CN109825437A

CN109825437A - A kind of micro-fluidic chip and cultural method for cell culture

Info

Publication number: CN109825437A
Application number: CN201910041695.0A
Authority: CN
Inventors: 楊士模; 尹棣; 杜冠儒; 殷磊
Original assignee: 100 Macau (tianjin) Biotechnology Co Ltd
Current assignee: 100 Macau (tianjin) Biotechnology Co Ltd
Priority date: 2018-02-09
Filing date: 2019-01-16
Publication date: 2019-05-31
Also published as: CN109868256A

Abstract

" a kind of micro-fluidic chip for cell culture " of the invention, belongs to microfluidic art.The micro-fluidic chip includes chip layer；The upper surface of the chip layer is provided with cell culture chamber, entrance pool, access road, outlet bath and exit passageway；The cell culture chamber is the biggish sunk area that chip layer upper surface is arranged in；The access road and exit passageway are the groove that chip layer upper surface is arranged in；The entrance pool and outlet bath are the lesser sunk area that chip layer upper surface is arranged in；The entrance pool and inlet passage, and be connected through the access road with the cell culture chamber；The cell culture chamber is connected through the exit passageway with the outlet bath；The entrance pool with fresh culture conveyance conduit for connecting；The outlet bath is used to connect with cell culture metabolism waste liquid discharge line.Carrying out cell culture using the chip of this law has many advantages, such as that amount of samples is few, culture efficiency is high, easy to operate, cell survival rate is high.

Description

A kind of micro-fluidic chip and cultural method for cell culture

Technical field

The invention belongs to microfluidic arts, and in particular to a kind of for the micro-fluidic chip of cell culture and culture side Method and a kind of cell culture apparatus.

Background technique

It survives to preferably imitate cell in human body ground microenvironment, only builds the system of various kinds of cell co-incubation The partial function of human organ could be recreated in vitro.Therefore a kind of new technology is urgently needed to carry out several cells Co-incubation.It is the important means for studying cell-cell interaction that cell, which co-cultures, co-cultures research for cell at present 1) mode is mainly include the following types: be mixed；2) conditioned medium culture；3) microcarrier cultivation；4) micropore counterdie covers ware Cultivation.There is the problems such as reagent consumption is big, flow control is inaccurate in all of above method, and be adapted only to research cell and exist Biological behaviour on two-dimensional surface is difficult the intracorporal complicated microenvironment of mimic biology, seriously affects experimental result.

For traditional cell culture processes such as by cell in culture dish or culture bottle, this method can not simulate cell reality Liquid flow environments under the situation of border, and need to be replaced frequently culture solution wasting manpower and material resources；And conventional micro-current controlled cell Cultural method needs using large scale equipments such as peristaltic pump, incubators, connects and generally requires to grow very much between chip and these equipment Pipeline, this very influences the progress of cell culture experiments.This patent proposes a kind of integrated cell culture apparatus, Ke Yiyou Effect ground replaces conventional cell training method and conventional micro-current controlled cell cultural method.

Therefore, this field needs to develop a kind of biological vivo environment of energy altitude simulation reduction, and reagent consumption is small, energy is quasi- The micro-fluidic chip and culture apparatus of the brand new of true coutroi velocity are used for co-incubation cell.

Summary of the invention

The present invention is provided a kind of based on the thin of micro-fluidic chip to solve above-mentioned deficiency present in the prior art Born of the same parents' co-incubation method.

Technical scheme is as follows:

A kind of micro-fluidic chip for cell culture, which is characterized in that including chip layer；

The upper surface of the chip layer is provided with cell culture chamber, entrance pool, access road, outlet bath and exit passageway；

The cell culture chamber is the biggish sunk area that chip layer upper surface is arranged in；

The access road and exit passageway are the groove that chip layer upper surface is arranged in；

The entrance pool and outlet bath are the lesser sunk area that chip layer upper surface is arranged in；

The entrance pool and inlet passage, and be connected through the access road with the cell culture chamber；

The cell culture chamber is connected through the exit passageway with the outlet bath；

The entrance pool with fresh culture conveyance conduit for connecting；

The outlet bath is used to connect with cell culture metabolism waste liquid discharge line.

The chip layer includes chip upper layer and chip lower layer；The upper surface on the chip upper layer tips upside down on chip facing downward In lower layer；The entrance pool and outlet bath on chip upper layer are engraved structure；The chip upper layer is additionally provided with and chip lower layer Entrance pool, the corresponding through-hole of outlet bath position size.

A kind of micro-fluidic chip for cell co-culture, including the micro-fluidic chip, which is characterized in that also wrap Porous layer is included, for separating the chip layer；The porous layer is between the chip upper layer and chip lower layer.

The chip layer further includes chip middle layer；The chip middle layer is between the chip upper layer and chip lower layer； Cell culture chamber, access road, exit passageway, entrance pool and the outlet bath in the chip middle layer are engraved structure；The core Porous layer is equipped between piece lower layer and chip middle layer；The chip middle layer offers and the entrance pool of chip lower layer, outlet bath position Set the corresponding through-hole of size.

The chip upper layer offers through-hole corresponding with the entrance pool in chip middle layer, outlet bath and lead to the hole site size.

Multiple chip middle layers are equipped between layer and chip lower layer on the chip；Wherein near chip lower layer Chip middle layer offer through-hole corresponding with the entrance pool of chip lower layer, outlet bath position size；Remaining is in this layer of chip Each layer of chip layer on layer offers entrance pool, outlet bath and the lead to the hole site size of lower layer chip layer adjacent thereto Corresponding through-hole；The entrance pool on chip upper layer and all through-holes corresponding with the entrance pool of other chip layers are all used for and culture Base conveyance conduit is connected；The outlet bath on chip upper layer and all through-holes corresponding with the outlet bath of other chip layers be all used for Cell culture is metabolized the connection of waste liquid discharge line.

The chip layer between any two all between be separated with porous layer；The porous layer is the structure with several micro-through-holes Layer, the through-hole can pass through cell culture medium but be not through cell.

The porous layer is porous polycarbonate film layer, biomembrane, polytetrafluoroethylene film or nitrocellulose filter etc..

Each chip layer, or, each chip layer and porous layer are superimposed from bottom to up forms the micro-fluidic chip；Each chip layer is big Small, shape is adapted；The size of cell culture chamber in each chip layer, shape, position are corresponding, but the entrance in each chip layer The axial position of pond, outlet bath on the micro-fluidic chip is not overlapped.

Each chip layer between any two or each chip layer and porous layer between any two, edge is sealing, to ensure culture medium It is not leaked out from the edge of chip.

The mode of the sealing is selected from one or more of following:

Each chip layer between any two or each chip layer and porous layer between any two, edge passes through hot press hot sealing； And/or each chip layer between any two or each chip layer and porous layer between any two, edge is sealed by viscose glue or tape-stripping； And/or each chip layer between any two or each chip layer and porous layer between any two, edge is fixed by clamp, uses screw Fit sealing.

The chip upper layer, the access road of chip lower layer are equal with the width of exit passageway；The chip middle layer enters The width of mouth channel and exit passageway is 900 μm -1100 μm, preferably 1000 μm；The chip upper layer, chip lower layer cell The width and the ratio between access road and the width of exit passageway of culturing room are as follows: 55:4~9:1.

The width and the ratio between access road and the width of exit passageway of the cell culture chamber in the chip middle layer are as follows: 9: 1~ 11:1。

The chip upper layer, the cell culture chamber of chip lower layer, access road, exit passageway cup depth be 80~ 120μm；The chip upper layer, the access road of chip lower layer, exit passageway width be 800~1000 μm, preferably 833 μ m.The cell culture chamber in the chip middle layer, access road, exit passageway cup depth be 180~220 μm.

