CN110305788A - The array chip and its preparation and operating method of cell capture and the culture of tumour ball - Google Patents
The array chip and its preparation and operating method of cell capture and the culture of tumour ball Download PDFInfo
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Abstract
The array chip of cell capture disclosed by the invention and the culture of tumour ball, including unicellular sorting layer and cell culture layer, cell culture layer is bonded on unicellular sorting layer, unicellular sorting layer includes at least one set of cell capture array, cell suspending liquid entrance and cell suspending liquid outlet, every group of cell capture array includes at least three column microarrays, each column microarray includes the unicellular capturing unit of laid out in parallel, unicellular capturing unit includes two micro-structures, screening tank is formed between two micro-structures, the both ends of screening tank are respectively formed the first hole and the second hole for capturing cell, the width of first hole is 2 μm bigger than the width of the second hole；Cell culture layer offers culture tank, and culture tank is corresponding with unicellular capturing unit.The invention also discloses the preparation method of the chip and operating method, have the characteristics that simple and quick, low consumed, can be widely applied for a variety of parallel high-throughput apply with multiple compound unicellular operation with analysis.
Description
Technical field
The invention belongs to cell biology and micro fluidic chip technical fields, and in particular to a kind of cell capture and tumour ball
The array chip of culture further relates to the preparation method of a kind of cell capture and the array chip of tumour ball culture, further relates to
A kind of operating method of cell capture and the array chip of tumour ball culture.
Background technique
Currently, the research and analysis based on individual cell level are widely used in biological study and clinical detection simultaneously
Grow into emerging an analysis method and detection platform.Microfluidic chip technology is a kind of miniature behaviour having just emerged in this century
Work and analysis method.It just shows extremely strong unicellular operation and analytic potential at the beginning of formulating.So far, based on micro-fluidic
The unicellular capture of chip mainly includes with operating method: micro-structure filtering, flow dynamics operate, magnetic operates, light operates,
Electric field operation and acoustic operation etc..Wherein, the method for unicellular capture and long-term cultivation being carried out based on micro-structure filter method,
It is swift to operate, can carry out high-throughput in real time and without assisting the advantages such as Other Instruments because its principle is simple, it has obtained extensive
Using and development.However, currently based on the chip of microstructure unit (such as: micro- dam and micropore) due to by principle itself
Limitation is difficult to carry out the research of long-term cultivation.And the detection (such as: stem cell culture, drug screening) based on cell long-period culture
Then it is forced not carrying out.The waste to capture cell is not only caused in this way, while largely being also limited and being captured
Single celled later period experimental study range.
Summary of the invention
The object of the present invention is to provide the array chips of a kind of cell capture and the culture of tumour ball, a kind of multiple by designing
Miscellaneous microfiltration structure realizes single different size and the single celled capture of morphotropism, so by and microcellular structure combination it is real
The single celled long-term cultivation that is now captured and subsequent detection, the cell chip for solving existing micro-filtration structure have a single function, nothing
Method carries out later period cell culture and causes the problem of capturing unicellular waste.
It is a further object of the present invention to provide the preparation method of a kind of cell capture and the array chip of tumour ball culture,
This method is easy to operate, is easily mastered.
Third object of the present invention is to provide the operation sides of a kind of cell capture and the array chip of tumour ball culture
Method.
The technical scheme adopted by the invention is that the array chip of cell capture and the culture of tumour ball, including it is unicellular
Layer, cell culture layer and the pipeline being embedded in unicellular sorting layer are sorted, is bonded with cell culture layer on unicellular sorting layer,
Unicellular sorting layer includes that at least one set of cell capture array, a cell suspending liquid entrance and multiple cell suspending liquids export,
Every group of cell capture array is exported by pipeline connection cell suspending liquid entrance and multiple cell suspending liquids, every group of cell capture battle array
Column include at least three column microarrays, and each column microarray includes multiple unicellular capturing units of laid out in parallel, each unicellular to catch
Obtaining unit includes two H-type micro-structures, screening tank of the formation for cell screening between two H-type micro-structures, and the two of screening tank
End is respectively formed the first hole and the second hole for capturing cell；
Interval is uniformly provided with multiple mutually independent culture tanks for cultivating cell on cell culture layer, and culture tank is in
Array arrangement, each culture tank and unicellular capturing unit correspond.
Other features of the invention also reside in,
Preferably, the spacing of adjacent two column microarray is 50 μm.
Preferably, the section of each H-type micro-structure is in the rectangle of middle part radial contraction, and the length of each H-type micro-structure is
200 μm~300 μm, the rectangle both ends width of each H-type micro-structure is 120 μm, and the width of middle part contraction place is 90 μm.
Preferably, wide both sides are narrow among screening tank, and the length of screening tank is 100 μm~150 μm, the width of intermediate the widest part
It is 40 μm~60 μm, the height of screening tank and the height of pipeline are identical, and the height of screening tank is 18 μm~28 μm.
Preferably, the two sides that unicellular sorting layer is located at cell capture array are provided with multiple microtrabeculaes, between each microtrabeculae
Distance be 25 μm~50 μm, for preventing pipeline from collapsing.
