CN110452869A - A kind of preparation method and application of the high-throughput micro-array chip for forming embryoid body - Google Patents
A kind of preparation method and application of the high-throughput micro-array chip for forming embryoid body Download PDFInfo
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Abstract
The present invention provides the preparation method and application of a kind of high-throughput micro-array chip for forming embryoid body, and this method is prepared for the PDMS polymer chip with the array micro-column structure of micro-meter scale using soft etching technology.The shape, size, homogeneity of the embryoid body of source of human stem cell are controlled by optimizing height and the spacing of micro-column structure.This method high-throughput can form embryoid body, and can be proliferated with in-situ dynamic observation embryoid body, the overall process of development.The chip have can effectively remove apoptotic cell and cell fragment, guarantee the characteristics of nutrition supply and signal between embryoid body transmit, it overcomes traditional embryoid body formation and suspends and cultivate the shortcomings that substep is realized, with simplified embryoid body operating procedure, advantage high-throughput, being formed in situ and break up, it, can be integrated with other technologies without special instrument and reagent.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of preparation side of the high-throughput micro-array chip for forming embryoid body
Method and application.
Background technique
Inductivity pluripotent stem cell and embryonic stem cell have the ability of self-renewing and Multidirectional Differentiation, in human development
Research is learned, disease model building, organizational project, cell therapy, the fields such as drug screening have important application value.Utilize this
Cell carries out in allelotaxis and cell induction atomization, and it is crucial step that embryoid body, which is formed,.Conventional method is using outstanding
Drop method and the pitting of commercialization form embryoid body.Sessile drop method have it is cumbersome, embryoid body formation efficiency is low, nutrition supply
The disadvantages of shortage.The pitting structure of commercialization is after embryoid body is formed, since the structure of pitting makes dead cell and cell broken
Piece is difficult to remove, these cell fragments will affect the induction efficiency of embryoid body.Therefore, embryoid body is formed using these methods
Afterwards, need for embryoid body to be transferred to continued growth and subsequent induction differentiation in the low culture plate sticked, so that experimental procedure
It is cumbersome.In addition, embryoid body is easy to receive the shearing force stimulation of larger fluid in transfer process, interferes the activity of cell and divide
The effect of change.
The material for preparing the chip of high throughput array micro-column structure is dimethyl silicone polymer
(polydimethylsiloxane, PDMS), PDMS are most materials in current micro Process and micro-fluidic field, are had saturating
The advantages such as bright, ventilative, inertia is good, hydrophobicity, easily molded, cell compatibility is good.At present it is reported in the literature prepare cell ball or
The method of embryoid body is all based on the pitting method of PDMS or geopolymer gel material, and further research needs metastatic cells microballoon
Stick culture systems, complex steps to newly low with embryoid body；A degree of damage will cause to stem cell in transfer process,
Cell debris does not allow the disadvantages of easy-clear.The method for currently forming embryoid body is the pitting chip and sessile drop method of microflow control technique,
Major defect is that dead cell fragment cannot be removed in time, needs to be transferred to and continues to cultivate in new low adherency culture dish and divide
To change, transfer process has different degrees of damage and loss to embryoid body, and cannot achieve embryoid body in situ and formed and cultivated,
Cannot the position observation same embryoid body in real time Development And Differentiation process.
In micro-column structure currently with micro-fluidic chip preparation, mainly based on miniature scale, opened up for studying surface
Structure is flutterred on unicellular ethological influence, there is no and form micro- culture space progress embryoid body formation and original position using larger microtrabeculae
Develop the application of research.
Therefore, present invention is generally directed to above-mentioned embryoid bodies to form limitation, prepare a kind of novel high-throughput embryoid bodily form
Application study at the chip with growth in situ culture, for stem cell field.
Summary of the invention
In view of the above-mentioned problems, a kind of preparation method of the high-throughput micro-array chip for forming embryoid body of the purpose of the present invention with
Using.
A kind of preparation method of the high-throughput micro-array chip for forming embryoid body, the specific steps are as follows:
(1) preparation of chip: preparing SU-8 template using soft etching technology, the sunk structure containing high-throughput cylinder,
Bottom is the curved surface of recess, low to template progress to stick modification, it is ensured that SU-8 is easily peeled off with PDMS polymer；
(2) the PDMS polymer chip of high throughput array micro-column structure is made: by PDMS polymer and initiator according to body
Product is mixed than 10~14:1, is poured into SU-8 template, froth in vacuum, 80 DEG C of 1~2h that are heating and curing, and room temperature removes SU-8 mould
Plate obtains the array micro-column structure PDMS chip of high-throughput constant spacing；
(3) the open culturing pond for forming the limitation of enclosure wall spline structure with PDMS module in PDMS chip perimeter, obtains high pass
Amount forms the micro-array chip of embryoid body.
Chip form is round or rectangular.
