CN103290145B - A kind of filling type bioreactor experimental technique and using appts thereof - Google Patents
A kind of filling type bioreactor experimental technique and using appts thereof Download PDFInfo
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- CN103290145B CN103290145B CN201310215963.9A CN201310215963A CN103290145B CN 103290145 B CN103290145 B CN 103290145B CN 201310215963 A CN201310215963 A CN 201310215963A CN 103290145 B CN103290145 B CN 103290145B
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- 238000002474 experimental method Methods 0.000 title abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 46
- 235000015097 nutrients Nutrition 0.000 claims description 80
- 239000012530 fluid Substances 0.000 claims description 58
- 239000012528 membrane Substances 0.000 claims description 13
- 238000001914 filtration Methods 0.000 claims description 10
- 238000009530 blood pressure measurement Methods 0.000 claims description 9
- 238000009434 installation Methods 0.000 claims description 7
- 230000002706 hydrostatic effect Effects 0.000 abstract description 33
- 238000004113 cell culture Methods 0.000 abstract description 21
- 230000000694 effects Effects 0.000 abstract description 11
- 230000003068 static effect Effects 0.000 abstract description 7
- 230000008859 change Effects 0.000 abstract description 6
- 238000010008 shearing Methods 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 5
- 230000005484 gravity Effects 0.000 abstract description 5
- 230000003321 amplification Effects 0.000 abstract description 3
- 238000013459 approach Methods 0.000 abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 3
- 230000000638 stimulation Effects 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 238000010998 test method Methods 0.000 abstract description 2
- 230000002572 peristaltic effect Effects 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 9
- 230000001105 regulatory effect Effects 0.000 description 9
- 238000000034 method Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 5
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 4
- 239000004926 polymethyl methacrylate Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000001360 synchronised effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 231100000652 hormesis Toxicity 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000005226 mechanical processes and functions Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
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Abstract
The present invention relates to the bioreactor technology field in medicine equipment, more specifically, relate to a kind of filling type bioreactor experimental technique and using appts thereof.The present invention proposes the experimental test procedures of the double influence of shearing force and the hydrostatic pressure caused by laminar flow for cell in filling type bioreactor, for in the amplification culture of filling type bioreactor, the impact of static pressure on cell cultures that test cultures liquid applies cell under gravity provides can the approach of quantitative examination.The present invention devises the device that quantitatively can apply hydrostatic pressure on set hydrodynamic shear, can under cell cultures be provided in the condition of hydrodynamic shear effect of setting, reoffer and to change by set rule or the physical stimulation factor of invariable hydrostatic pressure.
Description
Technical field
The present invention relates to the bioreactor technology field in medicine equipment, more specifically, relate to a kind of filling type bioreactor experimental technique and using appts thereof.
Background technology
Cell cultures is the key of organizational project, the science and technology basis of necessity of Ye Shi formative tissue engineering industry.The research of the existing filling type bioreactor for cell cultures is the condition based on small-scale cell cultures, when considering that the various physical force suffered by cell stimulate, all be the hypothesis of zero based on suffered hydrostatic pressure, namely be under the universal gravity constant of hypothesis liquid is the precondition of zero, the hydrodynamic shear of main consideration fluid under laminar model.
Existing for providing the major experimental instrument of accurate fluid shearing force condition to be parallel plate flow cavity device under research laminar model, its structural representation as shown in Figure 1, its function is: in the culture chamber 3 in Fig. 1, cultured cells provides the hormesis of accurate hydrodynamic shear, and the effect of research cell cultures under the stimulation of different fluid shearing force, with the quantitative relationship existed between test fluid flow shearing force and cell cultures effect.Its principle is the parallel plate flow cavity shown in Fig. 1, and the value of long L and wide b much larger than height h, then can think full-blown telescopic movement at the fluid of the middle portion of culture chamber 3, and the hydrodynamic shear that fluid produces can be calculated as by Poiseuille's law:
,
for fluid flow rate,
for the viscosity factor of nutrient solution, b is the width of culture chamber 3 in Fig. 1, and h is the height of culture chamber 3.
, b, h be constant, only nutrient solution need be regulated to enter the flow of culture chamber 3 from nutrient solution entrance 1, or flow Q regulating nutrient solution to flow out from nutrient solution outlet 2, the hydrodynamic shear needed for just obtaining.
