CN105907641A - Assembly type multi-condition parallel-culture microfluidic control device and using method thereof - Google Patents
Assembly type multi-condition parallel-culture microfluidic control device and using method thereof Download PDFInfo
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Abstract
The invention provides an assembly type multi-condition parallel-culture microfluidic control device which is composed of a microfluidic control chip and a cover plate, wherein the two parts can be flexibly combined, and are conveniently dismounted; the microfluidic control chip comprises a cell culture room array, a liquor inlet tank, a waste liquor tank and a microfluidic channel; the cell culture room array, the liquor inlet tank and the waste liquor tank communicate with one another through the microfluidic channel; and the cover plate detachably covers the cell culture room array. The microfluidic control device is strong in universality, is convenient to operate, and has a wide application prospect in the fields of cell treatment, tumor drug evaluation, and the like.
Description
Technical field
The invention belongs to organizational project-cell micro-environment research field, be specifically related to a kind of packaging, many condition parallel cultivation miniflow
Control device.
Background technology
Cell is very sensitive to the stimulation of physics various in internal microenvironment, chemical factor, and can by the latter be affected generation cell order
The transformation of fortune.Therefore, the research of cell micro-environment is taken seriously all the more.For most of cells in vivo, microenvironment factor bag
Include: cell heterogeneity, Cellular compartment, 3-d modelling, extracellular matrix protein, soluble factor, blood vessel and composition micro-loop
Other cell types in border.Present stage, for building the external model of bionical microenvironment, contribute to understanding internal microenvironment factor
For the impact of cell, at multiple biomedical sector such as drug screening, control and prevention of disease, demonstrate relatively conventional cell model and move
The huge advantage of object model.Existing method includes: uses and modifies the bioactive polymer (Conway with multivalent ligand or polypeptide
A,et al.Multivalent ligands control stem cell behaviour in vitro and in vivo.Nature nanotechnology.
2013;8:831-8.Dai X,et al.Peptide modified polymer poly(glycerol-dodecanedioate co-fumarate)
for efficient control of motor neuron differentiation.Biomed Mater.2015;10:065013.), utilize synthesis or
Hydrogel (Hsieh FY, the et al.3D bioprinting of neural stem cell-laden thermoresponsive of biogenetic derivation
biodegradable polyurethane hydrogel and potential in central nervous system repair.Biomaterials.
2015;71:48-57.Wang Y,et al.Combination of hyaluronic acid hydrogel scaffold and PLGA
microspheres for supporting survival of neural stem cells.Pharmaceutical research.
2011;28:1406-14.), use has different hardness or the micron of topological structure, nanometer three dimensional matrix (Li N, et al.
Three-dimensional graphene foam as a biocompatible and conductive scaffold for neural stem cells.
Scientific reports.2013;3:1604.Leipzig ND,et al.The effect of substrate stiffness on adult neural
stem cell behavior.Biomaterials.2009;30:6867-78.) etc..But, there is following defect in existing technologies: introduces
Ectogenic biomaterial or biotic factor, may cause immunologic rejection, low biocompatibility and low biodegradability;More
More pressing close to the accurate space-time control of real physiological microenvironment and effective biological assessment importantly, due to lack, cell is micro-
The external bionical structure of environment and the research to impact cell thereof are the most limited.
The microflow control technique that the nineties in last century grows up, also referred to as chip lab or micro-total analysis system, be considered
It is one of most important science and technology in this century, has and the basic operation unit such as conventional chemical and biology is integrated in chip piece
On ability, have that sample consumption is little, integrated level is high and can realize the features such as high flux, there is the strongest technical advance.By
This technology, can have the cell micro-environment of time-space resolution feature by controlled structure, be therefore widely used in cytobiology and turn
Change in the research of medical science.Wherein, in cell micro-environment is studied, utilize microfluidic chip technology, can be mutual to heterocyst
Effect, extracellular three dimensional matrix, cyto-dynamics process, vascularization, Some Circulating Factors gradient, biomechanical force stimulation etc. are micro-
Environmental factors carries out in-vitro simulated and Mechanism Study, for deepening cell and the research of the interphase interaction of microenvironment, and disease
Prevention, diagnose, treat and prognosis suffers from significance.