The template is low to stick modification specifically: uses low adhesion process reagent silanization treatment 10-15 minutes, 80 DEG C
Baking 1~2 hour, Temperature fall.
The low adhesion process reagent is trim,ethylchlorosilane or perfluor silane etc.；
The depth of the template recess pitting is between 500-1000 microns, and spacing is between 30-100 microns.
The microtrabeculae top is the curved-surface structure of protrusion, and diameter is at 500-1000 microns, and spacing is 30-100 microns, microtrabeculae
Spacing is identical, and wherein microtrabeculae size and microtrabeculae spacing are not limited to the range.
The initiator are as follows:184silicone elastomer curing agent.
The open culturing pond that the PDMS module is formed, feature are the shape according to chip, prepare corresponding hollow structure,
It is rectangular such as annular.By the structure plasma treatment or PDMS glue and chip sealing, purpose limits cell culture medium and overflows
Chip structure；
The high throughput refers to that the chip structure can infinitely amplify or compartmentalization.
A kind of application of the high-throughput micro-array chip for forming embryoid body, forms embryoid body simultaneously using said chip high throughput
Carry out the culture of embryoid body growth in situ, specific steps are as follows:
(1) chip sterilization: the micro-array chip that above-mentioned embryoid body is formed in situ is with 1~3 point of oxygen plasma treatment
Deionized water is added in clock；120~125 DEG C high pressure sterilization 20~30 minutes；Chip is placed in 4 DEG C of preservations in sterile culture dish
Or it is cooled to room temperature direct use.
(2) embryoid body is formed: taking out chip, mTeSR1 culture medium is added, is constantly blown and beaten with pipettor, removes small intercolumniation
Bubble makes microtrabeculae gap full of culture medium；By people's inductivity pluripotent stem cell (hiPSCs) with dispase digestion at slender
Born of the same parents, by density 5 × 106-10×106A hiPSCs/cm2Cell inoculation is added 1.5~2ml's into chip described in (1)
MTeSR1 culture medium, and 10~20 μM of Y27632 is added.Static gas wave refrigerator 24~48 hours, form embryoid body of the same size；
The control of embryoid body size, (5-10) x10 are realized according to cell inoculation quantity6/cm2150- may be implemented in the cell density of range
The cell ball of 300 micron diameters；
(3) embryoid body growth in situ: the 24-48 hour that embryoid body is formed replaces the fresh mTeSR1 without Y27632
Culture medium continues to cultivate, and removes dead cell and cell fragment, the growth of home position observation embryoid body and differentiation；
(4) being formed by embryoid body can be used for being sliced, tissue staining；
A kind of application of the high-throughput micro-array chip for forming embryoid body, application are not limited to people's inductivity pluripotency
Stem cell is equally applicable to other stem cells, embryonic stem cell including humans and animals (embryonic stem cells,
) and the inductivity pluripotent stem cell of animal origin ESCs.
The stem cell micro-assembly robot of different shapes is formed, and the tune of the density of cell and microtrabeculae spacing is mainly utilized
Section controls the effect of iuntercellular power, reaches balance and form micro-assembly robot of different shapes.
The effect of the Y27632 mainly reduces Apoptosis；
Before the removal cell debris method mainly utilizes replacement culture medium, swing chip gently is not influencing carefully
In the case where born of the same parents' ball, so that cell fragment floats, inclination chip to side gently siphons away culture medium, is gently added and trains in side
Base is supported, level is slowly placed, is placed in 37 ° of incubator cultures.
The home position observation growth and development positions each embryoid body by mark microtrabeculae sequence, every by micro- sem observation
The growing state of the embryoid body of a positioning.
The embryoid body slice, tissue staining refer to that the embryoid body on chip positions under the microscope, utilize pipettor hand
It is dynamic to take out, carry out immuning tissue's slice dyeing.
Using the embryoid body formed on chip, the embryoid body for embryonic stem cell is formed and grown cultures.Due to embryo
Stem cell is similar on characteristics of cell biology and function to inductivity pluripotent stem cell, and application range of the invention is equally suitable
It is formed for the embryoid body of embryonic stem cell and other inductivity pluripotent stem cells and class loading breaks up.
The micro-array chip method with culture is formed in situ in the high-throughput idiosome that the present invention establishes, and principle is enclosed using microtrabeculae
At the low adhesion characteristics of cell caused by the hydrophobicity of the identical three-dimensional space in four directions and PDMS material, by the ruler for adjusting microtrabeculae
Very little and spacing controls the shape and curvature in micro- space, and it is uniform that adjusting people's inductivity pluripotent stem cell quantity can form size
Ball shape structure.To guarantee nucleus culture medium in micro-column structure, array micro-column structure chip perimeter is impaled with PDMS module
Form the open culturing pond of limitation.PDMS chip hydrophilic treated first, using oxygen plasma treatment 1~3 minute, then by array
Microtrabeculae chip is immersed in deionized water, 120 °~125 ° high pressure sterilization 20~30 minutes, guarantee chip hydrophily.In embryoid body
In forming process, to prevent cell adhesion, restore hydrophobicity within chip static 3 days or more.In inoculating cell process, deionization is siphoned away
Water is added normal incubation medium replacement, removes bubble removing by piping and druming, can direct inoculating cell after small intercolumniation is hydraulically full.By core
Piece is placed in the culture dish of suitable size, regular growth culture.The chip repeated multiple times can use, individual chip can repeat
Using 30~50 times, the chip after use is cleaned with sterile PBS to remove cell debris, is immersed in sterile PBS solution, 4 °
Refrigerator Preservation in sterile condition replaces cell culture medium when reusing.
