CN104099247B - Unify method based on the pressurization cell culture system of micro-fluidic chip - Google Patents
Unify method based on the pressurization cell culture system of micro-fluidic chip Download PDFInfo
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- CN104099247B CN104099247B CN201310126330.0A CN201310126330A CN104099247B CN 104099247 B CN104099247 B CN 104099247B CN 201310126330 A CN201310126330 A CN 201310126330A CN 104099247 B CN104099247 B CN 104099247B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
Abstract
The present invention provides a kind of pressurization cell culture system based on micro-fluidic chip and unifies method, comprise micro-fluidic chip, pressure actuated device, connecting tube and pressure-detecting device, containing passage in described micro-fluidic chip, described pressure actuated device comprise mate mutually with it for holding the container of substratum, described container is connected with described micro-fluidic chip by connecting tube, described Power Drive Unit promotes the substratum in container to enter micro-fluidic chip, and described pressure-detecting device is connected with described micro-fluidic chip by connecting tube. This system can conveniently carry out the cultivation of cell under shearing force and pressure effect and correlative study, and this cell pressurization culture systems easy accessibility, is convenient to carry.
Description
Technical field
The present invention relates to cytobiology and cell mechanical technology field, specifically, it relates to a kind of cell based on micro-fluidic chip pressurization culture systems and method.
Background technology
Nineteen ninety, Switzerland scientist Manz and Widmer proposes the concept of micro-total analysis system (Micrototalanalysissystem), and its target is in a portable equipment or even one piece of small chip by the function i ntegration in whole analytical test laboratory. Therefore, micro-total analysis system is also referred to as in " laboratory (Lab-on-a-chip, i.e. LOC) on chip ". And micro-fluidic chip (Microfluidicchip) is the important component part of LOC, its main feature is the resulting structure (comprising passage, reactor and other functional units) of accommodation fluid is micron order size at least one dimension degree. Micro-fluidic chip is based on microcomputer EDM Technology, the materials such as silicon chip, glass or PDMS manufacture passage and integrated driving and inspection device, by the control of fluid in passage is prepared sample, is separated and detected, the basic operation units such as biology and chemical reaction are integrated on the chip of a piece several square centimeters (even less), in order to complete different biological or chemical reaction process, and its product is analyzed.
Conventional cell pressurization is cultivated as gas pressurization cultural method, such as by cell cultures in culture dish, is placed on by culture dish in pressure kettle equal pressure container, then is put in cell culture incubator by pressurized vessel. Patent CN1858203A discloses a kind of pneumatically controlled pressurizing cell cultivation instrument, comprises the container of band opening and the container cover mated mutually with it, described container or container cover are provided with inlet mouth, air outlet, tensimeter and safety valve. This kind of method operability is to be improved, and pressure is wayward.
Current micro-fluidic chip and the system comprising micro-fluidic chip have been widely used in multiple technical field, especially technical field of cell biology, the simulation correlative technology field such as hypertension then need to apply in cell cultivation process certain pressure with close proximity to body in physiological environment, the micro-fluidic chip culture systems up till now with pressurized operation is not appeared in the newspapers.
Summary of the invention
The present invention is directed to above problem, it is provided that a kind of pressurization cell culture system based on micro-fluidic chip is unified method.
The object of the present invention is achieved by following technique means:
A kind of pressurization cell culture system based on micro-fluidic chip, comprise micro-fluidic chip, pressure actuated device, connecting tube and pressure-detecting device, containing passage in described micro-fluidic chip, described pressure actuated device comprises the container for holding substratum mated mutually with it, described container is connected with described micro-fluidic chip by connecting tube, described Power Drive Unit promotes the substratum in container to enter micro-fluidic chip, and described pressure-detecting device is connected with described micro-fluidic chip by connecting tube.
Channel size in the micro-fluidic chip of pressurization cell culture system provided by the invention and cell size rank are suitable, and this passage in conjunction with perfusion training method closer to the physiological status in body; Use this micro-fluidic chip can conveniently control cell micro-environment, and this chip forms the environment of relative closure, reduce external environment to the interference of cell.
In pressurization cell culture system based on micro-fluidic chip provided by the invention, described pressure actuated device is syringe pump or peristaltic pump, the described container for substratum is syringe, described connecting tube is flexible pipe, and described flexible pipe can be rubber hose, plastic hose, PVC, PE, PP, various types of flexible pipe such as silicone tube, size is mated mutually with the outlet of the container for holding substratum, the entrance of micro-fluidic chip; Described pressure-detecting device is tensimeter or pressure warning unit.