The cell culture chamber of the chip layer, entrance pool, the chamfered shape of outlet bath are selected from round, oval, rectangular, water chestnut Shape, or, rule or irregular polygon；The preferred dimethyl silicone polymer of the material of the chip layer, acrylic board, sheet glass or Person's silicon wafer etc.；The width of the cell culture chamber is 9~11mm, preferably 10mm.

A kind of method of cell culture, which is characterized in that use the micro-fluidic chip, the thin of certain cell will be contained Born of the same parents' suspension injects the entrance pool on the chip upper layer.

The culture medium grown for cell is injected by the entrance pool.

A kind of cell co-culture method, which is characterized in that the micro-fluidic chip is used, it will be thin containing a kind of cell Born of the same parents' suspension is added in the entrance pool on chip upper layer；It will be in the cell suspension injection chip lower layer containing another cell or chip In the entrance pool of layer.

By the entrance pool on chip upper layer, and/or, by the entrance for corresponding to chip middle layer or chip lower layer on chip upper layer The through-hole in pond periodically injects the culture medium grown for cell.

On the other hand, the present invention provides a kind of kidney organ's chip, including at least 2 layers of chip layer and for by each chip layer point The porous layer separated；The porous layer is between each chip layer；

The upper surface of the chip layer is provided with cell culture chamber, entrance pool, access road, outlet bath and exit passageway； The cell culture chamber is the biggish sunk area that chip layer upper surface is arranged in, for providing three-dimensional training for the cell Support space；The access road and exit passageway are the groove that chip layer upper surface is arranged in；The entrance pool and outlet bath For the lesser sunk area that chip layer upper surface is arranged in；The entrance pool and inlet passage, and it is logical through the entrance Road is connected with the cell culture chamber；The cell culture chamber is connected through the exit passageway with the outlet bath；It is described Entrance pool with fresh culture conveyance conduit for connecting；The outlet bath is used to connect with cell culture metabolism waste liquid discharge line It connects.

The micro-fluidic chip includes 2 layers of chip layer；Chip layer positioned at the micro-fluidic chip top layer is on chip Layer；It is chip lower layer positioned at the undermost chip layer of the micro-fluidic chip；The porous layer is located under chip upper layer and chip Between layer；The upper surface on the chip upper layer is tipped upside down on facing downward in chip lower layer；The entrance pool and outlet bath on chip upper layer are equal For engraved structure；The chip upper layer is additionally provided with through-hole corresponding with the entrance pool of chip lower layer, outlet bath position size.

Kidney organ's chip includes 3 layers of chip layer；It is in chip between the chip upper layer and chip lower layer Layer；Cell culture chamber, access road, exit passageway, entrance pool and the outlet bath in the chip middle layer are engraved structure；It is described Porous layer is equipped between chip lower layer and chip middle layer；The chip middle layer offers and the entrance pool of chip lower layer, outlet bath The corresponding through-hole of position size.

Kidney organ's chip includes 3 layers or more of chip layer；It is equipped between layer and chip lower layer on the chip more A chip middle layer；Wherein offered and the entrance pool of chip lower layer, outlet bath near the chip middle layer of chip lower layer The corresponding through-hole of position size；Remaining each layer of chip layer on this layer of chip middle layer offers lower layer adjacent thereto Entrance pool, outlet bath and the corresponding through-hole of lead to the hole site size of chip layer；The entrance pool on chip upper layer and it is all with it is other The corresponding through-hole of the entrance pool of chip layer is all used to be connected with culture medium conveyance conduit；The outlet bath on chip upper layer and it is all with The corresponding through-hole of the outlet bath of other chip layers is all used to be metabolized waste liquid discharge line with cell culture and connect.

The chip layer between any two all between be separated with porous layer；The porous layer is the structure with several micro-through-holes Layer, the micro-through-hole can pass through cell culture medium but be not through cell；Preferably, the porous layer is porous polycarbonate film Layer, biomembrane, polytetrafluoroethylene film or nitrocellulose filter etc..

Each chip layer between any two or each chip layer and porous layer between any two, edge is sealing, to ensure culture medium It is not leaked out from the edge of chip；Preferably, the mode of the sealing is selected from one or more of following:

The chip upper layer, the access road of chip lower layer are equal with the width of exit passageway；The chip middle layer enters The width of mouth channel and exit passageway is 900 μm -1100 μm, preferably 1000 μm；The chip upper layer, chip lower layer cell The width and the ratio between access road and the width of exit passageway of culturing room are as follows: 55:4~9:1；Preferably, the chip middle layer The width and the ratio between access road and the width of exit passageway of cell culture chamber are as follows: 9: 1~11:1.

It is highly preferred that the recess depth on the chip upper layer, the cell culture chamber of chip lower layer, access road, exit passageway Degree is 80~120 μm；The chip upper layer, the access road of chip lower layer, exit passageway width be 800~1000 μm, it is excellent It is selected as 833 μm；Preferably, the cup depth of the cell culture chamber, access road, exit passageway in the chip middle layer be 180~ 220μm。

Preferably, the cell culture chamber of the chip layer, entrance pool, the chamfered shape of outlet bath be selected from round, ellipse, Rectangular, diamond shape, or, rule or irregular polygon；The preferred dimethyl silicone polymer of the material of the chip layer, acrylic board, Sheet glass or silicon wafer etc.；The width of the cell culture chamber is 9~11mm, preferably 10mm.

A kind of kidney organ's drug test model, which is characterized in that culture has kidney organ's chip of kidney organ's cell； Preferably, the culture includes that the cell suspension containing kidney organ's cell is injected the chip upper layer of kidney organ's chip Entrance pool；It is highly preferred that the culture further include: inject the culture medium grown for cell by the entrance pool.

Kidney organ's cell includes: human colon carcinoma cell line and pipe week capillary endothelial cell；Preferably, Culture has human colon carcinoma cell line, kidney organ's chip in the cell culture room of the top layer of kidney organ's chip Undermost cell culture room in culture have pipe week capillary endothelial cell；It is highly preferred that kidney organ's drug test Refer to: applying results of regular determination cell viability after drug on the kidney organ's cell cultivated in kidney organ's chip；Further preferably Ground, the drug is selected from CCK-8, or, DDP；The results of regular determination cell viability refers in the 1st day, the 4th day, the 7th for applying drug The OD of its measurement cell₄₅₀Value.

The first aspect of the invention provides a kind of micro-fluidic chip for cell culture.The micro-fluidic chip includes Chip layer；The upper surface of the chip layer is provided with cell culture chamber, entrance pool, access road, outlet bath and exit passageway；Institute Stating cell culture chamber is the biggish sunk area that chip layer upper surface is arranged in；The access road is to set with exit passageway Set the groove in chip layer upper surface；The entrance pool and outlet bath are the lesser depressed area that chip layer upper surface is arranged in Domain；The entrance pool and inlet passage, and be connected through the access road with the cell culture chamber；The cell training Room is supported to be connected through the exit passageway with the outlet bath；The entrance pool is used to connect with fresh culture conveyance conduit, The outlet bath is used to connect with cell culture metabolism waste liquid discharge line.

Cell culture chamber provides main attachment space for cell adherent growth；Access road is for being connected to cell culture Room and entrance pool；Exit passageway is for being connected to cell culture chamber and outlet bath；The setting of outlet bath can make in cell cultivation process The metabolism waste liquid of generation is discharged from chip in time；The setting of entrance pool can allow fresh culture by entrance pool in a steady stream not It is injected into the chip disconnectedly for cell grown cultures；In some specific operation schemes, entrance pool can be by described Pipeline is connect with syringe pump, for injecting fresh medium into chip at regular time and quantity, simulates the cell growth ring in human body Border.

