CN104560714A - Micro-fluidic chip for culturing and detecting lung cancer cells - Google Patents
Micro-fluidic chip for culturing and detecting lung cancer cells Download PDFInfo
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Abstract
The invention discloses a micro-fluidic chip for culturing and detecting lung cancer cells. The micro-fluidic chip sequentially comprises a cell culture layer, a micro-channel layer and a sealing cover layer from bottom to top, wherein the cell culture layer and the micro-channel layer are made of hydrogel materials; a cell culture chamber and a part a of a metabolite collection pool are formed in the cell culture layer; a fluid micro-channel for simulating the human lung structure and a part b of the metabolite collection pool are formed in the micro-channel layer; the sealing cover layer covers the micro-channel layer; a through hole communicated with an injection port is formed in one end of the sealing cover layer; and a detection window is formed in a position, which corresponds to the metabolite collection pool, on the sealing cover layer. According to the micro-fluidic chip disclosed by the invention, multiple groups of cell samples can be simultaneously cultured, a three-dimensional growth environment is provided for the cells, the human lung tissue structure is completely simulated, a growth environment close to a human body is provided for the cells, and the metabolite is very close to the metabolite of human cells, so that the detection data is closer to the human real conditions, and early diagnosis of lung cancer is promoted.
Description
Technical field
The present invention relates to a kind of micro-fluidic chip, being specifically related to a kind of for cultivating and the micro-fluidic chip of detection of lung cancer cell.
Background technology
Lung cancer has another name called bronchogenic carcinoma, and it betides tunica mucosa bronchiorum epithelium, and it is the fastest to be that M & M increases, to one of population health and the maximum malignant tumour of life threat.Because lung cancer early symptom is not obvious, reach an advanced stage when usually finding, therefore, early diagnosis is carried out to lung cancer and early treatment is the most effectual way of preventing and treating lung cancer and reducing case fatality rate.At present clinically, the common diagnosis method of lung cancer has several as follows: X-ray detects, CT scan (CT), magnetic resonance imaging,MRI (MRI), Sputum cytology, fiber segmental bronchus checks, Drug eluting stent etc., although this type of inspection method can reach the effect of expection pulmonary cancer diagnosis, but adopt the equipment of these methods huge, expensive, large to patient trauma, the professional of skilled operation is also needed to operate, and the result obtained also wants the medical worker of specialty to carry out analyzing and processing, greatly increase the burden of medical worker.
Micro-fluidic chip (microfluidic chip), also known as micro-total analysis system (micro total analysis system, ì-TAS) or chip lab (lab-on-a-chip), refer to sample preparation involved in the fields such as chemistry and biology, reaction, be separated, detect and cell cultures, sorting, the basic operation units such as cracking are integrated or be substantially integrated on the chip of a piece several square centimeters (even less), network is formed by microchannel, whole system is run through with controlled fluid, in order to replace a kind of technology platform of the various functions of conventional chemical or biology laboratory, it is high that it has separation efficiency, analysis speed is fast, clastotype is many, required sample is few, applied range, level of automation advantages of higher.But microfluidic chip technology is mainly also in the starting stage current, its cultivate for lung carcinoma cell and the correlation technique of detection field and method there is not been reported.
Summary of the invention
For above-mentioned deficiency of the prior art, main purpose of the present invention is to provide one can simulate human lung's weave construction, can cultivate single lung carcinoma cell, and carries out the micro-fluidic chip of monitoring in real time to its upgrowth situation.
To achieve these goals, the technical solution used in the present invention is as follows:
For cultivating and the micro-fluidic chip of detection of lung cancer cell, this micro-fluidic chip comprises cell cultures layer, microchannel layers and capping layer from the bottom to top successively, and cell cultures layer and microchannel layers are made up of hydrogel material.
Described cell cultures layer is provided with cell culture chamber and metabolite collecting tank a portion; Described cell culture chamber is the circular shrinkage hole to lower recess, and cell culture chamber is 60, and 60 cell culture chambers are 5 × 12 arrayed, and each cell culture chamber diameter is 1.5mm; Described metabolite collecting tank a portion is the rectangular groove structure of 16mm × 11mm, and side next-door neighbour's cell culture chamber in this metabolite collecting tank a portion, opposite side is near the end of chip.