But, realize large scale culturing cell, need consider that existing small-sized biological reactor is carrying out amplifying the rear problem produced, due to the amplification of scale, dark the produced hydrostatic pressure of liquid of nutrient solution is no longer negligible physical mechanics factor on the impact that cell cultures produces, and needs to study.Namely existing parallel plate flow cavity is as when in research filling type bioreactor, nutrient solution produces experimental technique and the instrument of mechanical stimulation factor, mainly has the following disadvantages:
(1), in existing research filling type bioreactor hydrodynamic interaction only considered the effect of hydrodynamic shear, does not consider the effect of hydrostatic pressure.In fact, when bio-reactor carries out amplification culture, and hydrostatic pressure that is that produce dark due to liquid can not be left in the basket again and disregard, and needs to consider that different hydrostatic pressures is on the impact of cell cultures;
(2), the existing single factors of accurate hydrostatic pressure that can provide is be placed on by cell in the dark culture tank of certain liquid to carry out quiescent culture on the experimental technique that cell cultures affects, although this standing cultural method can obtain hydrostatic pressure accurately deeply by controlling liquid, but the exchange of substance needed for cell can be made to be difficult to carry out smoothly because of not flowing of nutrient solution, the culture effect of cell can be had a strong impact on because exchange of substance difficulty, cannot the influencing factor of correct test liquid static pressure;
(3), existing parallel plate flow cavity can only provide hydrodynamic shear accurately, can not provide hydrostatic pressure accurately simultaneously.
(4), existing cell culture experiments method and apparatus, accurately controlled hydrodynamic shear and accurately controlled hydrostatic pressure can not be provided simultaneously, make these two reactive forces be independent of each other, can the power that coexist of each Self-controlled.
Summary of the invention
The present invention is for overcoming at least one defect described in above-mentioned prior art, a kind of filling type bioreactor experimental technique is provided, realization can provide accurately controlled hydrodynamic shear and accurately controlled hydrostatic pressure simultaneously, make these two reactive forces be independent of each other, can the power that coexist of each Self-controlled.Make cell cultures under the condition of nutrient solution flowing cultivation, the impact of Study of Liquid static pressure on cell cultures can be carried out.Further, provide its using appts, this apparatus structure is simple, easy to use.
For solving the problems of the technologies described above, the technical solution used in the present invention is: a kind of filling type bioreactor experimental technique, comprising following steps:
S1. according to the requirement of laminar flow hydrodynamic shear in filling type bioreactor, the fluid flow rate simultaneously regulating nutrient solution entrance and nutrient solution to export, makes the shearing force of parallel plate culture chamber inner fluid consistent with laminar fluid shearing force required in filling type bioreactor; Wherein, nutrient solution entrance and nutrient solution output fluid flow regulate by regulate be connected with nutrient solution entrance entrance peristaltic pump, export with nutrient solution the outlet peristaltic pump be connected and realize.
S2. keep the nutrient solution of nutrient solution outlet to flow out flow to immobilize, the nutrient solution increasing nutrient solution entrance flows into flow, make nutrient solution enter fluid pressure pipe, when the nutrient solution liquid level in fluid pressure pipe is raised to required height h, the nutrient solution of renewal cultivation liquid entrance flows into flow;
S3. in fluid pressure pipe, the liquid level of nutrient solution will maintain required height h, thus can be in parallel plate culture chamber and provide pressure to be
hydrostatic pressure,
for nutrient solution density, g is universal gravity constant, and h is the height in fluid pressure intraluminal fluid face;
S4. keep the nutrient solution of nutrient solution outlet to flow out flow to immobilize, regulate the nutrient solution of nutrient solution entrance to flow into flow, thus the height h of liquid level in regulates liquid static tube, thus regulate the size of hydrostatic pressure suffered in parallel plate culture chamber;
S5. the nutrient solution of synchronous same incremental adjustments nutrient solution entrance flows into the nutrient solution that flow and nutrient solution export and flows out flow, thus while the size of hydrodynamic shear suffered by adjustment parallel plate culture chamber, in maintenance liquid static tube, liquid level is constant.
Further, to the synchronous same incremental adjustments of inflow, effluent fluid in described step S1, be specially:
The flow that when regulating the inflow of nutrient solution, outflow flow, guarantee flows into, flow out is identical, synchronization regulation, and make the liquid level h in fluid pressure pipe be maintained 0, the hydrodynamic shear that parallel plate culture chamber inner fluid produces can be calculated as by Poiseuille's law:
,
for fluid flow rate,
for the viscosity factor of nutrient solution, W is the width of parallel plate culture chamber, and H is the height of parallel plate culture chamber,
, W, H be constant, only need regulate the flow Q of nutrient solution, the hydrodynamic shear needed for acquisition.