Using the present invention there is the micro-fluidic chip of above structure to carry out cell culture it has been confirmed by experiments that, can be cell In vitro culture provides a stable environment, and has the characteristics that easy to operate, quick and amount of samples is few, turns out simultaneously Cell survival rate it is high, 90% or more can be stably reached.

Further, the chip layer includes chip upper layer and chip lower layer；The upper surface on the chip upper layer is facing downward It tips upside down in chip lower layer；The entrance pool and outlet bath on chip upper layer are engraved structure；The chip upper layer be additionally provided with The entrance pool of chip lower layer, the corresponding through-hole of outlet bath position size.Chip upper layer, which tips upside down on, forms 1 closed three in lower layer Confining space is grown for cell, while the entrance pool on chip upper layer and outlet bath are made into engraved structure, is either buckled to still just To placement, culture medium can be injected into chip by entrance pool, it can also be by the metabolism waste liquid of cell culture from outlet It is discharged in pond.

The second aspect of the invention provides a kind of micro-fluidic chip for cell co-culture comprising described above Micro-fluidic chip and porous layer, the porous layer is for separating the chip layer；The porous layer is located on the chip Between layer and chip lower layer.Porous layer mainly cultivates cell, can be coated with collagen on it, promote the attachment of cell, On micropore size cell cannot penetrate, it is ventable；The setting of porous layer can make chip interior be partitioned into 2 or 2 or more Independent three-dimensional space is for cultivating different types of cell, and then height restores, simulates the physiological environment of inside of human body, especially It is usually worked together by two or more kinds of cells for certain human organs, is acted synergistically to maintain organ Normal operation, therefore chip of the invention can be used to simulate the organ working condition in human body, and can simulate as needed Organ and its cell type quantity, the mesh of the various types of cells co-incubation of different number is realized by adjusting the chip number of plies 's.

In a preferred embodiment, the chip layer further includes chip middle layer；The chip middle layer is on the chip Between layer and chip lower layer；The cell culture chamber in the chip middle layer, access road, exit passageway, entrance pool and outlet bath are equal For engraved structure；Porous layer is equipped between the chip lower layer and chip middle layer；The chip middle layer offers and chip lower layer Entrance pool, the corresponding through-hole of outlet bath position size.The chip upper layer offers and the entrance pool in chip middle layer, outlet bath Through-hole corresponding with lead to the hole site size.The effect that chip middle layer is designed to engraved structure is to make chip interior more up and down Keep connection between layer, entire chip made to form an organic whole, and the setting of porous layer ensure that liquid connection and incite somebody to action Different types of cell is kept apart, be more advantageous to simulation some intracorporal specific organ of people in different type cell work at the same time, Synergistic effect, respectively independent culture, growth and mutual liquid is such a physiological environment communicated.Meanwhile chip The through-hole that upper layer opens up corresponds to entrance pool, outlet bath and the through-hole in chip middle layer, and chip middle layer also offers same correspondence In the through-hole of the entrance pool outlet bath of chip lower layer, the advantages of this arrangement are as follows being not necessarily to after chip fixation is integral by each layer Addition injection culture medium respectively or export waste liquid are opened in dismantling, need to only be can be realized by each through-hole connecting pipe on chip upper layer The culture medium injection and waste liquid export of every layer of chip, operation when making using chip culture cell of the present invention is convenient, simply.

In other embodiments, multiple chip middle layers are equipped between layer and chip lower layer on the chip；Its In near the chip middle layer of chip lower layer offer through-hole corresponding with the entrance pool of chip lower layer, outlet bath position size； Remaining each layer of chip layer on this layer of chip middle layer offers the entrance pool of lower layer chip layer adjacent thereto, outlet Pond and the corresponding through-hole of lead to the hole site size.Similarly, in the case where the chip number of plies is greater than 3 layers, nethermost chip is removed Layer, each layer of chip can all open up next layer of upper inlet pond and outlet bath and the corresponding through-hole of through-hole adjacent thereto, Until the through-hole opened up on top layer's chip be it is most, respectively correspond entrance pool in several layers chip layer below, go out Mouth pond and through-hole, the chip layer from most are just able to satisfy liquid injection, export operation to all layers of chip layer.

In some embodiments, the chip layer between any two all between be separated with porous layer；The porous layer is with several The structure sheaf of micro-through-hole, the through-hole can pass through cell culture medium but be not through cell.The main function of porous layer be for Each layer in chip is effectively separated, porous layer mainly cultivates cell, can be coated with the reagent for promoting cell attachment.Core Can also more layers be arranged according to requirements in piece.During using chip culture cell of the invention, cell mainly adheres to In being grown on porous layer, separates every layer of chip layer and independently cultivate a certain cell, but porous layer not exclusively blocks liquid again Circulation between each chip layer of entire chip interior, in more preferable simulation human body, intraorganic physiological environment and cell work State.

Specifically, the porous layer is porous polycarbonate film, biomembrane, polytetrafluoroethylene film or nitrocellulose filter Deng.The common porous layer making material in above-mentioned this field, those skilled in the art can according to actual needs, for example, experiment purpose, Experimental implementation demand, product cost etc., the porous layer making material common to above-mentioned this field carry out conventional selection.

In more specific embodiment, each chip layer, or, each chip layer and porous layer are superimposed from bottom to up forms the miniflow Control chip；Each chip layer size, shape are adapted；The size of cell culture chamber in each chip layer, shape, position are corresponding, But the axial position of entrance pool, outlet bath on the micro-fluidic chip in each chip layer is not overlapped.Further embodiments Such as, the chip layer and porous layer are made as rectangular sheet structure；Each chip layer size, shape is compatible is advantageous in that shape At being easier to form when chip entirety, fixation procedure is simpler, and the process of components is simpler, reduces cost.

In preferred embodiment, each chip layer between any two or each chip layer and porous layer between any two, edge is Sealing, to ensure that culture medium is not leaked out from the edge of chip.Sealing is formed between layers, makes to inject chip interior Culture medium will not go out through the gap leakage at edge, advantageously reduce amount of samples, improve cell culture efficiency.

In further preferred scheme, the mode of the sealing is selected from one or more of following: each chip layer two Between two or each chip layer and porous layer between any two, edge passes through hot press hot sealing；And/or each chip layer two-by-two it Between or each chip layer and porous layer between any two, edge is sealed by viscose glue or tape-stripping；And/or each chip layer two-by-two it Between or each chip layer and porous layer between any two, edge is fixed by clamp, is sealed with fastened by screw.Chip of the present invention The mode of sealing includes but is not limited to above-mentioned several sealing means, and the purpose that those skilled in the art seal for chip may be used also Making chip edge closing seam not with use makes other seal means of fluid seepage.

In certain embodiments, the chip upper layer, the access road of chip lower layer are equal with the width of exit passageway；Institute The width of the access road and exit passageway of stating chip middle layer is 900 μm -1100 μm, preferably 1000 μm；The chip upper layer, The width and the ratio between access road and the width of exit passageway of the cell culture chamber of chip lower layer are as follows: 55:4~9:1.

In further embodiments, the width of the cell culture chamber in the chip middle layer and access road and exit passageway The ratio between width are as follows: 9: 1~11:1.

In some embodiments, the chip upper layer, the cell culture chamber of chip lower layer, access road, exit passageway it is recessed Falling into depth is 80~120 μm；The chip upper layer, the access road of chip lower layer, exit passageway width be 800~1000 μ M, preferably 833 μm.