Described microchannel layers is provided with flow microchannel and the metabolite collecting tank b portion of simulation human lung structure; Described flow microchannel comprises overall channel, the first subchannel, the second subchannel and microchannel, one end of overall channel is injection port, the other end is divided into two the first subchannels, each first subchannel is divided into two the second subchannels again, and the second subchannel is divided into multiple microchannel be interconnected again; Described microchannel is reticulated structure, and be provided with in Duo Tiao microchannel intersection and corresponding to the position of cell culture chamber the cell capture hole communicated with cell culture chamber, the diameter in this cell capture hole is 1.5mm; Described metabolite collecting tank b portion is engraved structure, and its shape and metabolite collecting tank a portion match and coincide with metabolite collecting tank a portion, and metabolite collecting tank a portion and metabolite collecting tank b portion form a metabolite collecting tank jointly.
Described capping layer covers in microchannel layers, the through hole communicated with injection port is provided with in one end of capping layer, detection window is provided with on capping layer and corresponding to the position of metabolism liquid collecting tank, this detection window is engraved structure, detection window is provided with the door that can cover this detection window, and this door and capping layer are slidably matched; Described door is provided with ventilating pit and pumping holes.
Further, described hydrogel material is made up of prepolymer and light trigger, wherein, prepolymer comprises PEGDA, HEMA and NVP, the volume ratio of PEGDA and HEMA is (1.5 ~ 2.33): 1, the volume of NVP is 10% ~ 15% of PEGDA and HEMA cumulative volume, and described light trigger adopts the quality of Irgacure2959, Irgacure2959 to be 0.3% ~ 0.45% of prepolymer quality.
Further, it is characterized in that, overall channel, the first subchannel, the second subchannel and microchannel are and seamlessly transit.
Further, the internal diameter of described overall channel is 2.5mm, and the maximum inner diameter of the first subchannel is 5mm, and the internal diameter of microchannel is 1mm; Overall channel injection port is 18mm to the slant range of microchannel entrance end, and microchannel entrance end is 35mm to the slant range of its exit end.
Further, in described prepolymer, the volume ratio of PEGDA and HEMA is the volume of 1.5:1, NVP is 10% of PEGDA and HEMA cumulative volume, and the quality of light trigger is 0.4% of prepolymer quality.
Further, in described prepolymer, the volume ratio of PEGDA and HEMA is the volume of 2.3:1, NVP is 15% of PEGDA and HEMA cumulative volume, and the quality of light trigger is 0.3% of prepolymer quality.
Relative to prior art, the present invention has following beneficial effect:
1, multiple cell culture chamber is set on cell cultures layer, 60 groups of cell samples can be cultivated at most simultaneously; The diameter of each cell culture chamber is set to 1.5mm, when condition of equivalent thickness, greatly can increases the volume of cell culture chamber, be conducive to cell attachment, be conducive to cell feeler, flagellum three-dimensional growth.
2, microchannel layers is provided with the flow microchannel of simulation human lung weave construction, cell suspending liquid or nutrient solution is injected by flow microchannel injection port, cell suspending liquid or nutrient solution are full of each cell culture chamber uniformly by the shunting action of flow microchannel, this flow microchannel simulates human lung's weave construction completely, for cell provides the growing environment closer to human body, its meta-bolites is in close proximity to the meta-bolites of human body cell, detection data are made more to be close to human body truth, for follow-up analytical work provides scientific basis more accurately, be beneficial to the early diagnosis to lung cancer.
3, metabolism liquid collecting tank is the rectangular groove structure of 16mm × 11mm, its cellular metabolism liquid collected is long-pending meets detection needs completely, porphyrin sensors visual array chip or other detecting instruments can be directly adopted to detect in real time the cellular metabolism liquid in metabolism liquid collecting tank, eliminate the step that the metabolism liquid in metabolism liquid collecting tank is collected, avoid because the otherness of each micro-fluidic chip cultured cells sample is on the impact detecting data.
4, the hydrogel material adopting formula of the present invention to make, there is good biocompatibility, cell adhesion forces, ready availability, low consumption, the hydrogel chip made further simulates inside of human body organizational environment, and the lung carcinoma cell metabolism fluid markers for next step detects and lays the first stone.
Accompanying drawing explanation
Fig. 1 is cell cultures Rotating fields schematic diagram;
Fig. 2 is microchannel layers structural representation;
Fig. 3 is capping layer structural representation.
Wherein, 1 is cell culture chamber, and 2 is metabolism liquid collecting tank a portion, and 3 is flow microchannel, and 4 is cell capture hole, 5 is metabolism liquid collecting tank b portion, and 6 is cell cultures layer, and 7 is microchannel layers, and 8 is capping layer, and 9 is door, 10 is detection window, and 11 is through hole, and 12 is ventilating pit, and 13 is pumping holes.