Preferably, the length of each culture tank be 80 μm~120 μm, width be 80 μm~120 μm, highly for 70 μm~
100μm。
Preferably, constant positioned at the size of the first hole of same row microarray and the constant dimension of the second hole, it is located at
The size of first hole of adjacent two column microarray differs 2 μm, positioned at the size difference 2 of the second hole of adjacent two column microarray
μm。
Another technical solution of the invention is the preparation method of the array chip of cell capture and the culture of tumour ball, tool
Body includes the following steps:
Step 1, unicellular sorting layer is prepared:
5:1 mixing PDMS matrix and curing agent in mass ratio, meanwhile, use unicellular point of trim,ethylchlorosilane steam treatment
Layer mold 5min~10min is selected, PDMS matrix and curing agent mixture are poured into trim,ethylchlorosilane treated unicellular point
It selects on layer mold, vacuumizes degassing and be placed in 80 DEG C~100 DEG C baking ovens the 0.5h~1h that is heating and curing, by the PDMS after solidification
It removes from mold, and is cut as required, and punching prepares screening tank, clean up spare；
Step 2, cell culture layer is prepared:
According to mass ratio 15:1 mixing PDMS matrix and curing agent, while using TMCS steam treatment cell culture layer mold
PDMS matrix and curing agent are poured into TMCS treated in cell culture layer mold, vacuumizes 80 DEG C~100 DEG C of degassing by 5min
Be heating and curing 1h~2h in baking oven, and the PDMS after solidification is removed from mold, and is cut as required, and punches preparation
Culture tank cleans up spare；
Step 3, the unicellular sorting layer that step 1 obtains and the cell culture layer that step 2 obtains are bonded bonding:
Unicellular sorting layer or cell culture layer are placed in the visual field of inverted fluorescence microscope, it is then that another layer is right
Qi Houyong adhesive tape is fixed and is placed it in 80 DEG C~100 DEG C baking ovens, obtains micro-control core after heating bonding 50h~100h
Piece takes out microcontroller chip, takes down adhesive tape, micro- along smearing closing is carried out with glue at the gap of the side of microcontroller chip
It is stand-by to control chip.
Preferably, the gap closing of the side of microcontroller chip can also be by pressing PDMS matrix and curing agent in step 3
According to being applied at the gap of microcontroller chip side after 8~15:1 of mass ratio mixing, it is placed in 80 DEG C~100 DEG C baking ovens, heats key
Closing 50h~100h makes its secure bond.
Third technical solution of the invention is the operating method of the array chip of cell capture and the culture of tumour ball,
Concrete operations include the following steps:
Step 1, under certain flow rate, cell suspension is more by the cell suspending liquid portal perfusion entrance of unicellular sorting layer
In a micro-structure, cell suspending liquid is by micro-structure, and the cell of maxicell or morphotropism difference passes through the first hole, by the second hole
Block capture, cellule or the strong cell of morphotropism pass through the first hole and the second hole without captured, to carry out
Into the screening tank of the smaller micro-structure of hole below；
Step 2, cytotostatic to be captured is distributed in capturing unit, by microcontroller chip overturning, it is static make it is captured thin
Born of the same parents are entered from unicellular trapping layer into the culture tank of cell culture layer in gravity, then will be with slug flow speed by cell
Culture solution, clinical medicine, detection reagent are irrigated by cell suspending liquid entrance, carry out cell culture.
The invention has the advantages that the array chip of cell capture and the culture of tumour ball, passes through unicellular point of design
Layer and cell culture layer are selected, the cell capture unit of array structure is devised in unicellular sorting layer, is devised in culture layer
The culture tank of array, mutually indepedent between each culture tank, realization is continuously finished single celled capture, culture and subsequent detection
Operation.Single celled operation is captured relative to the micro-structure in previous micro-fluidic chip, different size and deformation can be carried out
Property single celled capture and long-term cultivation, the preparation method and operating method of the array chip of cell capture and the culture of tumour ball
It is simple to operation, have the characteristics that simple and quick, sample and energy low consumption in terms of cell manipulation, can be widely applied
It is applied in a variety of parallel high-throughput and multiple compound unicellular operation with analysis.
Detailed description of the invention
Fig. 1 is the overall structure diagram of the array chip of cell capture and the culture of tumour ball of the invention；
The top view of cell capture array in the array chip of cell capture and the culture of tumour ball Fig. 2 of the invention；
Fig. 3 is micro-structure cross-sectional view in the array chip of cell capture and the culture of tumour ball of the invention；
Fig. 4 is the operating method flow diagram of the statement of microcontroller chip side structure of the invention；
Fig. 5 is to pour into the tumour cell with different size and morphotropism in embodiment 1 in microcontroller chip；Wherein, scheme
5A is the white light figure and fluorogram that Normal U251 cell is distributed in micro structure array, and Fig. 5 B is Induced U251 cell
The white light figure and fluorogram being distributed in micro structure array；
Fig. 6 be poured into microcontroller chip in embodiment 1 Normal U251 cell with different size and morphotropism and
Distribution statistics figure of the Induced U251 cell in micro-structure；Wherein, Fig. 6 A is Normal U251 cell and Induced
Percentage distribution statistics figure of the U251 cell in micro-structure；Fig. 6 B is Normal U251 cell and Induced U251 cell
Quantity statistics figure in micro-structure；Show that microstructure size is that unicellular sorting confluent monolayer cells capture the second hole of micro unit in figure
Size；
Individual cells form the white light figure of tumour ball in the different micro-structures of Fig. 7 embodiment 1；Wherein, scheme the number of top
Represent the size in the hole between adjacent microstructures；Fig. 7 A is the white light figure of Normal U251 cell；Fig. 7 B Induced
The white light figure of U251 cell；Show that microstructure size is that unicellular sorting confluent monolayer cells capture the big of the second hole of micro unit in figure
It is small；
Fig. 8 is Normal U251 cell and Induced U251 the cell unicellular source tumour in micro-structure in embodiment 1
The balling ratio of ball；
Fig. 9 is Normal U251 cell and Induced U251 the cell unicellular source tumour in micro-structure in embodiment 1
The size of ball；
Figure 10 A be in embodiment 1 different size and the single tumour of morphotropism forms that tumour ball formed in micro-structure at
Ball rate；Show that microstructure size is the size that unicellular sorting confluent monolayer cells capture the second hole of micro unit in figure；
Figure 10 B is that different size and the single tumour of morphotropism form the swollen of tumour ball formation in micro-structure in embodiment 1
The size of tumor ball；Show that microstructure size is the size that unicellular sorting confluent monolayer cells capture the second hole of micro unit in figure.