Microfluidic system cell micro-environment study in characteristic superiority, as fluid accurately manipulate, sample low consumption, high flux sieve
Choosing, unicellular manipulation, analyze and highly integrated, for external bionical structure and the research of cell micro-environment in real time, it is provided that novel,
Effective manner.First, characteristic based on microflow control technique time-space resolution, the bionical knot more pressing close to physiological environment can be built
Structure, truly simulation three-dimensional cell microenvironment, build the external model under micro-scaled structures;Secondly, micro-fluidic chip has spirit
Live controlled MCA and realize the features such as fluid accurately manipulation, it is provided that controlled biochemical conditions and biophysical conditions
Stimulate, specific fluid mechanical property the function of relevant cell is evaluated in simulation microenvironment;Finally, chip can enter
Row comprehensively in real time monitoring, it is achieved the real-time tracing of various kinds of cell behavior, can preferably observation analysis cell behavior, it is achieved right
The real-time monitored of cell and quantitative analysis.But, existing cell micro-environment based on micro-fluidic structure works, and is mostly focused on
Single factor test is studied or builds two dimension cell model in chip internal microchannel, not yet fully demonstrates the essential advantage of chip,
Lack comprehensive study cell fate determined for specific microenvironment and the screening of high-throughout optimal culture conditions.Meanwhile, high
Spend micro-fluidic chip integrated, convenient to operate and be provided to biologist or clinician, strengthen its practicality and clinic
Conversion ratio, solves biology or the practical problem of medical domain as early as possible.
Summary of the invention
For solving above-mentioned technical problem, the present invention designs a kind of packaging, many condition parallel cultivation micro fluidic device, and it is by spirit
The micro-fluidic chip that assembling alive, dismounting are made up of microchannel and cell culture chamber array, and nonbreakable glass cover plate, carried out simultaneously
The parallel cultivation of cell under many condition.The present invention is capable of building and the application of research field at organizational project-cell micro-environment,
It is the technical scheme is that
The present invention includes that two parts parts, whole micro fluidic device are made up of micro-fluidic chip and cover plate;Described micro-fluidic chip bag
Include cell culture chamber array, feed liquor pond and waste liquid pool, microchannel;Described cell culture chamber array, feed liquor pond and waste liquid pool are equal
For uncovered pond;Described microchannel is closed in inside micro-fluidic chip;Described cell culture chamber array, feed liquor pond and waste liquid pool are logical
Cross microchannel to be mutually communicated;Described cover plate is removably covered in above cell culture chamber array.
In technique scheme, the arrangement mode of described cell culture chamber array is not limited to the form that embodiment provides, can basis
Experiment condition, the factor such as experiment purpose, designs its arrangement mode, meets the cell parallel cultivation demand simultaneously carrying out under many condition.
Such as, each culturing room diameter 5mm, highly 2mm of described cell culture chamber array, interval 4mm, can realize and business
Industry multiple tracks liquid-transfering gun is combined, and improves experiment accuracy, simplifies experimental implementation process.
In technique scheme, described microchannel, for the culture fluid conveying of cell, meets the cell simultaneously carrying out under many condition
Parallel cultivation function, such as: include that the cell dispersion in the cultivation of two-dimension single layer cell, matrigel is cultivated, three-dimensional cell ball is cultivated
And the three-dimensional cell ball cultivation in matrigel, and static culture and perfusion training method can be simultaneously introduced.The cell that many condition is cultivated
Kind can be neural stem cell, also can be Malignant glioma cells;Matrigel can be endogenous collagen or Matrigel.
In technique scheme, described cover plate can meet following condition simultaneously: 1. covers all cell culture chamber array;
2. can fit tightly with described micro-fluidic chip, and can dismantle at any time according to specific requirement, it is simple to the collection of sample, thus real
Existing flexible combination;3. the size of cover plate, quantity can be selected flexibly according to the difference of condition of culture.
In the case of You Xuan, described cover plate is nonbreakable glass sheet or rigidity plastics sheet, rigid metal sheet;Labelling on described cover plate
Have can be corresponding with cell culture chamber array position mark.
Under most preferred case, micro-fluidic chip described in the embodiment of the present invention uses polydimethylsiloxane (PDMS) material, lid
Sheet uses polydimethylsiloxane (PDMS) coating, is realized two-part reversible by the surface tension effects between PDMS
Property sealing-in;By semiclosed for culturing room's array of micro-fluidic chip, it is achieved the assembling of device.Dismantle at any time also dependent on specific requirement,
It is easy to the collection of sample.Embodiment uses spin coating to have the nonbreakable glass sheet of polydimethylsiloxane (PDMS), with device miniflow
In control chip, culturing room's array supports the use, and plays the effect of semiclosed the latter.