Embodiment
Below in conjunction with accompanying drawing and example, the present invention is described in more detail.
See Fig. 1 ~ Fig. 3: for cultivate and the micro-fluidic chip of detection of lung cancer cell is the three-decker that three-layer tablet shape thing is formed by stacking, it comprises cell cultures layer 6, microchannel layers 7 and capping layer 8 from the bottom to top successively.Cell cultures layer 6 and microchannel layers 7 are made up of hydrogel material.The thickness of cell cultures layer 6 is 1mm, and cell cultures layer 6 is provided with cell culture chamber 1 and metabolism liquid collecting tank a portion 2.Cell culture chamber 1 is the circular shrinkage hole to lower recess.In the present embodiment, cell cultures layer 6 is provided with 60 cell culture chambers, 1,60 cell culture chambers 1 in arranged, often arranges 12, totally 5 rows.The diameter of each cell culture chamber 1 is 1.5mm.Side next-door neighbour's cell culture chamber 1 in metabolism liquid collecting tank a portion 2, opposite side is near the end of chip.Metabolism liquid collecting tank a portion 2 is rectangular groove structure, and its length is 16mm, and width is 11mm.Microchannel layers 7 is provided with flow microchannel 3 and the metabolism liquid collecting tank b portion 5 of simulation human lung structure.Flow microchannel 3 communicates with each cell culture chamber 1 be positioned on cell cultures layer 6.In the present embodiment, flow microchannel 3 comprises overall channel, and one end of overall channel is injection port, for injecting cell suspending liquid or nutrient solution; The other end is divided into two the first subchannels, each first subchannel is divided into two the second subchannels again, second subchannel is divided into multiple microchannel be interconnected again, microchannel is reticulated structure, the cell capture hole 4 communicated with cell culture chamber is provided with in Duo Tiao microchannel intersection and corresponding to the position of cell culture chamber 1, the diameter in this cell capture hole 4 is 1.5mm, and for catching cell, cell enters cell culture chamber 1 after being caught by cell capture hole 4 and cultivates.Namely between overall channel and the first subchannel overall channel, the first subchannel, the second subchannel and microchannel are and seamlessly transit, and, be seamlessly transit between the first subchannel and the second subchannel, between the second subchannel and microchannel.The internal diameter of overall channel is 2.5mm, and the maximum inner diameter of the first subchannel is 5mm, and the internal diameter of microchannel is 1mm; Overall channel injection port is 18mm to the slant range of microchannel entrance end, and microchannel entrance end is 35mm to the slant range of its exit end.Metabolism liquid collecting tank b portion 5 is engraved structure, its shape and metabolism liquid collecting tank a portion 2 match, metabolism liquid collecting tank a portion 2 and metabolism liquid collecting tank b5 portion form a complete metabolism liquid collecting tank jointly, and metabolism liquid collecting tank is connected with flow microchannel, for collecting cell metabolism liquid.Capping layer 8 covers in microchannel layers 7, is provided with the through hole 11 communicated with injection port in one end of capping layer 8, and on capping layer 8 and corresponding to the position of metabolism liquid collecting tank, be provided with detection window 10, this detection window 10 is engraved structure.Detection window 10 is provided with the door 9 that can cover this detection window 10, and this door 9 is slidably matched with capping layer 8, opens or closes detection window 10 by push-pull fashion.Door is provided with ventilating pit 12 and pumping holes 13.
Micro-fluidic chip is set to three-decker, and bottom is the cell cultures layer being provided with multiple cell culture chamber, for cell provides three dimensional growth environment, can cultivate 60 groups of cell samples at most simultaneously; The diameter of each cell culture chamber is set to 1.5mm, when condition of equivalent thickness, greatly can increases the volume of cell culture chamber, be conducive to cell attachment, be conducive to cell feeler, flagellum three-dimensional growth.Middle layer is microchannel layers, be provided with the flow microchannel of simulation human lung weave construction, cell suspending liquid or nutrient solution is injected by flow microchannel injection port, cell suspending liquid or nutrient solution are full of each cell culture chamber uniformly by the shunting action of flow microchannel, this flow microchannel simulates human lung's weave construction completely, for cell provides the growing environment closer to human body, its meta-bolites is in close proximity to the meta-bolites of human body cell, detection data are made more to be close to human body truth, for follow-up analytical work provides scientific basis more accurately, be beneficial to the early diagnosis to lung cancer.Metabolism liquid collecting tank can collect unnecessary cell suspending liquid and cellular metabolism liquid, because this micro-fluidic chip can cultivate 60 groups of lung carcinoma cells simultaneously, often organize lung cancer cell growth environment identical, in addition metabolism liquid collection bath is long-pending larger, the cellular metabolism liquid collected by metabolism liquid collecting tank is long-pending meets detection needs completely, can directly adopt porphyrin sensors visual array chip or other detecting instruments to detect in real time the cellular metabolism liquid in metabolism liquid collecting tank.Micro-fluidic chip of the present invention achieves cell cultures-separation-metabolism-detection integration, can accomplish, to the real-time monitoring of individual cells situation, can realize the Real-Time Monitoring to many parallel sampleses.