In figure, 1. unicellular sorting layers, 2. cell culture layers, 3. pipelines, 4. cell capture arrays, 5. microarrays, 6. sieves
Select slot, 7. unicellular capturing units, 8.H type micro-structure, 9. culture tanks, 10. first holes, 11. second holes.
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
The array chip of cell capture and the culture of tumour ball of the invention, as shown in Figure 1, Figure 2 and Figure 3, including it is slender
Born of the same parents sort layer 1, cell culture layer 2 and the pipeline 3 being embedded in unicellular sorting layer 1, are bonded on unicellular sorting layer 1 thin
Born of the same parents' culture layer 2, unicellular sorting layer include at least one set of 4, cell suspending liquid entrances of cell capture array and multiple cells
Suspension outlet, every group of cell capture array 4 are connected to cell suspending liquid entrance and the outlet of multiple cell suspending liquids by pipeline 3,
Every group of cell capture array 4 includes at least three column microarrays 5, and each column microarray 5 includes multiple unicellular captures of laid out in parallel
Unit 7, each unicellular capturing unit 7 include two H-type micro-structures 8, are formed between two H-type micro-structures 8 and are used for cell sieve
The screening tank 6 of choosing, the both ends of screening tank 6 are respectively formed the first hole 10 and the second hole 11 for capturing cell, the first hole
The width of gap 10 is 1 μm bigger than the width of the second hole 11~and 2 μm；
Interval is uniformly provided with multiple mutually independent culture tanks 9 for cultivating cell, culture on cell culture layer 2
Slot 9 is arranged in array, and each culture tank 9 is corresponded with unicellular capturing unit 7.
The spacing of adjacent two column microarray 5 is 50 μm.
The section of each H-type micro-structure 8 is in the rectangle of middle part radial contraction, and the length of each H-type micro-structure 8 is 200 μm
~300 μm, the rectangle both ends width of each H-type micro-structure is 120 μm, and the width of middle part contraction place is 90 μm.
Wide both sides are narrow among screening tank 6, and the length of screening tank 6 is 100 μm~150 μm, and the width of intermediate the widest part is 40 μ
The height of m~60 μm, screening tank 6 is identical as the height of pipeline 3, and the height of screening tank 6 is 18 μm~28 μm.
The two sides that unicellular sorting layer 1 is located at cell capture array are provided with the distance between multiple microtrabeculaes, each microtrabeculae
It is 25 μm~50 μm, for preventing pipeline from collapsing.
It is constant positioned at the size of the first hole 10 of same row microarray 5 and the constant dimension of the second hole 11, it is located at phase
The size of first hole 10 of adjacent two column microarrays 5 differs 2 μm, positioned at the size phase of the second hole 11 of adjacent two column microarray
Poor 2 μm.
The length of each culture tank 9 is 80 μm~120 μm, and it is highly 70 μm~100 μm, carefully that width, which is 80 μm~120 μm,
Culture tank 9 in born of the same parents' culture layer 2 is independent structure, does not have pipeline connection between each other, guarantees the unicellular culture that is captured
Independence.
The working principle of cell capture of the invention and the array chip of tumour ball culture is as follows: where unicellular point
The unicellular of unicellular capturing unit mainly the capture different size and morphotropism of layer is selected, culture tank is will be captured slender
Born of the same parents carry out long-term cultivation, and array structure is used for single celled capture and long-term cultivation.The structure of unicellular sorting layer of the invention
It avoids cellular damage and improves single celled separative efficiency.The micro- knot section of each of every group of cell capture array is radial in middle part
The rectangle of contraction, two adjacent micro-structures constitute a unicellular capturing unit, and there are two the holes for being used for cell capture for tool
Gap: the first hole and the second hole, wherein first the second hole of void ratio is 2 μm wide.The duct height of entire unicellular sorting layer
It is 18-28 μm, which includes most of mammalian cell size.Micro-structure makes in the rectangle of middle part radial contraction
Flow velocity flow velocity at two minimum sectional areas (the first hole and the second hole) by micro-structure reaches peak value, and in culture tank
The speed at middle part be gradually reduced, for the capture of cell or by providing a controllable fluid environment, avoid potential
Blocking risk and since cell and capturing unit cross the cellular damage caused by contacting for a long time.
Unicellular sorting layer is substantially carried out different size and the single celled capture of morphotropism, the culture tank in cell culture layer
Effect be unicellular to be cultivated captured.
The unicellular sorting layer of the present invention and the material of cell culture layer are PDMS polymer, by by different proportion
PDMS polymer carries out irreversible sealing-in, guarantee in chip the consistency of unicellular capture microchannel and cell culture micro unit and
Independence.
