and the drug screening of three-dimensional construction or histoorgan.The present invention also has favorable expandability, highly versatile, feature applied widely, has realized the stdn of tissue engineering product is cultivated, and has significantly reduced labour intensity, has improved production efficiency, has good promotion and application and is worth.
Obviously, all embodiment of foregoing description are a part of embodiment of the present invention, instead of whole embodiment.Based on embodiments of the invention, those of ordinary skill in the art are not making all other embodiment that obtain under creative work prerequisite, all belong to the category of the present invention's protection.
Claims (14)
1. be applicable to a bio-reactor for three-dimensional tissue's cell perfusion culture, comprise the reaction chamber of rectangular parallelepiped box body (1) composition of container for storing liquid (28), peristaltic pump (29) and lid (2) and at least one open top; Described box body (1) one end is provided with cut-off equipment (3), form static pressure groove (7) with box body (1), the other end is provided with overflow plate (8), form dredging flow groove (9) with box body (1), between static pressure groove (7) and dredging flow groove (9), be provided with and cut-off equipment (3) and all discontiguous screen bed bases (20) of overflow plate (8); On described reaction chamber, be provided with the inlet opening (18) that is communicated with static pressure groove (7) and liquid directly can be injected to static pressure groove (7), on the wall of box body (1), be provided with the fluid hole (19) that is communicated with dredging flow groove (9) and liquid directly can be discharged to dredging flow groove (9); The height of described cut-off equipment (3) is equal to or less than the degree of depth of box body (1), and the height of screen bed base (20) is lower than the height of cut-off equipment (3), and the height of overflow plate (8) is greater than or equal to the height of screen bed base (20) upper screen cloth (21); The fluid hole (19) of described reaction chamber, inlet opening (18), container for storing liquid (28) are connected by tubing (27) successively with peristaltic pump (29); It is characterized in that: on the sidewall of described static pressure groove (7), be provided with the overflow port (13) of can the interior liquid level of regulating static groove (7) stablizing static pressure to form, on the sidewall of box body (1), be provided with the unnecessary liquid that static pressure groove (7) can be overflowed through overflow port (13) and enter by inflow port (17) drainage trough (16) of dredging flow groove (9); Described cut-off equipment (3) longitudinally offers the some evenly distributed permeable holes (6) that the liquid in static pressure groove (7) at the uniform velocity evenly can be introduced to conversion zone in the same level of cross section; Regulate the flow velocity of liquid and the height of overflow port (13) and big or small to form stair-stepping poor position liquid level in static pressure groove (7), conversion zone and dredging flow groove (9) in inlet opening (18).
2. a kind of bio-reactor that is applicable to three-dimensional tissue's cell perfusion culture according to claim 1, is characterized in that: described screen bed base (20) is fixed on the two side of box body (1); On the face that described cut-off equipment (3), overflow plate (8) contact with box body (1) and the face of screen bed base (20) and box body (1) sidewall contact be provided with clamp (23), the sidewall of box body (1) with on diapire, be provided with fasten with clamp (23) engage groove (24).
3. a kind of bio-reactor that is applicable to three-dimensional tissue's cell perfusion culture according to claim 1, is characterized in that: described overflow port (13) comprise U-shaped groove and in static pressure groove U-shaped groove other arrange can by locating shaft (14) rotate cover U-shaped groove also can be by regulating U-shaped groove lower edge height to carry out the catch (15) of liquid level in regulating static groove (7).
4. a kind of bio-reactor that is applicable to three-dimensional tissue's cell perfusion culture according to claim 1, is characterized in that: described permeable hole (6) is identical bar hole, circular port, semi-circular hole or other profile hole.
5. a kind of bio-reactor that is applicable to three-dimensional tissue's cell perfusion culture according to claim 1, is characterized in that: the height of described overflow plate (8) is 0～4mm higher than the height of the screen cloth (21) on screen bed base (20).
6. a kind of bio-reactor that is applicable to three-dimensional tissue's cell perfusion culture according to claim 1, is characterized in that: the gate (4) that described cut-off equipment (3) is rectangular parallelepiped, permeable hole (6) is positioned at the middle and lower part of gate (4).
7. a kind of bio-reactor that is applicable to three-dimensional tissue's cell perfusion culture according to claim 1, it is characterized in that: described cut-off equipment (3) is the weir flow dam (5) that comprises dam body (10) and lower dam body (11), weir flow dam (5) is rectangular parallelepiped in static pressure groove (7) one sides, the earial drainage inclined-plane (12) of lower dam body (11) side adjacent with screen bed base (20) for flushing with bottom surface, weir flow dam (5); Described earial drainage inclined-plane (12) is 15 °～25 ° angles with box body (1) bottom surface, and the height of earial drainage inclined-plane (12) is 1/3～1/2 of weir flow dam (5) height; Described permeable hole (6) is positioned on dam body (10).
8. a kind of bio-reactor that is applicable to three-dimensional tissue's cell perfusion culture according to claim 1, it is characterized in that: on described screen cloth (21), be provided with the specification mould (22) of cell attachment, specification mould (22) is square, circular, trilateral or polymorphic structure.