Meanwhile, the embodiment of the present invention additionally provides and realizes described cover plate and another mode that micro-fluidic chip fits tightly, i.e.
It is aided with applying ambient pressure, the form that such as clip (stainless steel spring folder) compresses, strengthens closure effect, prevent leakage;Real
The assembling of existing device.
Described cover plate is used for carrying cell, and the cell culture chamber arranged in micro-fluidic chip is only used for carrying cell culture fluid etc.,
Therefore micro-fluidic chip is not in contact with cell sample, can Reusability, effectively reduce Financial cost.
Described packaging, the using method of many condition parallel cultivation micro fluidic device, it includes following operating procedure:
1. inoculate cell to be cultivated, the inoculation position of described cell and arrangement mode and cell culture chamber in cover plate PDMS coat side
Array is corresponding;
The cover plate that is vaccinated with cell to be cultivated is relative with cell culture chamber array 2., fit tightly;
3. add the culture fluid of cell to feed liquor pond, the culture fluid of described cell is delivered to each cell culture chamber battle array along microchannel
Row, by being saturated with or cell is cultivated by perfusion cell culture fluid, and discharge cell culture fluid eventually through waste liquid pool;
4. cultivate after terminating, gradually open from a contention of micro-fluidic chip and cover plate sealing surface, until these two parts are kept completely separate,
Take out wherein cell sample and carry out subsequent analysis.
Beneficial effects of the present invention:
1) micro fluidic device of the present invention is by the micro-fluidic chip having a microchannel and cell culture chamber array forms, and rigidity glass
Glass cover plate is constituted, these two parts can flexible combination, convenient dismounting, and Reusability can be realized, be efficiently modified traditional microfluidic dress
Put sample and reclaim the defects such as difficulty, condition of culture is single, recycling rate of waterused is low.
2) packaging, the many condition parallel cultivation micro fluidic device of the present invention, it is possible to provide a kind of reliable, highly versatile, operation side
Just model builds while external various kinds of cell microenvironment and compares with effect, is used for studying cell micro-environment at cell fate
In decisive action, the optimal culture conditions turned to high throughput format parllel screening certain destiny of beneficially cell.
3) present invention is during building cell micro-environment, is not introduced into ectogenic biomaterial, Some Circulating Factors or genetic modification,
More press close to internal true microenvironment, it is to avoid immunologic rejection and oncogenicity, there is higher biocompatibility and safety.
4) realize, through specific cultivation of this device, the cell that orientation converts, can be that clinical demand provides special cell products, and can be real
Now further commercialization;Especially, the cell to clinical patients, after In vitro culture processes, autologous feedback can be realized, keep away
Exempt from the immunologic rejection of patient, stopped because the disease that allosome input causes infects.
5) the method can be widely used in the application systems such as stem cell differentiation, tumour medicine evaluation.
Accompanying drawing explanation
Fig. 1 is for neural stem cell self renewal and the device schematic diagram of differentiation research;
Wherein: 1 is the nonbreakable glass sheet with PDMS film, 2 is micro-fluidic chip;11 is neural stem cell adhere-wall culture group
Unit cultivated by corresponding sheet glass, and 12 is the sheet glass cultivation unit that the unicellular encapsulated cultivation group of neural stem cell is corresponding, and 13 is god
The micro-fluidic chip corresponding through the unicellular encapsulated cultivation group of stem cell cultivates unit, and 14 is that neural stem cell adhere-wall culture group is corresponding
Micro-fluidic chip cultivates unit, and 15 is the micro-fluidic chip cultivation unit that neural molecular biology encapsulated cultivation group is corresponding, and 16 is neural
The micro-fluidic chip cultivation unit that stem cell ball cultivation group is corresponding, 17 is the sheet glass cultivation list that neural molecular biology cultivation group is corresponding
Unit, 18 is the sheet glass cultivation unit that neural molecular biology encapsulated cultivation group is corresponding;21 is cell culture chamber array, and 22 for entering
Liquid pool, 23 is waste liquid pool, and 24 is microchannel;
Under Fig. 2 difference condition of culture, the self-renewal capacity immunofluorescence of neural stem cell characterizes, and protein marker is Nestin;
Under Fig. 3 difference condition of culture, the self-renewal capacity quantitatively characterizing of neural stem cell, with Nestin positive expression cell hundred
Proportion by subtraction meter;
Under Fig. 4 difference condition of culture, the neuron of neural stem cell characterizes to differentiation capability immunofluorescence, and protein marker is
β-tubulin III;
Under Fig. 5 difference condition of culture, the self-renewal capacity quantitatively characterizing of neural stem cell, with β-tubulin III positive expression
Cell percentages meter.