Wherein, described micro-fluidic chip comprises entrance, outlet and passage. Channel height is 20-400 ��m. Passage can be various form, comprises straight channel, bending channel, single passage, many parallel channels etc. Preferably, described passage length is 3-6cm, and passage width is 1-3mm, is highly 50-200 ��m.
Wherein, described pressure actuated device and described pressure-detecting device lay respectively at the both sides of described micro-fluidic chip, and they are connected by the two ends of connecting tube with the passage in described micro-fluidic chip respectively.
In the pressurization cell culture system based on micro-fluidic chip that the preferred embodiment of the invention provides, described syringe pump lays respectively at the relative both sides of micro-fluidic chip with described pressure warning unit, they are connected by the two ends of flexible pipe with the passage in this micro-fluidic chip, when this cell culture system carries out pressure test after having assembled, syringe pump pushing syringe, medium flow in syringe enters the microchannel in micro-fluidic chip, flow out through this passage and flow to pressure warning unit direction follow-up continuing, in this kind of pattern simulation passage, cultured cells not only bears pressure but also bear the pattern of flowing shearing force.
Wherein, described pressure actuated device and described pressure-detecting device are positioned at the same side of micro-fluidic chip, and they are connected with one end of the passage in described micro-fluidic chip by connecting tube simultaneously, and the other end of described passage is closed.
In the pressurization cell culture system based on micro-fluidic chip that the preferred embodiment of the invention provides, described syringe pump and described pressure warning unit are positioned at the same side of micro-fluidic chip, it is connected with one end of the passage in micro-fluidic chip, the other end of this passage is connected with one end of flexible pipe, the other end of this flexible pipe is closed, when this cell culture system carries out pressure test after having assembled, liquid in syringe pump pushing syringe flows to pressure warning unit, and this kind of pattern simulation passage inner cell only bears pressure and do not bear the situation of shearing force.
Preferably, in the pressurization cell culture system based on micro-fluidic chip that the embodiment of the present invention provides, micro-fluidic chip is made up of one or more in following material: polymethylmethacrylate (Polymethylmethacrylate, PMMA), polydimethylsiloxane (Polydimethylsiloxane, PDMS), polyethylene terephthalate (Polyethyleneterephthalate, PET), polycarbonate (Polycarbonate, PC), glass, epoxy resin and hydrogel.
In the preferred embodiment of the invention, described micro-fluidic chip is made up of polydimethylsiloxane (PDMS) and glass, and PDMS is a kind of the macromolecule organic silicon compound, there is optics permeability, and be inertia under normal circumstances, and nontoxic, it is extensively for micro-fluidic chip field.
The another aspect of the present invention, it is provided that a kind of apply the above-mentioned pressurization cell culture system based on micro-fluidic chip carry out pressurize cell cultures method.
Specifically comprise the following steps:
1) attached cell is cultivated in the passage of micro-fluidic chip;
2) utilize pressure actuated device to promote cell culture medium to enter micro-fluidic chip with certain flow velocity, produce pressure;
3) pressure born by pressure-detecting device monitoring cell.
Wherein, described flow velocity is for being 0.01 �� l/min-10ml/min, it is preferable that, 5-50 �� l/min.
Pressurization cell culture system based on micro-fluidic chip provided by the invention has the following advantages: 1, the channel size of micro-fluidic chip is little, suitable with cell size; 2, passage and conventional perfusion training method are closer to the physiological status in body, simulate the growth conditions of cell under physiological status more really; 3, cell micro-environment can conveniently be controlled; 4, the multidimensional network structure of chip forms the environment of relative closure, reduces external environment to the stimulation of cell; 5, in real time the cell in chip channel can be observed, detect; 6, micro-fluidic chip volume is little, can save the consumption of cell and reagent, saves analysis time and cost; 7, the multiple monotechnics of chip can flexible combination, scale is integrated. 8, system bulk is little, easily builds, easy to assemble, flexibly, portable. 9, cut-in pressure detects device in systems in which, it is possible to pressure in Real-Time Monitoring microchannel.
Accompanying drawing explanation
Fig. 1 is the structural representation of the pressurization cell culture system based on micro-fluidic chip in one embodiment of the invention, and wherein syringe pump and pressure warning unit lay respectively at the both sides of micro-fluidic chip.
Fig. 2 is the structural representation of the pressurization cell culture system based on micro-fluidic chip in one embodiment of the invention, and wherein syringe pump and pressure warning unit are positioned at the same side of micro-fluidic chip.