The preparation method of the array chip of cell capture and the culture of tumour ball of the invention, specifically comprises the following steps:
Step 1, unicellular sorting layer is prepared:
5:1 mixing PDMS matrix and curing agent in mass ratio, meanwhile, use trim,ethylchlorosilane
The unicellular sorting layer mold 5min~10min of (Trimethylchlorosilan, TMCS) steam treatment, by PDMS matrix and admittedly
Agent mixture pours into trim,ethylchlorosilane treated on unicellular sorting layer mold, vacuumize degassing be placed in 80 DEG C~
Be heating and curing 0.5h~1h in 100 DEG C of baking ovens, and the PDMS after solidification is removed from mold, and is cut as required, and
Punching prepares screening tank, cleans up spare, wherein PDMS matrix and curing agent buying are numbered from Dow corning company:
SYLGARD 184。
Step 2, cell culture layer is prepared:
According to mass ratio 15:1 mixing PDMS matrix and curing agent, while using TMCS steam treatment cell culture layer mold
PDMS matrix and curing agent are poured into TMCS treated in cell culture layer mold, vacuumizes 80 DEG C~100 DEG C of degassing by 5min
Be heating and curing 1h~2h in baking oven, and the PDMS after solidification is removed from mold, and is cut as required, and punches preparation
Culture tank cleans up spare；
Step 3, the unicellular sorting layer that step 1 obtains and the cell culture layer that step 2 obtains are bonded bonding:
Since the alignment error of microwell chips is no more than 10 μm, using the higher inverted fluorescence microscope of precision into
Unicellular sorting layer or cell culture layer, are specifically placed in the visual field of inverted fluorescence microscope by row alignment, then will be another
It is fixed and is placed it in 80 DEG C~100 DEG C baking ovens with adhesive tape after one layer of alignment, obtained after heating bonding 50h~100h
Microcontroller chip takes out microcontroller chip, takes down adhesive tape, is smeared at the gap of the side of microcontroller chip with glue
It is stand-by to close microcontroller chip.
In step 3 gap closing of the side of microcontroller chip can also by by PDMS matrix and curing agent according to mass ratio
It is applied at the gap of microcontroller chip side, is placed in 80 DEG C~100 DEG C baking ovens after 8~15:1 mixing, heating bonding 50h~
100h makes its secure bond.
The operating method of the array chip of cell capture and the culture of tumour ball of the invention, as shown in figure 4, concrete operations
Include the following steps:
Step 1, micro-fluidic chip uses the preceding hydrophily for first increasing the surface PDMS using ultraviolet irradiation 2h.Then it pours into
Medicinal alcohol carries out disinfection, and finally pours into surfactant F127 so that cell can not be attached to the surface PDMS to keep cell outstanding
Floating life length.
Under certain flow rate, cell suspension is entered into multiple micro- knots by the cell suspending liquid portal perfusion of unicellular sorting layer
In structure, for cell suspending liquid by micro-structure, the cell of maxicell or morphotropism difference passes through the first hole, is blocked and catches by the second hole
It obtains, cellule or the strong cell of morphotropism pass through the first hole and the second hole without captured, to proceed to below
In the screening tank of the smaller micro-structure of hole；
Step 2, cytotostatic to be captured is distributed in capturing unit, by microcontroller chip overturning, it is static make it is captured thin
Born of the same parents are entered from unicellular trapping layer into the culture tank of cell culture layer in gravity, then will be with slug flow speed by cell
Culture solution, clinical medicine, detection reagent are irrigated by cell suspending liquid entrance, carry out cell culture.
Embodiment 1
The structure snd size of applicant of the present invention's lab design microcontroller chip, as shown in Fig. 2, A and B are respectively indicated
The width and length of micro-structure in unicellular sorting layer, value is 120 μm and 200 μm respectively, and the width of middle part contraction place is 90
μm, C indicates the distance between microarray, and value is 50 μm, D and E represent first hole between two neighboring micro-structure and
As in second pore-size same row capturing unit, but its size successively subtracts in the capturing unit of different microarrays
2 μm few (size of first hole is 16 to 8 μm i.e. in the embodiment, and the size of second hole is 14 to 6 μm), such as Fig. 3
Shown, the screening slot length formed between two neighboring micro-structure is 100 μm, and the width of intermediate the widest part is 40 μm, unicellular
The height L1 for sorting the screening tank of layer 1 is 18 μm, and the culture tank length and width of cell culture layer 2 are 80 μm, and height L2 is 70 μm, this
The micro-structure of a little culture tanks and unicellular sorting layer corresponds.
The preparation process of microcontroller chip is as follows:
Step 1, unicellular sorting layer is prepared:
5:1 mixing PDMS matrix and curing agent in mass ratio, meanwhile, using trim,ethylchlorosilane (Trimethylchlorosilan,
TMCS) the unicellular sorting layer mold 5min of steam treatment, PDMS matrix and curing agent mixture are poured at trim,ethylchlorosilane
On unicellular sorting layer mold after reason, vacuumizes degassing and be placed in 80 DEG C of baking ovens the 0.5h that is heating and curing, after solidification
PDMS is removed from mold cut punching preparation, cleans up spare, wherein curing agent buying is public from Dow corning
Department, number: SYLGARD 184.
Step 2, cell culture layer is prepared:
According to mass ratio 15:1 mixing PDMS matrix and curing agent, while using TMCS steam treatment cell culture layer mold
PDMS matrix and curing agent are poured into TMCS treated in cell culture layer mold, vacuumized in 80 DEG C of baking ovens of degassing by 5min
Be heating and curing 1h, and the PDMS after solidification is removed from mold and is cut, and punching cleans up spare；
Step 3, unicellular sorting layer or cell culture layer are placed in the visual field of inverted fluorescence microscope, it then will be another
It is fixed and is placed it in 80 DEG C of baking ovens with adhesive tape after one layer of alignment, obtain microcontroller chip after heating bonding 50h, it will be micro-
It controls chip to take out, takes down adhesive tape, close microcontroller chip along smear with glue at the gap of the side of microcontroller chip
?.
