CN104099247A - Micro-fluidic chip-based pressurized cell culturing system and method thereof - Google Patents
Micro-fluidic chip-based pressurized cell culturing system and method thereof Download PDFInfo
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Abstract
The invention provides a micro-fluidic chip-based pressurized cell culturing system and a method thereof. The system comprises a micro-fluidic chip, a pressure driving device, connecting pipelines and a pressure detecting device, the micro-fluidic chip contains a channel, the pressure driving device comprises a container used for containing a medium and matched with the pressure driving device, the container is connected with the micro-fluidic chip through one of the connecting pipelines, the pressure driving device pushes the medium in the container to enter the micro-fluidic chip, and the pressure detecting device is connected with the micro-fluidic chip through one of the connecting pipelines. The system can conveniently culture cells under the action of shearing force and a pressure and realizes relevant researches, and the pressurized cell culturing system is convenient to dismount and carry.
Description
Technical field
The present invention relates to cytobiology and cyto-mechanics technical field, specifically, relate to a kind of cell pressurization culture systems and method based on micro-fluidic chip.
Background technology
Nineteen ninety, Switzerland scientist Manz and Widmer have proposed the concept of micro-total analysis system (Micro total analysis system), and its target is that the function in whole analytical test laboratory is integrated in a portable equipment or even a small chip.Therefore, micro-total analysis system is also referred to as " laboratory on chip (Lab-on-a-chip, i.e. LOC) ".And micro-fluidic chip (Microfluidic chip) is the important component part of LOC, its principal character is that the resulting structure (comprising passage, reactor and other functional units) of containing fluid is micron order size at least one dimension.Micro-fluidic chip be take micro electronmechanical processing technology as basis, on the materials such as silicon chip, glass or PDMS, manufacture passage integrated driving and verifying attachment, by to the control of fluid in passage sample preparation, separation and detect, the basic operation units such as biology and chemical reaction are integrated on the chip of more than 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, culture dish is placed in pressure kettle equal pressure container, then pressurized vessel is put in cell culture incubator.Patent CN1858203A discloses a kind of pneumatically controlled pressurizing cell cultivation instrument, comprises with the container of opening and the container cover matching with it, is provided with inlet mouth, air outlet, tensimeter and safety valve in described container or container cover.This method operability is to be improved, and pressure is wayward.
Current micro-fluidic chip and the system that comprises micro-fluidic chip have been widely used in a plurality of technical fields, especially cytobiology technology field, at correlative technology fields such as simulation hypertension, need in cell cultivation process, apply certain pressure to approach body 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, a kind of pressurization cell culture system based on micro-fluidic chip method of unifying is provided.
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, in described micro-fluidic chip, contain passage, described pressure actuated device comprise match with it for holding the container of substratum, described container is connected with described micro-fluidic chip by connecting tube, the substratum that described Power Drive Unit promotes in container enters 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 is suitable with cell size rank, and this passage more approaches the physiological status in body in conjunction with perfusion training method; Use this micro-fluidic chip can control easily cell micro-environment, and this chip form the environment of relative closure, the interference of minimizing external environment to 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 pipes such as silicone tube, size with for holding the outlet of the container of substratum, the entrance of micro-fluidic chip matches; 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 μ m.Passage can be various forms, comprises straight channel, bending channel, single channel, many parallel channels etc.Preferably, described passage length is 3-6cm, and passage width is 1-3mm, is highly 50-200 μ 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 with the two ends of passage in described micro-fluidic chip by connecting tube 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 described pressure warning unit the both sides that micro-fluidic chip is relative, they are connected with the two ends of passage in this micro-fluidic chip by flexible pipe, 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, after flowing out, continues in this passage to flow to pressure warning unit direction, the pattern that in this kind of pattern simulation passage, cultured cells had not only been born pressure but also born Osima jacoti, Osima excavata.
Wherein, described pressure actuated device and described pressure-detecting device are positioned at the homonymy of micro-fluidic chip, and they are connected with one end of 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, be connected with one end of 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, one or more in following material of micro-fluidic chip are made: polymethylmethacrylate (Polymethylmethacrylate, PMMA), polydimethylsiloxane (Polydimethylsiloxane, PDMS), polyethylene terephthalate (Polyethylene terephthalate, PET), polycarbonate (Polycarbonate, PC), glass, epoxy resin and hydrogel.
In the preferred embodiment of the invention, described micro-fluidic chip is made by polydimethylsiloxane (PDMS) and glass, and PDMS is a kind of the macromolecule organic silicon compound, there is optical lens, and be generally inertia, and nontoxic, it is widely used in micro-fluidic chip field.