Further, fluid pressure liquid in pipe geo-stationary, the liquid in parallel plate culture chamber is full-blown telescopic movement, and static, dynamic liquid coexists, and is specially:
The liquid volume in parallel plate culture chamber is entered from nutrient solution entrance, with exported the liquid volume flowed out by nutrient solution, be completely equal, make the volume maintenance of parallel plate cultivation intracavity liquid constant, thus the liquid level in fluid pressure pipe can be made to remain unchanged;
In fluid pressure pipe, liquid bottom pipe participates in the liquid-flow in parallel plate culture chamber, liquid above bottom pipe does not participate in the liquid-flow in parallel plate culture chamber, the hydrostatic pressure that fluid pressure pipe provides is considered as the hydrostatic pressure under inactive liquid state, thus can by pressure formula
accurate hydrostatic pressure is provided, wherein,
for nutrient solution density, g is universal gravity constant, and h is the height in fluid pressure intraluminal fluid face.
Further, described step S4, S5 achieve hydrodynamic shear and hydrostatic pressure can carry out quantitatively regulating and controlling respectively, and can act on the experimental test procedures of cell cultures simultaneously, is specially:
By step S4, the hydrostatic pressure needed for can applying cell cultures, described hydrostatic pressure can carry out accurate quantitatively regulating, and can change according to set rule, also can maintain required steady state value constant;
By step S5, the hydrodynamic shear needed for can applying cell cultures, described hydrodynamic shear can carry out accurate quantitatively regulating, and can change according to set rule, also can maintain required steady state value constant.
A kind of filling type bioreactor experimental installation, comprise parallel plate culture chamber, the nutrient solution entrance be communicated with parallel plate culture chamber and nutrient solution to export, wherein, parallel plate culture chamber is also provided with fluid pressure pipe, fluid pressure pipe be axially perpendicular to parallel plate culture chamber, fluid pressure pipe is communicated with parallel plate culture chamber; Parallel plate culture chamber is also communicated with liquid pressure measurement interface; The top of fluid pressure pipe is provided with filtering membrane.Liquid pressure measurement interface can connect inspection units thus detect the liquid pressure in parallel plate culture chamber.
Further, described parallel plate culture chamber comprises culture chamber bottom, the culture chamber upper cover be located on culture chamber bottom, be located between culture chamber bottom and culture chamber upper cover culture chamber in room; Culture chamber bottom and culture chamber upper cover seal fixing by sealing-ring, the outlet of described nutrient solution entrance, nutrient solution, liquid pressure measurement interface are located on the outer wall of culture chamber bottom, are communicated with respectively with room in culture chamber; Described fluid pressure pipe is located on culture chamber and is covered, and it is axially perpendicular to culture chamber upper lid surface, is communicated with room in culture chamber.
Further, described culture chamber covers and is also provided with fixed screw holes, screw makes culture chamber upper cover and culture chamber bottom fix through fixed screw holes.
Concrete, described parallel plate culture chamber is rectangular structure, and its length is L=60mm, and width is W=20mm, is highly H=2.5mm.The long L of parallel plate culture chamber and the value of wide W are much larger than the value of its height, and described filtering membrane is the reticular membrane in 0.2 micron, aperture, and extraneous bacterium isolates by filtering membrane, and whole device material polymethylmethacrylate (PMMA) is made.
Compared with prior art, beneficial effect is:
1) the present invention take accurately controlled hydrodynamic shear and accurately controlled hydrostatic pressure be independent of each other, can the experimental technique of each Self-controlled, make to be undertaken strictly and accurately regulating and controlling by its respective mechanical function formula respectively.
2) the present invention takes accurately controlled hydrodynamic shear and the acting in conjunction simultaneously of accurately controlled hydrostatic pressure energy in the experimental technique of same point of application, the relation between the mechanical condition suffered by cell at this point of application place and cell cultures effect is made to carry out quantitative analysis accurately, and separable go out the impact of hydrostatic pressure single factors, for the impact of hydrostatic pressure in filling type bioreactor on cell cultures provides quantifiable accurate analysis approach.
3) experimental installation of the present invention, the experimental technique that can propose for the present invention provides the device of enforcement, accurately controlled hydrodynamic shear and accurately controlled hydrostatic pressure can be provided simultaneously, two reactive forces provided can be independent of each other, each Self-controlled of energy, and can act on same point of application.
Accompanying drawing explanation
Fig. 1 is existing parallel plate flow cavity structural representation.
Fig. 2 is the schematic flow sheet of experimental technique of the present invention.
Fig. 3 is the one-piece construction schematic diagram of experimental installation of the present invention.
Fig. 4 is the structure for amplifying schematic diagram of experimental installation of the present invention.