In some specific embodiments, the cell culture chamber in the chip middle layer, access road, the recess of exit passageway are deep Degree is 180~220 μm.

In more specific embodiment, the cell culture chamber of the chip layer, entrance pool, the chamfered shape of outlet bath are selected from circle Shape, ellipse, rectangular, diamond shape, or, rule or irregular polygon；The preferred dimethyl silicone polymer of the material of the chip layer, Acrylic board, sheet glass or silicon wafer etc.；The width of the cell culture chamber is 9~11mm, preferably 10mm.The present invention one Chip cell culture chamber shape approximate ellipse provided by specific embodiment, space is larger, can cultivate compared with many cells.It can also be with According to specific requirement, in addition similar culture chamber shape is set.

These above-mentioned dimensional values ranges, those skilled in the art can be according to actual cell culture purpose and experimental implementations Demand is made and being adjusted and selected accordingly.

The third aspect of the invention provides a kind of method of cell culture based on above-mentioned micro-fluidic chip, and feature exists In using the micro-fluidic chip, the cell suspension containing certain cell to be injected to the entrance pool on the chip upper layer.Using Any chip of the present invention, can be realized the culture of any type cell, inject cell by the entrance pool toward chip layer Suspension can reach the purpose of cell adherent growth in cell culture room within the regular hour.Meanwhile the present invention also by Repetition test confirms that, using chip progress cell culture of the invention, cell survival rate may be up to 90% or more.

In cell culture processes further embodiment, the culture medium grown for cell is injected by the entrance pool. Cell is persistently cultivated to realize, needs periodically continuously to inject fresh culture by entrance pool, to provide cell growth Required culture solution.And the present invention experiments verify that, using chip of the invention, compared with existing regular growth culture chip Compared with the liquid frequency of changing of regular growth chip is just to need to change liquid within 1-2 days, and chip of the invention stores 40ml's in centrifuge tube After culture solution, corresponding flow velocity is adjusted, can continuously cultivate cell 7 days or more without changing liquid.

The fourth aspect of the invention provides a kind of cell co-culture method, which is characterized in that uses and includes porous layer A kind of cell suspension containing cell is added in the entrance pool on chip upper layer by the micro-fluidic chip；It will contain another In the cell suspension injection chip lower layer of kind cell or the entrance pool in chip middle layer.Using the multilayer knot of present invention protection porous layer The purpose of 2 kinds or the co-cultivation of two or more cell can be achieved in any chip of structure.

In further specific embodiment, by the entrance pool on chip upper layer, and/or, by corresponding to core on chip upper layer The through-hole of the entrance pool of piece middle layer or chip lower layer periodically injects the culture medium grown for cell.

In cell culture apparatus of the invention, the entrance of chip connects with the outlet of peristaltic pump, the entrance access of peristaltic pump Store the liquid storage pipe outlet of cell culture fluid.The outlet of chip is connected with the entrance of liquid storage pipe, may be constructed closed wriggling and follows Loop system.It can efficiently solve in classical culture protocols, need the problem of manually frequently changing liquid.The revolving speed of vermiculator can be with It is adjusted around liquid storage pipe and is wrapped with heating device, it is heated by being contacted with liquid storage pipe, and be equipped with micro computer temperature Controller is spent, can effectively adjust temperature and by temperature control in the range of being suitble to cells survival.

Micro-fluidic chip provided by the invention can be used for studying cell and interact in three dimensions.

Microfluidic chip technology of the present invention can be used for biomedicine field, and provide a kind of based on micro-fluidic chip Cell co-culture method, can for cell three-dimensional grow support be provided.It is provided by the invention this novel bionical micro-fluidic Cell cultivation chip ensure that the stability of fluid velocity in cell culture chamber, pressure distribution and solution concentration, be cell three Dimension growth creates a stable microenvironment.

Micro-fluidic chip of the invention is easy to carry out a variety of because it is close with cell size matching, environment and physiological environment The features such as cell co-culture, can be used as the platform of bionic of new generation and cell research, have good biocompatibility, Light transmission ventilation, chip fabrication technique is simple, the cell culture and the various intracorporal physiology microenvironments of people of imitation being able to achieve in chip. It can guarantee good sealing after PDMS chip is surface-modified, microexamination, immunostaining etc. after being suitble to.

The present invention can carry out cell three-dimensional co-cultivation on one piece tens square centimeters of chip.Relative to traditional research Method provides one closer to intracorporal microenvironment for the growth and interaction of cell.And the present invention have it is easy to operate, The features such as reagent dosage is few.This is because traditional cell culture mode needs to change for one to two days time culture solution, and combine miniflow The dimensional culture mode of control technology, is passed through 40ml or so culture solution in centrifuge tube, can be recycled 7 days or more.Institute of the present invention The chip and cell culture processes of offer have important biomedical research value and economic value.The micro-current controlled cell simultaneously Culture chip can integrate the Batch Culture that cell is carried out in a chip system, and can be with micro- knot such as Micropump, micro-valve Structure, which combines, carries out relevant cell research, increases the authenticity of experimental result.

Cell culture apparatus according to the present invention can be used for the incubation of different types of cell, incubation time It is determined according to the upgrowth situation of the cell chamber size of chip and cell.The revolving speed of peristaltic pump can pass through in culture apparatus Such as built-in-potential device, external potentiometer, virtual instrument, development board is adjusted in various ways, by changing turning for peristaltic pump Speed can change the flow velocity of cell culture fluid, and simulation more meets actual cell culture environment.The entry/exit mouth of peristaltic pump can lead to It crosses converting interface to be connected from the pipeline of a variety of different inside/outside diameters or material equal-specification, can satisfy different cell culture experiments and want It asks.

Therefore, to make up defect present in current cell co-culture system, we devise a kind of based on micro-fluidic core The cell co-culture method of piece, accurate manipulation and control to fluid may be implemented in it, and cell is easily controllable in microchannel, The microenvironment of growth is close to internal microenvironment.Micro-fluidic chip more can be functions such as the cell culture and drug screening being related to It is integrated together, various biomedicine experiments is carried out on this small chip, chip itself needs to have good Perspectivity and bio-compatibility, the upgrowth situation of cell can be carried out in time, convenient observation.Pass through the cell of this patent Culture apparatus can be effectively integrated conventional microfluidic control cell culture processes, improve its originally existed disadvantage, train for cell Feeding experiment provides an efficient operation platform.

Detailed description of the invention

Fig. 1 is the schematic diagram of the cross-section structure of one embodiment of the invention.

Fig. 2 is the overlooking structure diagram on one embodiment of the invention chip upper layer.

Fig. 3 is the overlooking structure diagram of one embodiment of the invention porous polycarbonate film layer.

Fig. 4 is the overlooking structure diagram in one embodiment of the invention chip middle layer.

Fig. 5 is the overlooking structure diagram of one embodiment of the invention chip lower layer.

Fig. 6 is chip upper layer, chip middle layer and the chip lower layer of the micro-fluidic chip of 1 embodiment of the invention simultaneously more Projection is formed by chamfered shape schematic diagram in aperture layer.

Fig. 7 is a cell culture apparatus overlooking structure diagram of the invention.

Fig. 8 is a cell culture apparatus the schematic diagram of the section structure of the invention.

Fig. 9 is the cell survival rate testing result of the chip of the invention of experimental example 1；Wherein Dynamic refers to that there are shearing forces Group, Static refers to the group of no shearing force.