Fig. 3 is the structural representation of the passage in the micro-fluidic chip in one embodiment of the invention.
Fig. 4 is the structural representation of the passage in the micro-fluidic chip in one embodiment of the invention.
Wherein 1, syringe; 2, syringe pump; 3, flexible pipe; 4, micro-fluidic chip; 5, the aobvious pressure warning unit of number; 6, clip; 7, the passage in micro-fluidic chip
Embodiment
In order to make the object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is described in further detail. It is to be understood that specific embodiment described herein is only in order to explain the present invention, it is not intended to limit the present invention.
Embodiment 1 is unified method based on the pressurization cell culture system of micro-fluidic chip
As shown in Figure 1, wherein should comprise syringe pump, micro-fluidic chip and pressure warning unit based on the pressurization cell culture system of micro-fluidic chip, this syringe pump and pressure warning unit are connected with micro-fluidic chip by flexible pipe respectively, and this syringe pump and pressure warning unit lay respectively at the both sides of this micro-fluidic chip. When using the pressurization cell culture system of this mode of connection, in the passage of micro-fluidic chip, cultured cells not only bears pressure but also bear flowing shearing force.
This micro-fluidic chip is made up of PDMS and glass, is straight channel chip structure, wherein only has a passage, the long 3cm of this passage, wide 2mm, high 200 ��m.
First cell is seeded in passage, grows to after about 80% degree of converging until cell attachment, carry out pressure experiment. When using this cell culture system to carry out pressure experiment, syringe pump pushing syringe, substratum flows out from syringe, flow in the passage of micro-fluidic chip through connecting hose, pressure warning unit probe is flowed to through flexible pipe, manometer measures pressure value size after this passage flows out. First using higher flow velocity, such as 30-40 �� l/min, make pressure warning unit numerical value reach the pressure of 12KPa, in the research of vascular endothelial cell, this pressure is for simulating normal arterial pressure. Re-use lower flow velocity, such as 5-10 �� l/min, maintain force value within the scope of the deviation of �� 0.5KPa. Stop syringe pump after pressurization Therapy lasted for some time, the cell in substratum and pipeline can be carried out subsequent experimental and analysis.
Embodiment 2 is unified method based on the pressurization cell culture system of micro-fluidic chip
As shown in Figure 2, wherein should comprise syringe pump, micro-fluidic chip and pressure warning unit based on the pressurization cell culture system of micro-fluidic chip, this syringe pump and pressure warning unit are connected with two in three interfaces of a T connector respectively, the one end of the passage that another interface of this T connector is connected in this micro-fluidic chip, the other end of this passage and one hose connection, the other end clip of this flexible pipe is clamped and is made it close. When using this T connector pressurization cell culture system culturing cell, the cell in the passage of micro-fluidic chip only bears pressure.
This micro-fluidic chip is made up of PDMS and glass, has many parallel channels, the every long 2cm of bar passage, wide 1mm, high 75 ��m, as shown in Figure 3.
First cell is seeded in passage, grows to after about 80% degree of converging until cell attachment, carry out pressure experiment. When using this cell culture system to carry out pressure experiment, syringe pump pushing syringe, substratum flows out from syringe, flows to pressure warning unit probe, manometer measures pressure value size. First use bigger flow velocity, such as 40-50 �� l/min, make pressure warning unit numerical value reach the pressure of 18KPa, re-use lower flow velocity, such as 5-10 �� l/min, maintain force value within the scope of the deviation of �� 0.5KPa. Stop syringe pump after pressurization Therapy lasted certain time, the cell in substratum and pipeline can be carried out subsequent experimental and analysis.
Embodiment 3 is unified method based on the pressurization cell culture system of micro-fluidic chip
As shown in Figure 1, wherein should comprise syringe pump, micro-fluidic chip and pressure warning unit based on the pressurization cell culture system of micro-fluidic chip, this syringe pump and pressure warning unit are connected with micro-fluidic chip by flexible pipe respectively, and this syringe pump and pressure warning unit lay respectively at the both sides of this micro-fluidic chip.
This micro-fluidic chip is made up of PDMS and glass, is serpentine channel chip structure, wherein only has a passage, this channel width 100 ��m, high 20 ��m, as shown in Figure 4.