Embodiment
Accompanying drawing, only for exemplary illustration, can not be interpreted as the restriction to this patent; In order to better the present embodiment is described, some parts of accompanying drawing have omission, zoom in or out, and do not represent the size of actual product; To those skilled in the art, in accompanying drawing, some known features and explanation thereof may be omitted is understandable.
As shown in Figure 3,4, a kind of filling type bioreactor experimental installation, comprise parallel plate culture chamber 40, the nutrient solution entrance 10 be communicated with parallel plate culture chamber 40 and nutrient solution and export 30, wherein, parallel plate culture chamber 40 is also provided with fluid pressure pipe 50, fluid pressure pipe 50 be axially perpendicular to parallel plate culture chamber 40, fluid pressure pipe 50 is communicated with parallel plate culture chamber 40; Parallel plate culture chamber 40 is also communicated with liquid pressure measurement interface 20; The top of fluid pressure pipe 50 is provided with filtering membrane 60.Liquid pressure measurement interface 20 can connect inspection units thus detect the liquid pressure in parallel plate culture chamber 40.
Parallel plate culture chamber 40 comprises culture chamber bottom 41, the culture chamber upper cover 43 be located on culture chamber bottom 41, be located between culture chamber bottom 41 and culture chamber upper cover 43 culture chamber in room 42; Culture chamber bottom 41 seals fixing with culture chamber upper cover 43 by sealing-ring, nutrient solution entrance 10, nutrient solution outlet 30, liquid pressure measurement interface 20 are located on the outer wall of culture chamber bottom 41, are communicated with respectively with room in culture chamber 42; Fluid pressure pipe 50 is located on culture chamber upper cover 43, and it is axially perpendicular to culture chamber upper cover 43 surface, is communicated with room in culture chamber 42.
Culture chamber upper cover 43 is also provided with fixed screw holes 44, and screw makes culture chamber upper cover 43 fix with culture chamber bottom 41 through fixed screw holes 44.Parallel plate culture chamber 40 is rectangular structure, and its length is L=60mm, and width is W=20mm, is highly H=2.5mm.Filtering membrane 60 is the reticular membrane in 0.2 micron, aperture.The long L of parallel plate culture chamber and the value of wide W are much larger than the value of its height, and its size meets the requirement of culturing cell, and extraneous bacterium isolates by filtering membrane 60, and whole device material polymethylmethacrylate (PMMA) is made.
In device as shown in Figure 3,4, the glass slide of planting cell as shown in Figure 2, is placed into the middle position of room 42 in culture chamber by its method flow, is covered by culture chamber upper cover 43 on culture chamber bottom 41, between have O type circle to seal, then to be reinforced from fixed screw holes 44 with screw.Then the peristaltic pump with nutrient solution entrance 10 is only started, nutrient solution to be input in hold-up vessel in culture chamber in room 42, when filling nutrient solution in room in culture chamber 42, start and export 30 peristaltic pumps be connected with nutrient solution, regulate its flow, make fill nutrient solution in room 42 in culture chamber and do not inject nutrient solution in fluid pressure pipe 50.Then 2 peristaltic pumps being connected with nutrient solution entry and exit of synchronous same incremental adjustments, make the flux values that the fluid flow rate in culture chamber in room 42 reaches required
, the hydrodynamic shear with in room in culture chamber 42 needed for activity:
,
for fluid flow rate,
for the viscosity factor of nutrient solution, W is the width of parallel plate culture chamber 40, and H is the height of parallel plate culture chamber 40.In the experimental installation that the present invention proposes,
, W, H be constant, only need regulate the flow of nutrient solution
, the hydrodynamic shear needed for just obtaining.Now, the flow due to nutrient solution entry and exit remains consistent, and in culture chamber, in room 42, the volume of nutrient solution does not change, and makes to fill nutrient solution in room 42 in culture chamber and in fluid pressure pipe 50, the liquid level of nutrient solution is 0.
Then keep the flow exporting the peristaltic pump that 30 are connected with nutrient solution constant, only increase the flow of the peristaltic pump be connected with nutrient solution entrance 10, the fluid surface in fluid pressure pipe 50 is made to be elevated to required height h, and then make the flow of the peristaltic pump be connected with nutrient solution entrance 10 reduce to the flow exporting the peristaltic pump that 30 are connected with nutrient solution, thus it is constant to make the liquid level of nutrient solution in fluid pressure pipe 50 be maintained h.Due to the connection principle of liquid pressure, the liquid pressure be subject in room 42 in culture chamber is identical with the liquid pressure be subject to bottom fluid pressure pipe 50, is all
accurate hydrostatic pressure is provided, wherein,
for nutrient solution density, g is universal gravity constant, and h is the height of liquid level in fluid pressure pipe 50.Thus can be room 42 in culture chamber and provide pressure to be
hydrostatic pressure.