Detailed description of the invention
Below by way of specific embodiment, the present invention is described in detail, so as to illustrating operation principle and the working method of this device,
But it is not thereby limiting the invention.
Embodiment 1
Utilize a kind of packaging, many condition parallel cultivation micro fluidic device, carry out neural stem cell self renewal and differentiation research.
The design and fabrication of 1 micro-fluidic chip
1) the device schematic diagram of neural stem cell self renewal and differentiation research it is used for as shown in Figure 1.Whole device is by micro-fluidic
Chip 2, and two panels is coated with the nonbreakable glass sheet 1 of PDMS film and constitutes.Micro-fluidic chip 2 includes construction unit: cell is trained
Support room array 21 (4 × 4 array), feed liquor pond 22, waste liquid pool 23, microchannel 24.The cell culture chamber of four arrays is altogether
With a waste liquid pool 23.Two panels is coated with the nonbreakable glass sheet 1 of PDMS film, and the cell for different condition of culture is inoculated and right
The sealing of cell culture chamber array 21.
2) application negative photoresist SU-8, prepares anode membrane mould according to the soft lithography of standard, and inverts out with this formpiston
PDMS former.Cell culture chamber array 21 (4 × 4 arrays, each culturing room diameter 5 is pressed in this PDMS former relevant position
Mm, highly 2mm, interval 4mm), feed liquor pond 22, waste liquid pool 23 punch, punch position forms through former
Through-hole structure.With one block of clean sheet glass, irreversible with the PDMS former one side containing passage it is bonded, by all of for former logical
The bottom surface sealing of road and hole, forms micro-fluidic chip 2.This micro-fluidic chip 2, through autoclaving, makes overall hydrophilic carry
High, it is simple to liquid communication, and complete disinfecting process.
The 2 primary extraction of neural stem cell and cultivations
1) the primary SD SPF rat extracting from pregnant 13-14 days of neural stem cell, disconnected neck execution, abdominal part shaving, alcohol disinfecting.
Take out tire Mus, be placed in the culture dish filling PBS and penicillin, streptomycin and clean, peel off fetal membrane, cut off umbilical cord, by tire Mus
Moving on in another culture dish, cervical region breaks end, and takes out tire Mus brain, peels off meninges and blood vessel, takes forebrain cortex position, be placed in
In the DMEM/F12 of pre-cooling in advance.Shears shreds cerebral tissue.DMEM/F12 is moved in centrifuge tube with tissue mixed liquor,
Glass tubing is softly blown and beaten, and stands, and takes supernatant fluid and moves in another centrifuge tube centrifugal, abandons supernatant, by cell precipitation with
AccutaseTMCell separation liquid mixes, and moves to, in new culture dish, put into incubator and hatch.Take out culture dish, softly blow and beat,
Adding DMEM/F12 and terminate digestion, cell mixture moves in centrifuge tube centrifugal.Abandon supernatant, add Culture of neural stem cells
Base, cell counting, cell density is adjusted to 2 × 105Cell/ml, plants in culture bottle.Put into incubator to cultivate.Every 2 days semidefinites
Amount adds nerve stem cell culture medium, observes neural stem cell balling-up size.
2) when a diameter of 150 μm-200 μm of neural stem cell cell ball, can pass on.Cell suspension is moved in centrifuge tube
Centrifugal.Abandon supernatant, by cell precipitation and AccutaseTMCell separation liquid mixes, and moves to, in new culture dish, put into incubator and incubate
After educating, softly blowing and beating, add DMEM/F12 and terminate digestion, cell mixture moves in centrifuge tube centrifugal.Abandon supernatant, add
Enter nerve stem cell culture medium, cell counting, cell density is adjusted to 2 × 105Cell/ml, plants in culture bottle, puts into incubator
Cultivate.