First cell is seeded in passage, grows to after about 80% degree of converging until cell attachment, carry out pressure experiment. When using this cell culture system to carry out pressure experiment, syringe pump pushing syringe, substratum flows out from syringe, flow in the passage of micro-fluidic chip through connecting hose, pressure warning unit probe is flowed to through connecting hose, manometer measures pressure value size after this passage flows out. Higher flow velocity is first used to make pressure warning unit numerical value reach the pressure of 8KPa. Re-use lower flow velocity and maintain force value within the scope of the deviation of �� 0.5KPa. Stop syringe pump after pressurization Therapy lasted for some time, substratum and cell can be carried out subsequent experimental and analysis.
It can be seen that the various cells of various cultivation in micro-fluidic chip pipeline can be carried out continuing and constant pressure-controlling by the pressurization cell culture system based on micro-fluidic chip using the present invention from above embodiment.
Although, above the present invention is described in detail with a general description of the specific embodiments, but on basis of the present invention, it is possible to it being made some modifications or improvements, this will be apparent to those skilled in the art. Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (6)
1. the pressurization cell culture system based on micro-fluidic chip, comprise micro-fluidic chip, pressure actuated device, connecting tube and pressure-detecting device, containing passage in described micro-fluidic chip, described pressure actuated device comprise mate mutually with it for holding the container of substratum, described container is connected with described micro-fluidic chip by connecting tube, described pressure actuated device promotes the substratum in container to enter micro-fluidic chip, and described pressure-detecting device is connected with described micro-fluidic chip by connecting tube;
Described pressure actuated device is syringe pump or peristaltic pump, and the described container for holding substratum is syringe, and described connecting tube is flexible pipe, and described pressure-detecting device is tensimeter or pressure warning unit;
Passage length in described micro-fluidic chip is 3-6cm, and passage width is 1-3mm, is highly 50-200 ��m;
Described pressure actuated device and described pressure-detecting device lay respectively at the both sides of described micro-fluidic chip, and they are connected by the two ends of connecting tube with the passage in described micro-fluidic chip respectively; Or
Described pressure actuated device and described pressure-detecting device are positioned at the same side of micro-fluidic chip, they are connected with one end of the passage in described micro-fluidic chip by connecting tube simultaneously, the other end of described passage is closed, and described connecting tube joint is T connector.
2. the pressurization cell culture system based on micro-fluidic chip according to claim 1, it is characterized in that, described micro-fluidic chip is made up of one or more in following material: polymethylmethacrylate, polydimethylsiloxane, polyethylene terephthalate, polycarbonate, glass, epoxy resin and hydrogel.
3. a pressurization cell culture processes, it is characterised in that, based on the pressurization cell culture system of micro-fluidic chip described in application rights requirement 1 or 2.
4. method according to claim 3, it is characterised in that, comprise the following steps:
1) attached cell is cultivated in the passage of micro-fluidic chip;
2) utilize pressure actuated device to promote cell culture medium to enter micro-fluidic chip with certain flow velocity, produce pressure;
3) pressure born by pressure-detecting device monitoring cell.
5. method according to claim 4, it is characterised in that, step 2) described in flow velocity be 0.01 �� l/min-10ml/min.
6. method according to claim 5, it is characterised in that, step 2) described in flow velocity be 5-50 �� l/min.
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| CN108148749A (en) * | 2016-12-05 | 2018-06-12 | 中国科学院大连化学物理研究所 | Keep the micro-fluidic chip culture systems of culture solution dissolved oxygen concentration and acid-base value |
| CN106881160A (en) * | 2017-05-03 | 2017-06-23 | 山东大学 | A kind of candida albicans bacterium micro-fluidic chip |
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| CN109913372A (en) * | 2019-03-15 | 2019-06-21 | 清华大学 | A kind of culture systems and method of living tissue |
| CN112403539A (en) * | 2019-08-23 | 2021-02-26 | 无锡源清天木生物科技有限公司 | Micro-fluidic chip |
| CN110628621B (en) * | 2019-10-28 | 2023-12-22 | 合肥中科干细胞再生医学有限公司 | Device and method for obtaining tumor-specific T cells |
| CN112501020B (en) * | 2020-12-04 | 2022-09-06 | 陕西融光云生物科技有限公司 | Biological tissue culture system based on micro-fluidic chip and implementation operation method thereof |
| CN114134011B (en) * | 2021-11-24 | 2023-07-21 | 深圳先进技术研究院 | Device and mould for culturing monolayer bacteria |
| CN115193497A (en) * | 2022-07-19 | 2022-10-18 | 哈尔滨工业大学 | Portable micro-fluidic chip operating device with complicated electric field regulation and control function |
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