Another aspect of the present invention, provides a kind of pressurize method of cell cultures of the above-mentioned pressurization cell culture system based on micro-fluidic chip of applying.
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 with certain flow velocity and enter micro-fluidic chip, produce pressure;
3) by pressure-detecting device, monitor the pressure that cell bears.
Wherein, described flow velocity is for being 0.01 μ l/min-10ml/min, preferred, 5-50 μ 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 more approach the physiological status in body, simulate more really the growth conditions of cell under physiological status; 3, can control easily cell micro-environment; 4, the multidimensional network structure of chip forms the environment of relative closure, reduces the stimulation of external environment to cell; 5, can in real time the cell in chip channel be observed, be detected; 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, and scale is integrated.8, system bulk is little, easily builds, easy to assemble, flexibly, portable.9, cut-in pressure proofing unit in system, can Real-Time Monitoring microchannel in pressure.
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, digital display pressure warning unit; 6, clip; 7, the passage in micro-fluidic chip
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is described in further detail.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
The pressurization cell culture system of embodiment 1 based on the micro-fluidic chip method of unifying
As shown in Figure 1, wherein should comprise syringe pump, micro-fluidic chip and pressure warning unit by the pressurization cell culture system based on micro-fluidic chip, this syringe pump is connected with micro-fluidic chip by flexible pipe respectively with pressure warning unit, and this syringe pump and pressure warning unit lay respectively at the both sides of this micro-fluidic chip.While using the pressurization cell culture system of this mode of connection, in the passage of micro-fluidic chip, cultured cells is not only born pressure but also bear Osima jacoti, Osima excavata.
This micro-fluidic chip is made by PDMS and glass, is straight channel chip structure, wherein only has a passage, the long 3cm of this passage, wide 2mm, high 200 μ m.
First cell is seeded in passage, after cell attachment grows to approximately 80% degree of converging, carries out pressure experiment.When using this cell culture system to carry out pressure experiment, syringe pump pushing syringe, substratum flows out from syringe, through connecting hose, flow in the passage of micro-fluidic chip, through this passage, flow out by flexible pipe and flow to pressure warning unit probe, manometry force value size.First use higher flow velocity, as 30-40 μ l/min, make pressure warning unit numerical value reach the pressure of 12KPa, in the research of vascular endothelial cell, this pressure is used for simulating normal arterial pressure.Re-use lower flow velocity, as 5-10 μ l/min, maintain force value in the deviation range of ± 0.5KPa.Pressurization Therapy lasted stops syringe pump after for some time, can carry out subsequent experimental and analysis to the cell in substratum and pipeline.
The pressurization cell culture system of embodiment 2 based on the micro-fluidic chip method of unifying
As shown in Figure 2, wherein should comprise syringe pump, micro-fluidic chip and pressure warning unit by the pressurization cell culture system based on micro-fluidic chip, this syringe pump and pressure warning unit are connected with in three interfaces of a T connector two respectively, another interface of this T connector is connected to one end of the passage in this micro-fluidic chip, the other end of this passage and one hose connection, the other end of this flexible pipe is clamped and is made its sealing with clip.While 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 by PDMS and glass, has many parallel channels, every long 2cm of passage, and wide 1mm, high 75 μ m, as shown in Figure 3.
First cell is seeded in passage, after cell attachment grows to approximately 80% degree of converging, carries 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, manometry force value size.First use larger flow velocity, as 40-50 μ l/min, make pressure warning unit numerical value reach the pressure of 18KPa, re-use lower flow velocity, as 5-10 μ l/min, maintain force value in the deviation range of ± 0.5KPa.After pressurization Therapy lasted certain hour, stop syringe pump, can carry out subsequent experimental and analysis to the cell in substratum and pipeline.
The pressurization cell culture system of embodiment 3 based on the micro-fluidic chip method of unifying
As shown in Figure 1, wherein should comprise syringe pump, micro-fluidic chip and pressure warning unit by the pressurization cell culture system based on micro-fluidic chip, this syringe pump is connected with micro-fluidic chip by flexible pipe respectively with pressure warning unit, 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 by PDMS and glass, is serpentine channel chip structure, wherein only has a passage, this channel width 100 μ m, and high 20 μ m, as shown in Figure 4.
First cell is seeded in passage, after cell attachment grows to approximately 80% degree of converging, carries out pressure experiment.When using this cell culture system to carry out pressure experiment, syringe pump pushing syringe, substratum flows out from syringe, through connecting hose, flow in the passage of micro-fluidic chip, through this passage, flow out by connecting hose and flow to pressure warning unit probe, manometry force value size.First use higher flow velocity to make pressure warning unit numerical value reach the pressure of 8KPa.Re-use lower flow velocity and maintain force value in the deviation range of ± 0.5KPa.Pressurization Therapy lasted stops syringe pump after for some time, can carry out subsequent experimental and analysis to substratum and cell.