Cell cultures layer and microchannel layers adopt hydrogel material to make, hydrogel material is made up of prepolymer and light trigger, wherein, prepolymer comprises PEGDA(polyethyleneglycol diacrylate), HEMA(hydroxyethyl methylacrylate) and NVP(N-vinyl pyrrolidone), the volume ratio of PEGDA and HEMA is the volume of (1.5 ~ 2.33): 1, NVP is 10% ~ 15% of PEGDA and HEMA cumulative volume.Light trigger adopts the quality of Irgacure2959, Irgacure2959 to be 0.3% ~ 0.45% of prepolymer quality.
PEGDA(polyethyleneglycol diacrylate) at room temperature polyethyleneglycol diacrylate hydrogel can be aggregated into rapidly under light trigger and action of ultraviolet light.HEMA(hydroxyethyl methylacrylate), effectively can improve the wetting ability of chip surface further, obviously can improve the cellular affinity of polyethyleneglycol diacrylate hydrogel material.NVP(N-vinyl pyrrolidone) there is better curing characteristics, increase the toughness of hydrogel chip.Adopt the hydrogel material made of as above filling a prescription, there is good biocompatibility, cell adhesion forces, ready availability, low consumption, the hydrogel chip made further simulates inside of human body organizational environment, and the lung carcinoma cell metabolism fluid markers for next step detects and lays the first stone.
Below specifically to fill a prescription, the formula of hydrogel material is illustrated:
Formula 1: prepolymer comprises PEGDA, HEMA and NVP, wherein, PEGDA is 1.8ml, HEMA be 1.2ml, NVP is 0.3ml, and the total mass of prepolymer is 3.5g, adds Irgacure2959, and its quality is 0.0140g, stirs.The micro-fluidic chip good biocompatibility adopting this formula hydrogel material to make, cell adhesion forces is strong, consumable material is less, holds swollen rate little, has good toughness, reusable more than 5 times.
Formula 2: prepolymer comprises PEGDA, HEMA and NVP, wherein, PEGDA is 2.1ml, HEMA be 0.9ml, NVP is 0.3ml, and the total mass of prepolymer is 3.4g, add Irgacure2959 with, its quality is 0.0135g, stirs.The micro-fluidic chip good biocompatibility adopting this formula hydrogel material to make, cell adhesion forces is strong, consumable material is less, holds swollen rate little, has good toughness, reusable more than 5 times.
Formula 3: prepolymer comprises PEGDA and HEMA, and wherein, PEGDA is 2ml, HEMA is 2ml, and the total mass of prepolymer is 3.3g, adds Irgacure2959, and its quality is 0.012g, stirs.The micro-fluidic chip biology adopting this formula hydrogel material to make is comparatively soft, holds swollen rate large, and rate of water absorption is fast, puts water 30min into and degrades, only can use once.
Formula 4: prepolymer comprises PEGDA, HEMA and NVP, wherein, PEGDA is 2.1ml, HEMA be 0.7ml, NVP is 0.6ml, and the total mass of prepolymer is 3.84g, adds Irgacure2959, and its quality is 0.016g, stirs.The micro-fluidic chip hardness adopting this formula hydrogel material to make is higher, and poor biocompatibility cannot use.
Can be found out by above-mentioned formula, the micro-fluidic chip best performance be made up of the hydrogel material of formula 1 and formula 2, more can simulated human tissue environment, be more conducive to the cultivation of cell.