9. according to a kind of bio-reactor that is applicable to three-dimensional tissue's cell perfusion culture described in claim 1 to 8 any one, it is characterized in that: multiple box bodys (1) dock the fixing stacked box body (30) that forms by reverse stack, the box body (1) that lid (2) covers at top above and with top box body (1) forms top reaction chamber, the fluid hole (19) of the adjacent pack (1) of stacked box body (30) is connected with inlet opening (18), container for storing liquid (28) and peristaltic pump (29) are connected between the fluid hole (19) and the inlet opening (18) of top reaction chamber of stacked box body (30) bottom box body (1) by tubing (27).
10. according to a kind of bio-reactor that is applicable to three-dimensional tissue's cell perfusion culture described in claim 9 any one, it is characterized in that: in described stacked box body, the quantity of box body is 2 to 20.
11. a kind of bio-reactors that are applicable to three-dimensional tissue's cell perfusion culture according to claim 9, it is characterized in that: the outside of the sidewall upper surface of described box body (1) is provided with step-like flange (25), on box body (1) diapire, offer can with the gomphosis groove (26) of flange (25) tabling, multiple box bodys (1) are oppositely and forms stacked box body (30) by flange (25) with docking of gomphosis groove (26).
12. a kind of bio-reactors that are applicable to three-dimensional tissue's cell perfusion culture according to claim 9, it is characterized in that: the fluid hole (19) of bottom box body (1) is positioned on the sidewall of box body (1) in described stacked box body (30), the fluid hole (19) of other box bodys (1) is arranged on the diapire of box body (1) and the liquid of dredging flow groove (9) directly can be inputted to bottom box body (1) static pressure groove (7).
13. a kind of bio-reactors that are applicable to three-dimensional tissue's cell perfusion culture according to claim 9, it is characterized in that: the outside of described stacked box body (30) is provided with the reaction box being made up of casing (31) and case lid (32), on reaction box, offer the duct (34) that can make tubing (27) pass through, container for storing liquid (28) and peristaltic pump (29) are positioned at outside reaction box.
14. a kind of bio-reactors that are applicable to three-dimensional tissue's cell perfusion culture according to claim 13, is characterized in that: described reaction box is arranged with cyclic water jacket (33) outward, container for storing liquid (28) and peristaltic pump (29) are positioned at outside cyclic water jacket (33).
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410218150.XA CN103966095B (en) | 2014-05-22 | 2014-05-22 | A kind of bio-reactor being applicable to three-dimensional tissue's Cell infusion and cultivating |
| PCT/CN2015/079330 WO2015176653A1 (en) | 2014-05-22 | 2015-05-20 | Bioreactor for three-dimensional tissue perfusion culture |
| US15/328,460 US9902929B2 (en) | 2014-05-22 | 2015-05-20 | Bioreactor for three-dimensional tissue perfusion culture |
| EP15795352.2A EP3147349B1 (en) | 2014-05-22 | 2015-05-20 | Bioreactor for three-dimensional tissue perfusion culture |
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| CN201410218150.XA CN103966095B (en) | 2014-05-22 | 2014-05-22 | A kind of bio-reactor being applicable to three-dimensional tissue's Cell infusion and cultivating |
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| CN103966095B CN103966095B (en) | 2016-01-20 |
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| WO2015176653A1 (en) * | 2014-05-22 | 2015-11-26 | 第三军医大学第三附属医院 | Bioreactor for three-dimensional tissue perfusion culture |
| CN105754856A (en) * | 2016-04-12 | 2016-07-13 | 中国航天员科研训练中心 | Drawer type three-dimensional quasi-vascularization tissue culture organ chip and establishment method thereof |
| CN105950468A (en) * | 2016-05-23 | 2016-09-21 | 西安交通大学 | Bioreactor for perfusion and bioreactor for permeation |
| CN106940333A (en) * | 2017-04-17 | 2017-07-11 | 山西医科大学 | The outer Electrophysiology research perfusion system of isolated cells |
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| CN108749274A (en) * | 2018-04-30 | 2018-11-06 | 浙江聚众柔印科技有限公司 | Flexible platemaking machine Chinese medicine device water-dispersed |
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| CN105754856B (en) * | 2016-04-12 | 2018-06-22 | 中国航天员科研训练中心 | A kind of drawer type three-dimensional class blood vessel tissue culture organ chip and its construction method |
| CN105754856A (en) * | 2016-04-12 | 2016-07-13 | 中国航天员科研训练中心 | Drawer type three-dimensional quasi-vascularization tissue culture organ chip and establishment method thereof |
| CN105950468A (en) * | 2016-05-23 | 2016-09-21 | 西安交通大学 | Bioreactor for perfusion and bioreactor for permeation |
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| CN106940333A (en) * | 2017-04-17 | 2017-07-11 | 山西医科大学 | The outer Electrophysiology research perfusion system of isolated cells |
| CN108060081A (en) * | 2018-02-05 | 2018-05-22 | 中山大学 | A kind of multi-layer planar filling type bioreactor |
| CN108749274A (en) * | 2018-04-30 | 2018-11-06 | 浙江聚众柔印科技有限公司 | Flexible platemaking machine Chinese medicine device water-dispersed |
| KR102381523B1 (en) * | 2020-10-15 | 2022-03-31 | 주종일 | Three-dimensional cell culture chip with automatic supplying cell culture media |
| CN113512497A (en) * | 2021-08-12 | 2021-10-19 | 浙江大学 | 3D printing culture tank, culture system and detection system for in-vitro intestinal living body culture and application |
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