The operating method of the culture cell of the array chip of cell capture and the culture of tumour ball is as shown in figure 4, use
Tumour cell is that human glioma cell (U251) is obtained from Shanghai Research Institute, the Chinese Academy of Sciences, two different tumour cells
(Normal U251 cell and Induced U251 cell) is respectively the side using DMEM/F12+10%FBS (fetal calf serum)
The plain edition U251 (Normal U251 cell) that method is cultivated, and using addition B27 (1 ×), recombined human in DMEM/F12
Epidermal growth factor (20ng/mL), basic fibroblast growth factor (20ng/mL), LIF ELISA (10ng/
ML the induction type U251 cell (Induced U251)) cultivated；
It is 40 μ l/min in driving flow velocity, it will be in cell sample (Normal U251 cell and Induced U251 cell)
Under conditions of cell density is 10 000cells/mL, cell sample is poured by the cell suspending liquid entrance of unicellular sorting layer
In microcontroller chip, 20s makes cell sample by equally distributed pipeline, and perfusion enters in multiple micro-structures respectively；In 100 μ l/
By micro structure array so that cell sample is unicellular according to different sizes and morphotropism progress by micro structure array under min
Capture and separation.Perhaps the cell of morphotropism difference can be blocked and small or change by front end gap by rear end gap greatly
The strong cell of shape can be by two gaps without captured, to proceed in the smaller micro structure array of following slot
It goes；Then, cytotostatic to be captured is distributed in micro structure array, by microcontroller chip overturning, static makes captured cell
By gravity, the micro- culture tank entered from unicellular trapping layer to cell culture layer forms tumour ball, then with one
Cell culture fluid, clinical medicine, detection reagent etc. are irrigated by 10 μ l/min of slug flow speed by entrance.
As shown in figure 5, cell concentration is that 10000cells/mL is irrigated latter two in cell sample in 100 μ l/min
Point of different size and morphotropism tumour cell (Normal U251 cell and Induced U251 cell) in micro structure array
Cloth, as can be seen from the figure the distribution of two kinds of cells is significantly different.
As shown in fig. 6, cell concentration is that 10000cells/mL is irrigated latter two in cell sample in 100 μ l/min
Point of different size and morphotropism tumour cell (Normal U251 cell and Induced U251 cell) in micro structure array
Cloth, as can be seen from the figure the distribution of Normal U251 cell (A) and Induced U251 cell (B) is significantly different.
As shown in fig. 7, the tumour cell different to Normal U251 cell (A) and Induced U251 cell (B) is micro-
It is distributed in array of structures after stablizing, cell culture fluid (DMEM/F12+FBS 10%) is irrigated culture carefully with 10 μ l/min
Born of the same parents are observed and are taken pictures to certain time (0 day, 5 days and 10 days).From figure from the growth that can be seen that with incubation time,
Gradually proliferation forms tumour ball to single tumor cell in micro- culture tank, cultivates by the 10th day, virtually all formation tumour ball.
As shown in Figure 8 and Figure 9, from result it can be seen that the tumor formation rate of Induced U251 cell is higher than Normal
U251 cell；See that single Induced U251 cell is formed by tumour ball and is greater than single Normal U251 cell.
As shown in Figure 10, show the balling ratio of cellule or/and the big cell of morphotropism by comparing and form tumour ball
Size be above maxicell or/and the weak cell of morphotropism.
Embodiment 2
The structure snd size of applicant of the present invention's lab design microcontroller chip, as shown in Fig. 2, A and B are respectively indicated
The width and length of micro-structure in unicellular sorting layer, value is 120 μm and 250 μm respectively, and the width of middle part contraction place is 90
μm, C indicates the distance between microarray, and value is 50 μm, D and E represent first hole between two neighboring micro-structure and
As in second pore-size same row capturing unit, but its size successively subtracts in the capturing unit of different microarrays
2 μm few (size of first hole is 16 to 8 μm i.e. in the embodiment, and the size of second hole is 14 to 6 μm), such as Fig. 3
Shown, the screening slot length formed between two neighboring micro-structure is 120 μm, and the width of intermediate the widest part is 50 μm, unicellular
The height L1 for sorting the screening tank of layer 1 is 25 μm, and the culture tank length and width of cell culture layer 2 are 100 μm, and height L2 is 75 μm,
The micro-structure of these culture tanks and unicellular sorting layer corresponds.
Step 1, unicellular sorting layer is prepared:
5:1 mixing PDMS matrix and curing agent in mass ratio, meanwhile, use trim,ethylchlorosilane
The unicellular sorting layer mold 7min of (Trimethylchlorosilan, TMCS) steam treatment, PDMS matrix and curing agent are mixed
It closes object to pour into trim,ethylchlorosilane treated on unicellular sorting layer mold, vacuumizes degassing and be placed in 90 DEG C of baking ovens and heat
Solidify 0.7h, the PDMS after solidification is removed from mold and cut punching preparation, is cleaned up spare, wherein PDMS base
Matter and curing agent buying are numbered: SYLGARD 184 from Dow corning company.
Step 2, cell culture layer is prepared:
According to mass ratio 15:1 mixing PDMS matrix and curing agent, while using TMCS steam treatment cell culture layer mold
PDMS matrix and curing agent are poured into TMCS treated in cell culture layer mold, vacuumized in 90 DEG C of baking ovens of degassing by 5min
Be heating and curing 1.5h, and the PDMS after solidification is removed from mold and is cut, and punching cleans up spare；
Step 3, unicellular sorting layer or cell culture layer are placed in the visual field of inverted fluorescence microscope, it then will be another
It is fixed and is placed it in 90 DEG C of baking ovens with adhesive tape after one layer of alignment, obtain microcontroller chip after heating bonding 80h, it will be micro-
It controls chip to take out, takes down adhesive tape, close microcontroller chip along smear with glue at the gap of the side of microcontroller chip
?.