Figure 10 is that the albumin in the cell metabolite of the chip culture renal tubule RPTEC cell of the invention of experimental example 3 contains Measure testing result.

Figure 11 be the micro-fluidic chip culture RPTECs that is provided using another embodiment of the present invention on day 1, the 4th day, The CCK-8 comparing result of 7th day and static group RPTECs, wherein fluidic refer to micro-fluidic chip culture RPTECs's as a result, Static refers to the result of static group RPTECs.

Figure 12 is that influence of the DDP of various concentration to the cell survival rate cultivated in static group cell and chip compares knot Fruit, wherein fluidic refers to the nephrocyte of micro-fluidic chip culture as a result, static refers to static group nephrocyte result；The chip The cell of middle culture refers to the nephrocyte using micro-fluidic chip culture provided by one embodiment of the invention.

Wherein, appended drawing reference lists as follows: 1,2- porous layer；3- chip upper layer；4- chip middle layer；5- chip lower layer；6- The entrance pool on chip upper layer；The cell culture chamber on 7- chip upper layer；The outlet bath on 8- chip upper layer；The entrance on 9- chip upper layer is logical Road；The exit passageway on 10- chip upper layer；The entrance pool in 11- chip middle layer；The cell culture chamber in 12- chip middle layer；13- chip The outlet bath in middle layer；The access road in 14- chip middle layer；The exit passageway in 15- chip middle layer；The entrance pool of 16- chip lower layer； The cell culture chamber of 17- chip lower layer；The outlet bath of 18- chip lower layer；The access road of 19- chip lower layer；20- chip lower layer Exit passageway.A- cell chip；B- pump, B1- pump discharge, B2- peristaltic pump driving plate；C- fluid reservoir；D- heating component；E- Rack；E1- rack guide hole, E2- rack interface, E3- chip window, E31- chip cover board, E4- digital display screen window.

Specific embodiment

Specific implementation process of the invention, the specific embodiments described herein are further illustrated below in conjunction with attached drawing It is only used to explain the present invention, be not intended to limit the present invention.

The source of biomaterial

Renal tubule RPTEC cell, pipe week capillary HPCECs cell used in the embodiment of the present invention are purchased from Shanghai The odd biological Co., Ltd of match.

1st group of embodiment, cell cultivation chip of the invention

This group of embodiment provides a kind of micro-fluidic chip for cell culture.This organize all embodiments all have it is as follows Feature: the micro-fluidic chip includes chip layer；The upper surface of the chip layer is provided with cell culture chamber, entrance pool, entrance Channel, outlet bath and exit passageway；The cell culture chamber is the biggish sunk area that chip layer upper surface is arranged in；It is described Access road and exit passageway are the groove that chip layer upper surface is arranged in；The entrance pool and outlet bath are to be arranged in chip The lesser sunk area of layer upper surface；The entrance pool and inlet passage, and through the access road and the cell Culturing room is connected；The cell culture chamber is connected through the exit passageway with the outlet bath；The entrance pool be used for The connection of fresh culture conveyance conduit, the outlet bath are used to connect with cell culture metabolism waste liquid discharge line.Using being based on Wriggling pump discharge can connect with entrance pool, culture medium is input to core by peristaltic pump by the culture apparatus of micro-current controlled cell In piece.Outlet bath can be connected with liquid storage tube inlet, and extra cell culture medium is recovered to liquid storage pipe.By what is connect with chip After the connection of wriggling circulation line, chip can be placed on chip heating sheet, cover chip cover board.Pass through temperature controller It can control the temperature of chip heating sheet Yu liquid storage pipe heating sheet, to maintain the temperature of chip and cell culture fluid in suitable cell In the range of culture.

In some specific operations, the entrance pool (such as can be centrifuged by culture medium conveyance conduit and device for storing liquid Pipe) or syringe pump, Micropump or micro-valve be connected, for quantitatively control into chip convey fresh culture for cell grow；Together When, outlet bath can also be by waste liquid discharge line and waste collecting device, and the exhaust device through negative pressure valve or other forms is by core Because the metabolism waste liquid that cell growth generates is discharged in cell cultivation process in piece.

In a further embodiment, the chip layer includes chip upper layer and chip lower layer；The chip upper layer it is upper Surface is tipped upside down on facing downward in chip lower layer；The entrance pool and outlet bath on chip upper layer are engraved structure；The chip upper layer It is additionally provided with through-hole corresponding with the entrance pool of chip lower layer, outlet bath position size.Chip upper layer, which tips upside down on, forms 1 in lower layer A three closed confining spaces are grown for cell, while the entrance pool on chip upper layer and outlet bath are made into engraved structure, either Culture medium, can be injected into chip by back-off or positive placement by entrance pool, can also be by the metabolism of cell culture Waste liquid is discharged from outlet bath.

2nd group of embodiment, cell of the invention co-culture chip

This group of embodiment provides a kind of micro-fluidic chip for cell co-culture.This is organized all embodiments and all has Following common feature: it includes the 1st group of embodiment any micro-fluidic chip and porous layer, institute that the cell, which co-cultures chip, Porous layer is stated for separating the chip layer；The porous layer is between the chip upper layer and chip lower layer.Porous layer Cell is mainly cultivated, collagen can be coated on it, promote the attachment of cell, micropore size cell thereon cannot be saturating It crosses, it is ventable；The setting of porous layer can make chip interior be partitioned into 2 or 2 or more independent three-dimensional space for cultivating Different types of cell, and then height restores, simulates the physiological environment of inside of human body, particularly with certain human organs Speech is usually worked together by 2 kinds or cell of more than two kinds, is acted synergistically to maintain the normal operation of organ, therefore this hair Bright chip can be used to simulate the organ working condition in human body, and the organ and its cell type number that can be simulated as needed Amount, the purpose of the various types of cells co-incubation of different number is realized by adjusting the chip number of plies.

In some specific operations, the entrance pool on the chip upper layer and corresponding with other chip layer entrance pools It is all connected with culture medium conveyance conduit on through-hole, and passes through culture medium conveyance conduit and device for storing liquid (such as centrifuge tube) or injection Pump, Micropump or micro-valve are connected, and fresh culture are conveyed into chip for cell growth for quantitatively controlling, and by accurate Control the input quantity of culture medium quantitatively to simulate the microenvironment of the intracorporal cell growth of people；Meanwhile the outlet bath on chip upper layer with And waste liquid discharge line is all connected on through-hole corresponding with other chip layer outlet baths, and fill by pipeline and waste collection It sets, the exhaust device through negative pressure valve or other forms will give up in the cell cultivation process in chip because cell grows the metabolism generated Liquid discharge.

Specifically, the porous layer is porous polycarbonate film layer, biomembrane, polytetrafluoroethylene film or nitrocellulose filter Deng.The common porous layer making material in above-mentioned this field, those skilled in the art can according to actual needs, for example, experiment purpose, Experimental implementation demand, product cost etc., the porous layer making material common to above-mentioned this field carry out conventional selection.

In more specific embodiment, each chip layer, or, each chip layer and porous layer are superimposed from bottom to up forms the miniflow Control chip；Each chip layer size, shape are adapted；The size of cell culture chamber in each chip layer, shape, position are corresponding, But the axial position of entrance pool, outlet bath on the micro-fluidic chip in each chip layer is not overlapped.Further embodiments Red, the chip layer and porous layer are made as rectangular sheet structure；Each chip layer size, shape is compatible is advantageous in that shape At being easier to form when chip entirety, fixation procedure is simpler, and the process of components is simpler, reduces cost.