What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (6)
1., for cultivating and the micro-fluidic chip of detection of lung cancer cell, it is characterized in that, this micro-fluidic chip comprises cell cultures layer, microchannel layers and capping layer from the bottom to top successively, and cell cultures layer and microchannel layers are made up of hydrogel material;
Described cell cultures layer is provided with cell culture chamber and metabolite collecting tank a portion; Described cell culture chamber is the circular shrinkage hole to lower recess, and cell culture chamber is 60, and 60 cell culture chambers are 5 × 12 arrayed, and each cell culture chamber diameter is 1.5mm; Described metabolite collecting tank a portion is the rectangular groove structure of 16mm × 11mm, side next-door neighbour's cell culture chamber in this metabolite collecting tank a portion, and opposite side is near the end of chip;
Described microchannel layers is provided with flow microchannel and the metabolite collecting tank b portion of simulation human lung structure; Described flow microchannel comprises overall channel, the first subchannel, the second subchannel and microchannel, one end of overall channel is injection port, the other end is divided into two the first subchannels, each first subchannel is divided into two the second subchannels again, and the second subchannel is divided into multiple microchannel be interconnected again; Described microchannel is reticulated structure, and be provided with in Duo Tiao microchannel intersection and corresponding to the position of cell culture chamber the cell capture hole communicated with cell culture chamber, the diameter in this cell capture hole is 1.5mm; Described metabolite collecting tank b portion is engraved structure, and its shape and metabolite collecting tank a portion match and coincide with metabolite collecting tank a portion, and metabolite collecting tank a portion and metabolite collecting tank b portion form a metabolite collecting tank jointly;
Described capping layer covers in microchannel layers, the through hole communicated with injection port is provided with in one end of capping layer, detection window is provided with on capping layer and corresponding to the position of metabolism liquid collecting tank, this detection window is engraved structure, detection window is provided with the door that can cover this detection window, and this door and capping layer are slidably matched; Described door is provided with ventilating pit and pumping holes.
2. according to claim 1 for cultivating and the micro-fluidic chip of detection of lung cancer cell, it is characterized in that, described hydrogel material is made up of prepolymer and light trigger, wherein, prepolymer comprises PEGDA, HEMA and NVP, and the volume ratio of PEGDA and HEMA is the volume of (1.5 ~ 2.33): 1, NVP is 10% ~ 15% of PEGDA and HEMA cumulative volume, described light trigger adopts the quality of Irgacure2959, Irgacure2959 to be 0.3% ~ 0.45% of prepolymer quality.
3. according to claim 1 and 2 for cultivating and the micro-fluidic chip of detection of lung cancer cell, it is characterized in that, overall channel, the first subchannel, the second subchannel and microchannel are and seamlessly transit.
4. according to claim 3 for cultivating and the micro-fluidic chip of detection of lung cancer cell, it is characterized in that, the internal diameter of described overall channel is 2.5mm, and the maximum inner diameter of the first subchannel is 5mm, and the internal diameter of microchannel is 1mm; Overall channel injection port is 18mm to the slant range of microchannel entrance end, and microchannel entrance end is 35mm to the slant range of its exit end.
5. according to claim 2 for cultivating and the micro-fluidic chip of detection of lung cancer cell, it is characterized in that, in described prepolymer, the volume ratio of PEGDA and HEMA is 1.5:1, the volume of NVP is 10% of PEGDA and HEMA cumulative volume, and the quality of light trigger is 0.4% of prepolymer quality.
6. according to claim 2 for cultivating and the micro-fluidic chip of detection of lung cancer cell, it is characterized in that, in described prepolymer, the volume ratio of PEGDA and HEMA is 2.3:1, the volume of NVP is 15% of PEGDA and HEMA cumulative volume, and the quality of light trigger is 0.3% of prepolymer quality.
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Cited By (3)
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| CN106811414A (en) * | 2015-12-01 | 2017-06-09 | 中国科学院大连化学物理研究所 | One kind visualization Transwell chips and preparation method thereof |
| CN112063510A (en) * | 2020-07-29 | 2020-12-11 | 北京大学 | Structure of high-flux cell culture chip and manufacturing and using method thereof |
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Cited By (4)
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| CN106811414A (en) * | 2015-12-01 | 2017-06-09 | 中国科学院大连化学物理研究所 | One kind visualization Transwell chips and preparation method thereof |
| CN105907641A (en) * | 2016-05-19 | 2016-08-31 | 大连医科大学附属第医院 | Assembly type multi-condition parallel-culture microfluidic control device and using method thereof |
| CN105907641B (en) * | 2016-05-19 | 2018-06-29 | 大连医科大学附属第一医院 | A kind of packaging, many condition parallel culture micro fluidic device and its application method |
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