Embodiment 3
The structure snd size of applicant of the present invention's lab design microcontroller chip, applicant of the present invention's lab design
The structure snd size of microcontroller chip, as shown in Fig. 2, A and B respectively indicate the width and length of micro-structure in unicellular sorting layer,
Value is 120 μm and 300 μm respectively, and the width of middle part contraction place is 90 μm, and C indicates the distance between microarray, and value is 50 μ
M, D and E are represented in first hole and second pore-size same row capturing unit between two neighboring micro-structure
, but its size successively reduces by 2 μm of (sizes of first hole i.e. in the embodiment in the capturing unit of different microarrays
It is 16 to 8 μm, the size of second hole is 14 to 6 μm), as shown in figure 3, the screening tank formed between two neighboring micro-structure
Length is 150 μm, and the width of intermediate the widest part is 60 μm, and the height L1 of the screening tank of unicellular sorting layer 1 is 28 μm, cell
The culture tank length and width of culture layer 2 are 120 μm, and height L2 is 100 μm, the micro-structure one of these culture tanks and unicellular sorting layer
One is corresponding.
Step 1, unicellular sorting layer is prepared:
5:1 mixing PDMS matrix and curing agent in mass ratio, meanwhile, use trim,ethylchlorosilane
The unicellular sorting layer mold 10min of (Trimethylchlorosilan, TMCS) steam treatment, by PDMS matrix and curing agent
Mixture pours into trim,ethylchlorosilane treated on unicellular sorting layer mold, vacuumizes degassing and is placed in 100 DEG C of baking ovens
Be heating and curing 1h, and the PDMS after solidification is removed from mold and cut punching preparation, is cleaned up spare, wherein PDMS
Matrix and curing agent buying are numbered: SYLGARD 184 from Dow corning company.
Step 2, cell culture layer is prepared:
According to mass ratio 15:1 mixing PDMS matrix and curing agent, while using TMCS steam treatment cell culture layer mold
PDMS matrix and curing agent are poured into TMCS treated in cell culture layer mold, vacuumized in 100 DEG C of baking ovens of degassing by 5min
Be heating and curing 2h, and the PDMS after solidification is removed from mold and is cut, and punching cleans up spare；
Step 3, unicellular sorting layer or cell culture layer are placed in the visual field of inverted fluorescence microscope, it then will be another
It is fixed and is placed it in 100 DEG C of baking ovens with adhesive tape after one layer of alignment, obtain microcontroller chip after heating bonding 100h, it will
Microcontroller chip takes out, and takes down adhesive tape, closes micro-control core along smear with glue at the gap of the side of microcontroller chip
Piece.
Embodiment 4
The structure snd size of applicant of the present invention's lab design microcontroller chip, applicant of the present invention's lab design
The structure snd size of microcontroller chip, as shown in Fig. 2, A and B respectively indicate the width and length of micro-structure in unicellular sorting layer,
Value is 120 μm and 300 μm respectively, and the width of middle part contraction place is 90 μm, and C indicates the distance between microarray, and value is 50 μ
M, D and E are represented in first hole and second pore-size same row capturing unit between two neighboring micro-structure
, but its size successively reduces by 2 μm of (sizes of first hole i.e. in the embodiment in the capturing unit of different microarrays
It is 16 to 8 μm, the size of second hole is 14 to 6 μm), as shown in figure 3, the screening tank formed between two neighboring micro-structure
Length is 150 μm, and the width of intermediate the widest part is 60 μm, and the height L1 of the screening tank of unicellular sorting layer 1 is 28 μm, cell
The culture tank length and width of culture layer 2 are 120 μm, and height L2 is 100 μm, the micro-structure one of these culture tanks and unicellular sorting layer
One is corresponding.
Step 1, unicellular sorting layer is prepared:
5:1 mixing PDMS matrix and curing agent in mass ratio, meanwhile, use trim,ethylchlorosilane
The unicellular sorting layer mold 10min of (Trimethylchlorosilan, TMCS) steam treatment, by PDMS matrix and curing agent
Mixture pours into trim,ethylchlorosilane treated on unicellular sorting layer mold, vacuumizes degassing and is placed in 100 DEG C of baking ovens
Be heating and curing 1h, and the PDMS after solidification is removed from mold and cut punching preparation, is cleaned up spare, wherein PDMS
Matrix and curing agent buying are numbered: SYLGARD 184 from Dow corning company.
Step 2, cell culture layer is prepared:
According to mass ratio 15:1 mixing PDMS matrix and curing agent, while using TMCS steam treatment cell culture layer mold
PDMS matrix and curing agent are poured into TMCS treated in cell culture layer mold, vacuumized in 100 DEG C of baking ovens of degassing by 5min
Be heating and curing 2h, and the PDMS after solidification is removed from mold and is cut, and punching cleans up spare；
Step 3, unicellular sorting layer or cell culture layer are placed in the visual field of inverted fluorescence microscope, it then will be another
It is fixed and is placed it in 100 DEG C of baking ovens with adhesive tape after one layer of alignment, obtain microcontroller chip after heating bonding 100h, it will
Microcontroller chip takes out, and takes down adhesive tape, is applied to microcontroller chip after PDMS matrix and curing agent are mixed according to mass ratio 8:1
It at the gap of side, is placed in 80 DEG C of baking ovens, heating bonding 50h makes its secure bond.