In more specific embodiment, the cell culture chamber of the chip layer, entrance pool, the chamfered shape of outlet bath are selected from circle Shape, ellipse, rectangular, diamond shape, or, rule or irregular polygon；The preferred dimethyl silicone polymer of the material of the chip layer, Acrylic board, sheet glass or silicon wafer etc.；The width of the cell culture chamber is 9~11mm, preferably 10mm.The present invention one Chip cell culture chamber shape approximate ellipse provided by specific embodiment, space is larger, can cultivate compared with many cells.It can also root According to specific requirement, in addition similar culture chamber shape is set.

3rd group of embodiment, cell culture processes of the invention

This group of embodiment provides a kind of cell culture processes based on above-mentioned micro-fluidic chip.All embodiments are organized at this In, the cell culture processes all have following feature: using any reality of the 1st group of embodiment and/or the 2nd group of embodiment Micro-fluidic chip provided by example is applied, the cell suspension containing certain cell is injected to the entrance pool on the chip upper layer.Using Any chip of the present invention, can be realized the culture of any type cell, inject cell by the entrance pool toward chip layer Suspension can reach the purpose of cell adherent growth in cell culture room within the regular hour.Meanwhile the present invention also by Repetition test confirms that, using chip progress cell culture of the invention, cell survival rate may be up to 90% or more.

In this group of cell culture processes further embodiment, the culture grown for cell is injected by the entrance pool Base.Cell is persistently cultivated to realize, needs periodically continuously to inject fresh culture by entrance pool, it is raw to provide cell Long required culture solution.And the present invention experiments verify that, using chip of the invention, with existing regular growth culture chip phase Compare, the liquid frequency of changing of regular growth chip is just to need to change liquid within 1-2 days, and chip of the invention is in the culture solution for being passed through 40ml Later, cell 7 days or more can continuously be cultivated without changing liquid.

4th group of embodiment, cell co-culture method of the invention

A kind of cell co-culture method is provided, which is characterized in that the micro-fluidic chip comprising porous layer is used, it will A kind of cell suspension containing cell is added in the entrance pool on chip upper layer；By the cell suspension injection containing another cell In the entrance pool in chip lower layer or chip middle layer.Using any chip of the multilayered structure of present invention protection porous layer, The purpose of 2 kinds or the co-cultivation of two or more cell can be achieved.

In a particular embodiment, by the entrance pool on chip upper layer, and/or, by corresponding to chip middle layer on chip upper layer Or the through-hole of the entrance pool of chip lower layer, periodically inject the culture medium grown for cell.

Micro-fluidic chip provided by any embodiment does not limit in 1st group of embodiment of the invention and/or the 2nd group of embodiment It, can also be using micro-fluidic chip culture zooblast of the invention, plant cell, micro- in being used to cultivate the cell from human body Biological cell etc..

5th group of embodiment, cell culture apparatus of the invention

This group of embodiment provides a kind of cell culture apparatus based on microflow control technique.This group of embodiment has following common Feature: the cell culture apparatus includes cell culture fluid circulation；The cell culture fluid circulation is including that can be thin Born of the same parents provide the cell chip A, pump B and the fluid reservoir C that can store cell culture fluid of culture space；The entrance of the cell chip A It is connected with the outlet B1 of pump B by pipeline；The entrance of the pump B is connected by pipeline with the outlet of the fluid reservoir C；Institute The outlet for stating cell chip is connected by pipeline with the entrance of fluid reservoir.Form such circulation, cell chip outlet discharge Waste liquid return in fluid reservoir again, mix with the fresh medium in fluid reservoir, can't pollute in this way because General experimental situation can be considered germ-free condition in the process time of experiment, and can all have penicillin medicine in culture solution Product have bactericidal effect.And culture solution is made to form circulation, effect is: since the amount of cell metabolism is very little, if using perfusion If, when collecting cell secreta, amount can be very little, if using circulation, then cell secreta can tire out in circulation fluid Product can obtain preferable signal in measurement.

In the particular embodiment, the pump is peristaltic pump；The cell chip is micro-fluidic chip.

Preferably, the peristaltic pump lower part is provided with peristaltic pump driving plate B2, for controlling start and stop and the revolving speed of peristaltic pump.

In a further embodiment, the cell culture apparatus further includes heating component D；The cell chip bottom and Heating component D is provided with inside the fluid reservoir.

In a preferred embodiment, the heating component D is connected by route with the microcomputer temperature controller E.

In a still further embodiment, as shown in Figure 7 and Figure 8, the cell culture apparatus further includes rack E；Cell Chip A, pump B, fluid reservoir C and heating component D are integrated in inside the rack E, and benefit is: it is easy to use, space is saved, and Product design for aesthetic；The rack top surface be provided with hollow out for the rack guide hole E1 of external pipe, rack interface E2, use In the digital display screen window E4 for the chip window E3 and the digital display screen for exposing the microcomputer temperature controller for exposing cell chip. The rack interface E2 and rack guide hole E1 are arranged respectively to 2；The wherein external pipeline on 1 rack interface E2 One end of (not shown) is connected with the pump discharge B1 in the rack E of the cell culture apparatus, other end connection wherein 1 Rack guide hole E1, the rack guide hole E1 are connected with the entrance pool of cell chip A；Another 1 rack guide hole E1 and cell The outlet bath of chip A is connected, and one end of the rack guide hole E1 external pipe, external another 1 machine of the other end of the pipeline Bridge joint mouth E2 is simultaneously returned in fluid reservoir C, to form culture solution circulation loop.

In some specific embodiments, openable and closable chip cover board E31 is provided on the chip window, for covering Chip window E3.

The micro-fluidic chip includes at least 1 layer of chip layer；The upper surface of the chip layer is provided with cell culture chamber, enters Mouth pond, access road, outlet bath and exit passageway；The cell culture chamber is the biggish recess that chip layer upper surface is arranged in Region, for providing three-dimensional culture space for the cell；The access road and exit passageway are to be arranged in chip layer The groove of upper surface；The entrance pool and outlet bath are the lesser sunk area that chip layer upper surface is arranged in；The entrance Pond and inlet passage, and be connected through the access road with the cell culture chamber；Described in the cell culture chamber warp Exit passageway is connected with the outlet bath；The entrance pool with fresh culture conveyance conduit for connecting；The outlet bath For being connect with cell culture metabolism waste liquid discharge line.

The micro-fluidic chip includes 2 layers of chip layer；Chip layer positioned at the micro-fluidic chip top layer is on chip Layer；It is chip lower layer positioned at the undermost chip layer of the micro-fluidic chip；The upper surface on the chip upper layer is buckled to facing downward Under the die on layer；The entrance pool and outlet bath on chip upper layer are engraved structure；The chip upper layer is additionally provided with and chip The entrance pool of lower layer, the corresponding through-hole of outlet bath position size.

The micro-fluidic chip further includes porous layer, for separating the chip layer；The porous layer is located at the core Between piece upper layer and chip lower layer.

The micro-fluidic chip includes 3 layers of chip layer；It is in chip between the chip upper layer and chip lower layer Layer；Cell culture chamber, access road, exit passageway, entrance pool and the outlet bath in the chip middle layer are engraved structure；It is described Porous layer is equipped between chip lower layer and chip middle layer；The chip middle layer offers and the entrance pool of chip lower layer, outlet bath The corresponding through-hole of position size；The chip upper layer offers and the entrance pool in chip middle layer, outlet bath and lead to the hole site size Corresponding through-hole；

Preferably, the micro-fluidic chip includes 3 layers or more of chip layer；On the chip between layer and chip lower layer Equipped with multiple chip middle layers；Wherein near the chip middle layer of chip lower layer offer with the entrance pool of chip lower layer, The corresponding through-hole of outlet bath position size；Remaining each layer of chip layer on this layer of chip middle layer offers adjacent thereto Lower layer chip layer entrance pool, outlet bath and the corresponding through-hole of lead to the hole site size；The entrance pool on chip upper layer and all Through-hole corresponding with the entrance pool of other chip layers is all used to be connected with culture medium conveyance conduit；The outlet bath on chip upper layer and All through-holes corresponding with the outlet bath of other chip layers are all used to be metabolized waste liquid discharge line with cell culture and connect.