So far, achieved the cell cultures place of room 42 in culture chamber, provide two reactive forces simultaneously, a reactive force is hydrostatic pressure
, regulate its size by fluid pressure intraluminal fluid face height h; Another one reactive force is hydrodynamic shear
, by regulating the flow of nutrient solution in room 42 in culture chamber
regulate its size.
Pressure in culture chamber in room 42, carries out real-time monitored by the liquid pressure sensor be connected with liquid pressure measurement interface, to obtain the data of hydrostatic pressure accurately.
When gas in nutrient solution flows in room 42 in culture chamber, can be discharged in air from the filtering membrane 60 Fig. 3, make the pressure in culture chamber in room 42 not influenced by air release.Filtering membrane 60 is the mesh film of 0.2 micron for aperture, by gas, and plays isolate bacteria.
The method of adjustment h size is: keep the flow exporting the peristaltic pump that 30 are connected with nutrient solution constant, only change the flow of the peristaltic pump be connected with nutrient solution entrance 10, the fluid surface in fluid pressure pipe 50 is made to be elevated to required height h, and then make the flow of the peristaltic pump be connected with nutrient solution entrance 10 return to the flow exporting the peristaltic pump that 30 are connected with nutrient solution, thus it is constant to make the liquid level of nutrient solution in fluid pressure pipe 50 be maintained required h.
Described adjustment
the method of size is: 2 peristaltic pumps be synchronously connected with nutrient solution entry and exit with incremental adjustments, make the flux values that the fluid flow rate in culture chamber in room 42 reaches required
.
The method of described adjustment h, while adjustment h, can keep
constant; Described adjustment
method, adjustment
while, h can be kept constant.
Described hydrostatic pressure, it is the reactive force of geo-stationary, in fluid pressure pipe 50, only have a small amount of nutrient solution in bottom can change because of the flowing of nutrient solution in room in culture chamber 42, most of nutrient solution more than fluid pressure pipe 50 inner bottom part is geo-stationary, it is to the hydrostatic pressure provided in room in culture chamber 42, can more strictly according to
calculate.
The experimental technique that the present invention proposes, achieve while hydrostatic pressure effect is provided, cell can be cultivated under the environment of laminar flow, overcome rest on exchange of substance when cultivating in culture tank unfavorable factor on the impact of cell cultures effect, quantize experimental approach accurately for the assessment of static pressure liquid in filling type bioreactor to cell cultures effect provides science.
Obviously, the above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.All any amendments done within the spirit and principles in the present invention, equivalent to replace and improvement etc., within the protection domain that all should be included in the claims in the present invention.
Claims (1)
1. a filling type bioreactor experimental installation, comprise parallel plate culture chamber (40), the nutrient solution entrance (10) be communicated with parallel plate culture chamber (40) and nutrient solution to export (30), it is characterized in that, parallel plate culture chamber (40) is also provided with fluid pressure pipe (50), fluid pressure pipe (50) be axially perpendicular to parallel plate culture chamber (40), fluid pressure pipe (50) is communicated with parallel plate culture chamber (40); Parallel plate culture chamber (40) is also communicated with liquid pressure measurement interface (20); The top of fluid pressure pipe (50) is provided with filtering membrane (60);
Described parallel plate culture chamber (40) comprises culture chamber bottom (41), the culture chamber upper cover (43) be located on culture chamber bottom (41), be located between culture chamber bottom (41) and culture chamber upper cover (43) culture chamber in room (42); Culture chamber bottom (41) seals fixing with culture chamber upper cover (43) by sealing-ring, described nutrient solution entrance (10), nutrient solution outlet (30), liquid pressure measurement interface (20) are located on the outer wall of culture chamber bottom (41), are communicated with respectively with room in culture chamber (42); Described fluid pressure pipe (50) is located on culture chamber upper cover (43), and it is axially perpendicular to culture chamber upper cover (43) surface, is communicated with room in culture chamber (42);
Described culture chamber upper cover (43) is also provided with fixed screw holes (44), and screw makes culture chamber upper cover (43) fix with culture chamber bottom (41) through fixed screw holes (44);
Described parallel plate culture chamber (40) is rectangular structure, and its length is L=60mm, and width is W=20mm, is highly H=2.5mm;
The reticular membrane that described filtering membrane (60) is 0.2 micron, aperture.
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