Embodiment 5
The structure snd size of applicant of the present invention's lab design microcontroller chip, applicant of the present invention's lab design
The structure snd size of microcontroller chip, as shown in Fig. 2, A and B respectively indicate the width and length of micro-structure in unicellular sorting layer,
Value is 120 μm and 200 μm respectively, and the width of middle part contraction place is 90 μm, and C indicates the distance between microarray, and value is 50 μ
M, D and E are represented in first hole and second pore-size same row capturing unit between two neighboring micro-structure
, but its size successively reduces by 2 μm of (sizes of first hole i.e. in the embodiment in the capturing unit of different microarrays
It is 16 to 8 μm, the size of second hole is 14 to 6 μm), as shown in figure 3, the screening tank formed between two neighboring micro-structure
Length is 150 μm, and the width of intermediate the widest part is 60 μm, and the height L1 of the screening tank of unicellular sorting layer 1 is 25 μm, cell
The culture tank length and width of culture layer 2 are 120 μm, and height L2 is 75 μm, the micro-structure one of these culture tanks and unicellular sorting layer
One is corresponding.
Step 1, unicellular sorting layer is prepared:
5:1 mixing PDMS matrix and curing agent in mass ratio, meanwhile, use trim,ethylchlorosilane
The unicellular sorting layer mold 10min of (Trimethylchlorosilan, TMCS) steam treatment, by PDMS matrix and curing agent
Mixture pours into trim,ethylchlorosilane treated on unicellular sorting layer mold, vacuumizes degassing and is placed in 100 DEG C of baking ovens
Be heating and curing 1h, and the PDMS after solidification is removed from mold and cut punching preparation, is cleaned up spare, wherein PDMS
Matrix and curing agent buying are numbered: SYLGARD 184 from Dow corning company.
Step 2, cell culture layer is prepared:
According to mass ratio 15:1 mixing PDMS matrix and curing agent, while using TMCS steam treatment cell culture layer mold
PDMS matrix and curing agent are poured into TMCS treated in cell culture layer mold, vacuumized in 100 DEG C of baking ovens of degassing by 5min
Be heating and curing 2h, and the PDMS after solidification is removed from mold and is cut, and punching cleans up spare；
Step 3, unicellular sorting layer or cell culture layer are placed in the visual field of inverted fluorescence microscope, it then will be another
It is fixed and is placed it in 100 DEG C of baking ovens with adhesive tape after one layer of alignment, obtain microcontroller chip after heating bonding 100h, it will
Microcontroller chip takes out, and takes down adhesive tape, is applied to micro-control core after PDMS matrix and curing agent are mixed according to mass ratio 10:1
It at the gap of piece side, is placed in 90 DEG C of baking ovens, heating bonding 100h makes its secure bond.
Embodiment 6
The structure snd size of applicant of the present invention's lab design microcontroller chip, applicant of the present invention's lab design
The structure snd size of microcontroller chip, as shown in Fig. 2, A and B respectively indicate the width and length of micro-structure in unicellular sorting layer,
Value is 120 μm and 200 μm respectively, and the width of middle part contraction place is 90 μm, and C indicates the distance between microarray, and value is 50 μ
M, D and E are represented in first hole and second pore-size same row capturing unit between two neighboring micro-structure
, but its size successively reduces by 2 μm of (sizes of first hole i.e. in the embodiment in the capturing unit of different microarrays
It is 16 to 8 μm, the size of second hole is 14 to 6 μm), as shown in figure 3, the screening tank formed between two neighboring micro-structure
Length is 120 μm, and the width of intermediate the widest part is 50 μm, and the height L1 of the screening tank of unicellular sorting layer 1 is 28 μm, cell
The culture tank length and width of culture layer 2 are 100 μm, and height L2 is 100 μm, the micro-structure one of these culture tanks and unicellular sorting layer
One is corresponding.
Step 1, unicellular sorting layer is prepared:
5:1 mixing PDMS matrix and curing agent in mass ratio, meanwhile, use trim,ethylchlorosilane
The unicellular sorting layer mold 10min of (Trimethylchlorosilan, TMCS) steam treatment, by PDMS matrix and curing agent
Mixture pours into trim,ethylchlorosilane treated on unicellular sorting layer mold, vacuumizes degassing and is placed in 100 DEG C of baking ovens
Be heating and curing 1h, and the PDMS after solidification is removed from mold and cut punching preparation, is cleaned up spare, wherein PDMS
Matrix and curing agent buying are numbered: SYLGARD 184 from Dow corning company.
Step 2, cell culture layer is prepared:
According to mass ratio 15:1 mixing PDMS matrix and curing agent, while using TMCS steam treatment cell culture layer mold
PDMS matrix and curing agent are poured into TMCS treated in cell culture layer mold, vacuumized in 100 DEG C of baking ovens of degassing by 5min
Be heating and curing 2h, and the PDMS after solidification is removed from mold and is cut, and punching cleans up spare；
Step 3, unicellular sorting layer or cell culture layer are placed in the visual field of inverted fluorescence microscope, it then will be another
It is fixed and is placed it in 100 DEG C of baking ovens with adhesive tape after one layer of alignment, obtain microcontroller chip after heating bonding 100h, it will
Microcontroller chip takes out, and takes down adhesive tape, is applied to micro-control core after PDMS matrix and curing agent are mixed according to mass ratio 15:1
It at the gap of piece side, is placed in 100 DEG C of baking ovens, heating bonding 85h makes its secure bond.