The performance detection of experimental example 1, micro-fluidic chip of the present invention

The present invention to micro-fluidic chip and existing common chip of the invention (such as: application for a patent for invention The micro-fluidic chip recorded in 201410191283,201510860959) research experiment that has carried out aspect of performance itself, is removed Chip is different, and other conditions all, including use identical cell (specifically human colon carcinoma cell line RPTECs), In identical condition of culture, (culture medium used in specific cell cultivation process, method and condition of culture are referring to commercially available The product description of RPTECs carries out) under carry out cell culture and obtain following correlation data:

Chip type	Sample initial content	Change the liquid frequency	Cell survival rate
				Chip of the present invention	30ml	7 days or more	80~95%
Existing common chip	15ml	1-2 days	40~50%

The chip that will have been cultivated 3 days, 6 days and 9 days takes out, and configures CCK-8 solution, and microplate reader detects cell survival rate, As a result as shown in Figure 9.Human body mesonephric tubule cell is in always under the shearing force of blood.Research has shown that shearing force can promote cell Skeleton rearranges, and changes the transhipment of expression transport protein and adjusting sodium potassium ion etc..Experimental result is also shown, and there are shearing forces Group (Dynamic) grow more preferably than group (Static) cell without shearing force.

In view of the special construction of chip of the present invention, using micro-fluidic chip of the present invention to carry out cell culture can be to provide cell Three-dimensional growing space is provided, is similar to the intracorporal cell growing environment of machine, thus the obtained survival rate of cell of culture compared with Height, and use existing common chip culture cell, only two-dimentional cultural method, obtained cell survival rate is not high, cannot Height reduction and simulation inside of human body environment well；Meanwhile the structure of chip of the present invention determines in cell cultivation process Amount of samples is generally reduced than conventional method, changes the reduction of the liquid frequency, it is more convenient to operate；As can be seen from the above table, no matter Amount of samples is changed in liquid operation, or in terms of cell survival rate, chip of the present invention all achieves very compared with prior art It is significantly progressive.

Experimental example 2, kidney organ's chip and drug test model of the invention

(1) kidney organ's chip

As shown in Figures 1 to 5: a kind of micro-fluidic chip, it includes porous polycarbonate film layer 1, in porous polycarbonate The two sides of film layer 1 is respectively arranged with chip upper layer 2 and chip lower layer 3, i.e. the porous polycarbonate film layer 1 is folded in chip Between upper layer 2 and chip lower layer 3, and the chip upper layer 2 and chip lower layer 3 is by (or sub- gram of dimethyl silicone polymer Power plate) it is made；

Upper inlet pond (6), the upper cell culture chamber (7) of diamond shape, upper outlet pond are offered on the chip upper layer (3) (8), middle entrance pool (11), middle outlet pond (13), lower inlet pond (16) and lower outlet bath (18) and penetratingly are offered, it is upper enter Be communicated with first passage (9) between mouthful pond (6) and upper cell culture chamber (7), upper outlet pond (8) and upper cell culture chamber (7) it Between be communicated with second channel (10), and the width of upper cell culture chamber (7) be H, the width of first passage (9) and second channel (10) Spend equal h；Layer opens up the middle cell culture chamber (12) of middle entrance pool (11), diamond shape penetratingly in the chips, in middle entrance pool (11) It is communicated between middle cell culture chamber (12) third channel (14), connects between middle outlet pond (13) and middle cell culture chamber (12) Be connected with fourth lane (15), and the width of middle cell culture chamber be H, the equal n of width of third channel (14) and fourth lane (15), H:n=9:1~11:1；Lower inlet pond (16), the lower cell culture chamber (17) of diamond shape and lower outlet are offered on layer under the die Pond (18) is communicated with Five-channel (19) between lower inlet pond (16) and lower cell culture chamber (17), lower outlet bath (18) with The 6th channel (20) is communicated between lower cell culture chamber (17), and the width of lower cell culture chamber (17) is H, Five-channel (19) and the width in the 6th channel (20) is h.

The value range of the H is 9~11mm, preferred value 10mm.The value range of the h is 800~ 1000 μm, preferred value is 833 μm.It is the first passage (9), upper cell culture chamber (7), second channel (10), the 5th logical The height in road (19), lower cell culture chamber (17) and fourth lane (15) is M, and the value range of M is 80~120 μm, excellent Choosing value is 100 μm.The height of third channel (14), middle cell culture chamber (12) and fourth lane (15) is Q, and the height of Q is 180~220 μm, preferred value is 200 μm.

The upper inlet pond (4), first passage (7), upper cell culture chamber (5), second channel (8), upper outlet pond (6), profile such as Fig. 2 institute is collectively formed in middle entrance pool (11), middle outlet pond (13), lower inlet pond (16) and lower outlet bath (18) Show, profile is collectively formed in the middle entrance pool (11), third channel (14), lower cell culture chamber (12), fourth lane (15) As shown in figure 4, lower outlet bath (16), lower inlet pond (18), Five-channel (19), lower cell culture chamber (17), the 6th channel (20) profile is collectively formed with lower outlet bath (18) as shown in figure 5, the profile of Fig. 2, Fig. 4 and the profile of Fig. 5 are jointly porous poly- The profile that projection in carbonic ester film layer is formed is as shown in Figure 6.

Below by renal tubule RPTEC cell, pipe week capillary cells co-incubation for, to how utilize the chip Cell culture is carried out to be illustrated.

The course of work of the micro-fluidic chip of the embodiment of the present invention is as follows: first passage 9, upper cell on chip upper layer 3 Culturing room 7 and second channel 10 simulate renal tubule jointly；Third channel 14, middle cell culture chamber 12 on chip middle layer 4 and Four-way 15 simulates the cytoplasm between renal tubule and kidney end capillary jointly；It is Five-channel 19 in chip lower layer 5, lower thin Urinary cast week capillary is simulated in born of the same parents culturing room 17 and the 6th channel 20 jointly.

Chip upper layer 3 is allowed first upward, RPTEC cell suspension is injected by entrance pool 6, liquid will during flowing RPTEC cell delivery is at upper cell culture chamber 7.RPTEC cell stays in cell culture chamber 7 later, and in porous poly- carbon It is grown in acid esters film layer 1, it is not open close with culture medium；After RPTEC cell is adherent, then from the middle entrance pool on chip upper layer 3 Culture medium is injected in 11, simulates the environment of cytoplasm in human body.Then the injection pipe week from the lower inlet pond 16 on chip upper layer Capillary cells are inverted chip, so that pipe week capillary adherent growth, not open close with culture medium.To RPTEC cell Culture 3 days or more, after forming the single layer converged, then syringe pump is connected from the entrance pool 6 on chip upper layer 2, utilize syringe pump essence Really control fluid flow, makes the hemodynamic microenvironment of reabsorption in its fluid mode analogue body；Above-mentioned work is completed Afterwards, the model for meeting renal tubule anatomical structure in human body is just constructed.

The micro-fluidic renal tubule chip of the invention provides the microenvironment closer to cell growth in vivo for the growth of cell, The growth distribution of cell in the chip can be controlled well simultaneously.The bionical micro-current controlled cell culture chip can be used for it is some with The relevant research of cell, such as the low cost quickly screening of cell drug.