From above embodiment, can find out, use the pressurization cell culture system based on micro-fluidic chip of the present invention to continue and constant pressure-controlling by various cells in micro-fluidic chip pipeline various cultivations.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. the pressurization cell culture system based on micro-fluidic chip, comprise micro-fluidic chip, pressure actuated device, connecting tube and pressure-detecting device, in described micro-fluidic chip, contain passage, described pressure actuated device comprise match with it for holding the container of substratum, described container is connected with described micro-fluidic chip by connecting tube, the substratum that described Power Drive Unit promotes in container enters micro-fluidic chip, and described pressure-detecting device is connected with described micro-fluidic chip by connecting tube.
2. the pressurization cell culture system based on micro-fluidic chip according to claim 1, it is characterized in that, described pressure actuated device is syringe pump or peristaltic pump, described is syringe for holding the container of substratum, described connecting tube is flexible pipe, and described pressure-detecting device is tensimeter or pressure warning unit.
3. the pressurization cell culture system based on micro-fluidic chip according to claim 1, is characterized in that, the channel height in described micro-fluidic chip is 20-400 μ m.Preferably, described passage length is 3-6cm, and passage width is 1-3mm, is highly 50-200 μ m.
4. the pressurization cell culture system based on micro-fluidic chip according to claim 1, it is characterized in that, one or more in following material of described micro-fluidic chip are made: polymethylmethacrylate, polydimethylsiloxane, polyethylene terephthalate, polycarbonate, glass, epoxy resin and hydrogel.
5. according to the pressurization cell culture system based on micro-fluidic chip described in claim 1-4 any one, it is characterized in that, described pressure actuated device and described pressure-detecting device lay respectively at the both sides of described micro-fluidic chip, and they are connected with the two ends of passage in described micro-fluidic chip by connecting tube respectively.
6. according to the pressurization cell culture system based on micro-fluidic chip described in claim 1-4 any one, it is characterized in that, described pressure actuated device and described pressure-detecting device are positioned at the homonymy of micro-fluidic chip, they are connected with one end of passage in described micro-fluidic chip by connecting tube simultaneously, the other end of described passage is closed, preferably, described connecting tube joint is T connector.
7. a pressurization cell culture processes, is characterized in that, application rights requires the pressurization cell culture system based on micro-fluidic chip described in 1-6 any one.
8. according to the method for claim 7, it is characterized 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 with certain flow velocity and enter micro-fluidic chip, produce pressure;
3) by pressure-detecting device, monitor the pressure that cell bears.
9. method according to Claim 8, is characterized in that step 2) described in flow velocity for being 0.01 μ l/min-10ml/min, preferred, 5-50 μ l/min.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106881160A (en) * | 2017-05-03 | 2017-06-23 | 山东大学 | A kind of candida albicans bacterium micro-fluidic chip |
| CN107134208A (en) * | 2017-07-14 | 2017-09-05 | 安疗生命科学（武汉）有限公司 | A kind of external intervention embolization simulation system |
| 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 |
| CN109425733A (en) * | 2017-08-25 | 2019-03-05 | 国家纳米科学中心 | Flexible biological probe, its application method and application apparatus |
| CN109913372A (en) * | 2019-03-15 | 2019-06-21 | 清华大学 | A kind of culture systems and method of living tissue |
| CN110628621A (en) * | 2019-10-28 | 2019-12-31 | 合肥中科干细胞再生医学有限公司 | Equipment and method for obtaining tumor specific T cells |
| 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 |
| CN112501020A (en) * | 2020-12-04 | 2021-03-16 | 西北大学 | Biological tissue culture system based on micro-fluidic chip and implementation operation method thereof |
| CN114134011A (en) * | 2021-11-24 | 2022-03-04 | 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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| CN109913372A (en) * | 2019-03-15 | 2019-06-21 | 清华大学 | A kind of culture systems and method of living tissue |
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| CN110628621A (en) * | 2019-10-28 | 2019-12-31 | 合肥中科干细胞再生医学有限公司 | Equipment and method for obtaining tumor specific T cells |
| 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 |
| CN112501020A (en) * | 2020-12-04 | 2021-03-16 | 西北大学 | Biological tissue culture system based on micro-fluidic chip and implementation operation method thereof |
| CN114134011A (en) * | 2021-11-24 | 2022-03-04 | 深圳先进技术研究院 | Device and mould for culturing monolayer bacteria |
| 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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