Claims (10)
1. the array chip of cell capture and the culture of tumour ball, which is characterized in that trained including unicellular sorting layer (1), cell
It supports layer (2) and is embedded in the unicellular pipeline (3) sorted in layer (1), be bonded with cell on the unicellular sorting layer (1)
Culture layer (2), the unicellular sorting layer (1) include at least one set of cell capture array (4), a cell suspending liquid entrance and
The outlet of multiple cell suspending liquids, cell capture array (4) described in every group by pipeline (3) be connected to the cell suspending liquid entrance and
Multiple cell suspending liquids outlets, cell capture array (4) described in every group include at least three column microarrays (5), described in each column
Microarray (5) includes multiple unicellular capturing units (7) of laid out in parallel, and each unicellular capturing unit (7) includes two H-types
Micro-structure (8) forms the screening tank (6) for being used for cell screening between two H-type micro-structures (8), the screening tank (6)
Both ends are respectively formed the first hole (10) and the second hole (11) for capturing cell；
Interval is uniformly provided with multiple mutually independent culture tanks (9) for cultivating cell, institute on the cell culture layer (2)
It states culture tank (9) to be arranged in array, each culture tank (9) and the unicellular capturing unit (7) correspond.
2. the array chip of cell capture as described in claim 1 and the culture of tumour ball, which is characterized in that adjacent two column institute
The spacing for stating microarray (5) is 50 μm.
3. the array chip of cell capture as described in claim 1 and the culture of tumour ball, which is characterized in that each H
The section of type micro-structure (8) is in the rectangle of middle part radial contraction, and the length of each H-type micro-structure (8) is 200 μm~300 μ
M, the rectangle both ends width of each H-type micro-structure are 120 μm, and the width of middle part contraction place is 90 μm.
4. the array chip of cell capture as claimed in claim 3 and the culture of tumour ball, which is characterized in that the screening tank
(6) intermediate wide both sides are narrow, and the length of the screening tank (6) is 100 μm~150 μm, and the width of described intermediate the widest part is 40 μm
~60 μm, the height of the screening tank (6) is identical as the height of the pipeline (3), and the height of the screening tank (6) is 18 μm~
28μm。
5. the array chip of cell capture as described in claim 1 and the culture of tumour ball, which is characterized in that described unicellular
The two sides that sorting layer (1) is located at the cell capture array are provided with multiple microtrabeculaes, the distance between each microtrabeculae for 25 μm~
50 μm, for preventing the pipeline from collapsing.
6. the array chip of cell capture as described in claim 1 and the culture of tumour ball, which is characterized in that each training
The length for supporting slot (9) is 80 μm~120 μm, and it is highly 70 μm~100 μm that width, which is 80 μm~120 μm,.
7. the array chip of cell capture as described in claim 1 and the culture of tumour ball, which is characterized in that be located at same row
The size of the first hole (10) of the microarray (5) and the constant dimension of the second hole (11) are constant, are located at adjacent two column institute
The size for stating the first hole (10) of microarray (5) differs 2 μm, the second hole (11) for arranging the microarray positioned at adjacent two
Size differs 2 μm.
8. such as the preparation method of the described in any item cell captures of claim 1-7 and the array chip of tumour ball culture,
It is characterized in that, specifically comprises the following steps:
Step 1, unicellular sorting layer is prepared:
5:1 mixing PDMS matrix and curing agent in mass ratio, meanwhile, use the unicellular sorting layer of trim,ethylchlorosilane steam treatment
PDMS matrix and curing agent mixture are poured into trim,ethylchlorosilane treated unicellular sorting layer by mold 5min~10min
It on mold, vacuumizes degassing and is placed in 80 DEG C~100 DEG C baking ovens the 0.5h~1h that is heating and curing, by the PDMS after solidification from mold
Upper removing, and cut as required, and punching prepares screening tank, it cleans up spare；
Step 2, cell culture layer is prepared:
According to mass ratio 15:1 mixing PDMS matrix and curing agent, while using TMCS steam treatment cell culture layer mold
PDMS matrix and curing agent are poured into TMCS treated in cell culture layer mold, vacuumizes 80 DEG C~100 DEG C of degassing by 5min
Be heating and curing 1h~2h in baking oven, and the PDMS after solidification is removed from mold, and is cut as required, and punches preparation
Culture tank cleans up spare；
Step 3, the unicellular sorting layer that step 1 obtains and the cell culture layer that step 2 obtains are bonded bonding:
Unicellular sorting layer or cell culture layer are placed in the visual field of inverted fluorescence microscope, it then will be after another layer of alignment
It is fixed and is placed it in 80 DEG C~100 DEG C baking ovens with adhesive tape, obtain microcontroller chip after heating bonding 50h~100h, it will
Microcontroller chip takes out, and takes down adhesive tape, closes micro-control core along smear with glue at the gap of the side of microcontroller chip
Piece is stand-by.
9. the preparation method of cell capture as claimed in claim 8 and the array chip of tumour ball culture, which is characterized in that
In the step 3 gap closing of the side of microcontroller chip can also by by PDMS matrix and curing agent according to mass ratio 8~
It is applied at the gap of microcontroller chip side, is placed in 80 DEG C~100 DEG C baking ovens after 15:1 mixing, heating bonding 50h~100h
Make its secure bond.
10. such as the operating method of the described in any item cell captures of claim 1-7 and the array chip of tumour ball culture,
It is characterized in that, concrete operations include the following steps:
Step 1, under certain flow rate, by cell suspension by it is unicellular sorting layer cell suspending liquid portal perfusion enter it is multiple micro-
In structure, for cell suspending liquid by micro-structure, the cell of maxicell or morphotropism difference passes through the first hole, is blocked by the second hole
Capture, cellule or the strong cell of morphotropism pass through the first hole and the second hole without captured, thus after proceeding to
In the screening tank of the smaller micro-structure of face gap；
Step 2, cytotostatic to be captured is distributed in capturing unit, by microcontroller chip overturning, static captured cell is made to exist
Gravity is entered from unicellular trapping layer into the culture tank of cell culture layer, then will be with slug flow speed by cell culture
Liquid, clinical medicine, detection reagent are irrigated by cell suspending liquid entrance, carry out cell culture.
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