(2) kidney organ's drug test model

Specific chip operation process: firstly, the one side of film repopulating cell is applied to using ECM collagen, it is thin to help Born of the same parents preferably adhere to.Cell (fluidic) operates the static group (static) as described above, as control in kidney chip, will be thin Born of the same parents' density is 5 × 10⁴RPTECs the and PCECs cell of/ml takes 100ul to be uniformly planted in film surface, is placed in 6 orifice plates.To thin After born of the same parents cultivate adherency in 2 hours, addition newly rarely has blood meida to continue culture for 24 hours.The entrance and exit of kidney chip is connected respectively to In centrifuge tube containing DMEM culture medium, and upper cell is exposed in 0.2dyn/cm²Fluid shear stress under.

It selects at three time points, the upgrowth situation of cell to be analyzed over 1 day, 4 days and 7 days.When cell culture this period, CCK-8 solution (CCK-8 reagent: having blood meida=1:10) is added in the film containing RPTECs and is incubated for 2 hours.Later, Solution with CCK-8 is sucked in 96 orifice plates (every hole 120-150ul), and passes through the wavelength at microplate reader measurement 450nm.

As a result as shown in figure 11, on day 1, cultivated in the 4th day and the 7th day static group RPTECs (static) and chip RPTECs (fluidic) CCK-8 result.(*P<0.05.) OD value represents cell viability, OD value is higher, represents living cells and gets over It is more.In addition to shear stress, fluid group and static group are under the same conditions.Cell quantity in 7 days growth cycles, in two groups It is continuously increased.The results show that living cells quantity in the chips has very big mention compared with the 4th day and the 7th day static group It is high.This also turns out that the survival activity of cell can be improved in the environment with flowing.Chip based on microflow control technique can be used as thin The platform of born of the same parents' long-term cultivation, and can be in the test for carrying out related drugs function above.

Experimental example 3, renal tubule RPTEC cell albumin are metabolized analyte detection

The increase of diabetes (DM) and secondary injury of kidney illness rate results in diabetic nephropathy (DN).Early nephropathy definition Albumin detection for microalbuminuria (30-300mg/ days), therefore in kidney organ's metabolin is just particularly important.

Nephrocyte metabolism analyte detection is to prove the well-grown efficiency index of cell.Use albumin detection reagent box (bromocresol green colorimetric method) measures the albumin content in cell metabolite under microplate reader.As shown in Figure 10, culture medium is detected In original albumin content be 2.22mg/ml, postalbumin content is 2.353mg/ml within 3 days, and postalbumin content is within 6 days 2.704mg/ml, 9 days postalbumin contents are 2.745mg/ml.Cell is most with the 6th day albumin metabolism amount on day 3, carefully Intracellular growth situation is preferable, metabolic stability.

Experimental example 4, nephrotoxic drugs DDP (neoplatin) detection model

Cell (fluidic) and static group cell (static) are as described in experimental example 2 in chip, and continuous culture is for 24 hours.Later By configured drug DDP (0,10,20,30,40 μm of ol/L) each lead into chip with cultivated for 24 hours in culture dish.For 24 hours it Afterwards, CCK-8 solution (CCK-8 reagent: having blood meida=1:10) is added in the film containing RPTECs and is incubated for 2 hours.It Afterwards, the solution with CCK-8 is sucked in 96 orifice plates (every hole 120-150ul), and passes through the wave at microplate reader measurement 450nm It is long.

As shown in figure 12, the DDP of various concentration is compared to the cell cultivated in static group cell (static) and chip (fluidic) influence of survival rate.Due to the growth of the environmental stimulus cell flowed in chip, the cell viability on chip is equal Higher than in static group.In addition, cell viability also constantly reduces with the increase of drug concentration.Embody DDP medicine renal toxicity The trend being positively correlated with its dosage.It can be seen from the figure that the cell viability without DDP medicine group core on piece is about 1.55 (OD450), when DDP concentration being increased to 40 μm of ol/L, cell mortality and their cell viability is reduced to 0.81 (OD450)。

Claims

1. a kind of micro-fluidic chip for cell culture, which is characterized in that including chip layer；

The entrance pool with fresh culture conveyance conduit for connecting；

2. micro-fluidic chip according to claim 1, which is characterized in that the chip layer includes under chip upper layer and chip Layer；The upper surface on the chip upper layer is tipped upside down on facing downward in chip lower layer；The entrance pool and outlet bath on chip upper layer are to engrave Hollow structure；The chip upper layer is additionally provided with through-hole corresponding with the entrance pool of chip lower layer, outlet bath position size.

3. a kind of micro-fluidic chip for cell co-culture, including micro-fluidic chip described in claim 1, feature exist In further including porous layer, for separating the chip layer；The porous layer be located at the chip upper layer and chip lower layer it Between.

4. micro-fluidic chip according to claim 3, which is characterized in that the chip layer further includes chip middle layer；It is described Chip middle layer is between the chip upper layer and chip lower layer；

Cell culture chamber, access road, exit passageway, entrance pool and the outlet bath in the chip middle layer are engraved structure；

Porous layer is equipped between the chip lower layer and chip middle layer；

The chip middle layer offers through-hole corresponding with the entrance pool of chip lower layer, outlet bath position size；

5. micro-fluidic chip according to claim 4, which is characterized in that set between layer and chip lower layer on the chip There are multiple chip middle layers；

It is wherein offered near the chip middle layer of chip lower layer corresponding with the entrance pool of chip lower layer, outlet bath position size Through-hole；Remaining each layer of chip layer on this layer of chip middle layer offers the entrance of lower layer chip layer adjacent thereto Pond, outlet bath and the corresponding through-hole of lead to the hole site size；

The entrance pool on chip upper layer and all through-holes corresponding with the entrance pool of other chip layers are all used for and culture medium delivery pipe Road is connected；

The outlet bath on chip upper layer and all through-holes corresponding with the outlet bath of other chip layers are all used to be metabolized with cell culture The connection of waste liquid discharge line.

6. according to micro-fluidic chip as claimed in claim 3 to 5, which is characterized in that the chip layer is all spaced between any two There is porous layer；The porous layer is the structure sheaf with several micro-through-holes, and the through-hole can pass through cell culture medium but impermeable Cross cell.

7. micro-fluidic chip according to claim 6, which is characterized in that the porous layer be porous polycarbonate film layer, Biomembrane, polytetrafluoroethylene film or nitrocellulose filter etc..

8. according to any micro-fluidic chip of claim 3-6, which is characterized in that each chip layer, or, each chip layer and more Aperture layer is superimposed forms the micro-fluidic chip from bottom to up；

Each chip layer size, shape are adapted；The size of cell culture chamber in each chip layer, shape, position are corresponding, but each The axial position of entrance pool, outlet bath on the micro-fluidic chip in chip layer is not overlapped.

9. according to any micro-fluidic chip of claim 3-8, which is characterized in that each chip layer is between any two or each core Between any two, edge is sealing, to ensure that culture medium is not leaked out from the edge of chip for lamella and porous layer.

10. micro-fluidic chip according to claim 9, which is characterized in that the mode of the sealing in following one Kind is several:

Each chip layer between any two or each chip layer and porous layer between any two, edge passes through hot press hot sealing；And/or

Each chip layer between any two or each chip layer and porous layer between any two, edge is sealed by viscose glue or tape-stripping； And/or

Each chip layer between any two or each chip layer and porous layer between any two, edge is fixed by clamp, tight with screw Gu sealing.