The inoculation of 3 neural stem cell and the combination with micro-fluidic chip
1) four groups of different Culture of neural stem cells conditions are set: neural stem cell adhere-wall culture group, neural molecular biology cultivation group,
The unicellular encapsulated cultivation group of neural stem cell and neural molecular biology encapsulated cultivation group.Cell inoculation all uses third generation nerve trunk thin
Born of the same parents, by nerve ball AccutaseTMBeing separated into single cell suspension, inoculum density is 1 × 106cell/ml.Four groups of different conditions
The seeded process of Culture of neural stem cells needs three days altogether.
2) first day, two panels was coated with the nonbreakable glass sheet 1 of PDMS film through ultraviolet radiation disinfection 1h.Neural stem cell list
Cell suspension drips in the most a piece of upper surface, dripping position as shown in Figure 1 16,15, with micro-fluidic chip two of which erect
Cell culture chamber 17,18 alignment of row, totally two row.Owing to being coated with the surface hydrophobic of the nonbreakable glass sheet of PDMS film,
Two row droplet arrays are defined on its surface.With adhesive plaster, sheet glass being sticked at culture dish lid, culture dish lid reverses carefully, is buckled in
At the bottom of culture dish containing PBS, being put in incubator, neural stem cell, through precipitation, gathering, forms cell ball.Meanwhile, poly bird
Propylhomoserin (PO) drips and is coated with the nonbreakable glass sheet position 11 of PDMS film in another, dropping position and micro-fluidic chip wherein 1
The cell culture chamber 14 of individual perpendicular row aligns, and is placed in room temperature, is coated overnight.
3) second day, replace PO with laminin,LN (LN), put into incubator and hatch.By single cell suspension after PBS
Drip the position being coated in LN, be put in incubator overnight, as neural stem cell adhere-wall culture group.
4) the 3rd day, the preparation of neural stem cell adhere-wall culture group and neural molecular biology cultivation group was complete.Use glue
Former hydrogel is as neural stem cell extracellular matrix (ECM) culture medium, with PBS and distilled water diluting collagen stock solution, hydrogen
Sodium oxide regulation pH value.In the unicellular encapsulated cultivation group of neural stem cell: by resuspended for unicellular collagen diluent, drip in
It is coated with the nonbreakable glass sheet position 12 of PDMS film, aligns with the cell culture chamber 13 of micro-fluidic chip 2 wherein another perpendicular row.
Cultivation group encapsulated for neural molecular biology: suck neural ball surrounding media in position 18, drips collagen on neural ball.Bag
Containing the collagen system of cell and cell ball that dissipates through 37 DEG C of environment of incubator, crosslink reaction, formed outside the born of the same parents of neural stem cell
Matrix environment.
5) the two panels PDMS nonbreakable glass sheet 1 containing 4 × 4 arrays (11,12,17,18), faces down containing cell, respectively
The cell culture chamber array (14,13,16,15) of row perpendicular with four of micro-fluidic chip 2 aligns, and is placed on micro-fluidic chip 2
On, under PDMS-PDMS surface tension effects, three parts fit together, and the micro-fluidic chip becoming a semi-hermetic is thin
Born of the same parents' cultivating system, by two parts of sealing stainless steel spring folder clamping, strengthens closure effect, prevents leakage.Only stay feed liquor pond
22 and waste liquid pool 23 be open aperture, for introducing and the outer row of liquid.
In 4 micro fluidic devices, the perfusion of neural stem cell is cultivated and static culture
1) static culture: use liquid-transfering gun culture medium to be added from feed liquor pond 22, the bubble in emptying chip, make liquid be full of
Passage and cell culture chamber, submergence 4 × 4 array all neural stem cell sample.Chip is partially placed into incubator, and every day is changed
Culture medium.
2) perfusion is cultivated: the same static culture of pouring liquid step in chip;Aseptic connecting tube is used to connect the feed liquor pond of chip
22 are controlled culture medium perfusion rate with the microsyringe equipped with culture medium, this syringe by syringe pump.Chip is partially placed into training
Support case.
5 immunofluorescence dyeings identify neural stem cell self renewal and differentiation capability
After static or perfusion cultivation terminate, alligator clamp is taken off, from micro-fluidic chip 2 and the one of nonbreakable glass sheet 1 sealing surface
Contend and gradually open, until these two parts are kept completely separate, reclaim the cell sample under different condition of culture on nonbreakable glass sheet 1.Many
Polyformaldehyde is fixed, and adds Triton-100 after PBS, adds BSA and close after PBS.It is separately added into Nestin, β-tubulin
III mono-is anti-overnight.Antibody diluent is abandoned in suction, adds two anti-diluents after PBS, and lucifuge is hatched.Two anti-diluents are abandoned in suction,
Add core dyestuff Hoechst lucifuge after PBS to hatch.Core dyestuff is abandoned in suction, sees after PBS under confocal fluorescent microscope
Examine.30 aspects of each scan sample, take wherein 5 equally spaced scanned pictures of aspect, statistics positive expression cell number and thin
Born of the same parents' sum, calculates positive expression cell proportion.The self-renewal capacity immunofluorescence of neural stem cell and quantitatively characterizing such as figure
Shown in 2 and Fig. 3;The neuron of neural stem cell is to differentiation capability immunofluorescence and quantitatively characterizing as shown in Figures 4 and 5.
By the use of this micro fluidic device, successfully construct a kind of by dimensional culture, factor intervention and biomechanics stimulation height
Integrated integrated system, it is possible to assess simultaneously multiple condition of culture to neural stem cell self renewal, breed and the cell such as differentiation
Learn the impact of destiny, the physics in analogue body, chemistry and physiological signal more accurately.Compared to existing investigative technique, the present invention
Providing a kind of microenvironment and cell fate guides the high flux Effective selection platform of research, its comparative benefits is embodied in: be not introduced into
Ectogenic biomaterial or the biotic factor of immunologic rejection may be caused, thus improve system biocompatibility;Training mode
Expanded to the parallel cultivation of many condition by single limitation, integration degree is high, operate simple and convenient;What is more important, due to structure
Build and more pressed close to the accurate space-time control of real physiological microenvironment and effective biological assessment, be more beneficial for cell micro-environment body
Outer bionical structure and the research to impact cell thereof.
Claims (8)
1. packaging, a many condition parallel cultivation micro fluidic device;It is characterized in that: described device is by micro-fluidic chip and lid
Sheet is constituted;Described micro-fluidic chip includes cell culture chamber array, feed liquor pond and waste liquid pool, microchannel;Described cell is cultivated
Room array, feed liquor pond and waste liquid pool are uncovered pond;Described microchannel is closed in inside micro-fluidic chip;Described cell is cultivated
Room array, feed liquor pond and waste liquid pool are mutually communicated by microchannel;Described cover plate is removably covered in cell culture chamber array
Top.
Micro fluidic device the most according to claim 1, it is characterised in that: described micro-fluidic chip material is poly dimethyl silicon
Oxygen alkane.
Micro fluidic device the most according to claim 1, it is characterised in that: described cover plate is nonbreakable glass sheet, rigidity plastics
Sheet or rigid metal sheet.
Micro fluidic device the most according to claim 1, it is characterised in that: it is marked with on described cover plate and cell culture chamber battle array
Arrange corresponding position mark.
Micro fluidic device the most according to claim 1, it is characterised in that: described cover plate uses polydimethylsiloxane coating.
Micro fluidic device the most according to claim 1, it is characterised in that: culturing room's diameter of described cell culture chamber array
5mm, highly 2mm;Culturing room interval 4mm.
Micro fluidic device the most according to claim 1, it is characterised in that: also include described cover plate and micro-fluidic chip pressure
Tight clip.
The using method of the most packaging, many condition parallel cultivation micro fluidic device, it is characterised in that
Including following operating procedure:
1. cell to be cultivated, the inoculation position of described cell and arrangement mode and cell culture chamber are inoculated in cover plate PDMS coat side
Array is corresponding;
2. the cover plate that is vaccinated with cell to be cultivated is relative with cell culture chamber array, fit tightly;
3. add the culture fluid of cell to feed liquor pond, the culture fluid of described cell is delivered to each cell culture chamber battle array along microchannel
Row, by being saturated with or cell is cultivated by perfusion cell culture fluid, and discharge cell culture fluid eventually through waste liquid pool;
4. cultivate after terminating, gradually open from a contention of micro-fluidic chip and cover plate sealing surface, until these two parts are kept completely separate,
Take out wherein cell sample and carry out subsequent analysis.
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