Design chips array micro-column structure of the present invention is compared to the pitting structure of recess, and cell debris is during routinely changing liquid
It can largely be removed, reduce the influence to the activity and function of embryoid body.Further, since the restriction effect of microtrabeculae and thin
The biomechanical characterization of intercellular so that each embryoid body can with fixation in situ in micro-structure, will not because replacement culture medium and
The case where being moved to other positions, observing the development of single embryoid body convenient for long-term dynamics, while decreasing and being sent out between embryoid body
Raw adhesion.
Main application range of the invention: being the formation and induction differentiation in situ of embryoid body, the development of simulation human organ and
Function is organoid (organoids) formation of people's inductivity pluripotent stem cell, human organ auxology research, disease mould
Type building, medicament research and development, toxicity assessment and cell/tissue/organ replacement therapy provide new controllable technological means.
Creativeness of the invention is: by adjusting microtrabeculae size and microtrabeculae spacing and cell density, height may be implemented
The embryoid body of the size adjustable of flux.
The present invention has the advantages that high-throughput form embryoid body and can carry out induction differentiation in situ, be conducive to sight in situ
The dynamic process of embryoid body development is examined, guarantees nutrition supply and signal transmitting between embryoid body, it is thin apoptosis can be effectively removed
Born of the same parents and cell fragment.The shortcomings that culture induction differentiation substep that formed and suspended the method overcome traditional embryoid body is realized, has
Simplify embryoid body operating procedure, advantage high-throughput, being formed in situ and cultivate can be with other without special instrument and reagent
Integration ofTechnology.
Detailed description of the invention
Fig. 1 is the array micro-column structure that high-throughput embryoid body forms with directly induction differentiation in situ in specific embodiment 1
PDMS polymer chip preparation process；Wherein 1 is the exposure mask with figure, and 2 be SU-8, and 3 be substrate of glass, and 4 be uv-exposure
Process, 5 be casting PDMS (match (10~14) with initiator: 1 mixes), and 6 be the PDMS chip of cured band structure.
Fig. 2 is bowing for the PDMS polymer chip for the array micro-column structure that high-throughput embryoid body is formed in specific embodiment 1
View and sectional view；
Fig. 3 is in Fig. 24 enlarged drawing, for describing the specific size of micro-structure；
In Fig. 2, Fig. 3,1 is micro-column structure, and 2 be microtrabeculae spacing, and 3 be chip periphery dam structure, and 4 be that there are four micro-column structures
The embryoid body forming region surrounded；5 be the diameter of micro-column structure, and length is between 800 microns, and 6 be microtrabeculae spacing, and 7 be embryoid
Body forming region.
Fig. 4 is the formation and grown cultures of the embryoid body in people's inductivity pluripotent stem cell source in specific embodiment 2,
In 1 be microtrabeculae, 2 be embryoid body, and 3 be microtrabeculae spacing.
Specific embodiment
Above scheme is described further below in conjunction with specific embodiment.It should be understood that these embodiments are for illustrating
The present invention and be not limited to the scope of the present invention.Implementation condition used in the examples can be done according to the condition of specific producer into one
Successive step, the implementation condition being not specified are usually the condition in routine experiment.
Agents useful for same of the present invention is commercially available.
Embodiment 1
The preparation of array micro-column structure PDMS polymer chip
SU-8 polymer template is made using optical etching technology, it is small that template contains the circle that diameter is 500~800 microns
Hole.Preparation process is as shown in Figure 1.Wherein 1 is the exposure mask with figure, and 2 be SU-8, and 3 be substrate of glass, and 4 be uv-exposure mistake
Journey, 5 be casting PDMS (match (10~14) with initiator: 1 mixes), and 6 be the PDMS chip of cured band structure.Chip system
Standby process is as follows: the SU-8 polymer template that will be prepared uses silanization treatment 15 minutes, be put into heating 1 in 80 degree of baking ovens~
2h, Temperature fall.PDMS ((10~14): 1 mixing) is poured into SU-8 template, is added in 80 degree of baking ovens after froth in vacuum
Heat 40~60 minutes, after cooling, by cured PDMS lift-off stencil.In blank glass on piece according to the speed of 3000 revolution per seconds
One layer of PDMS (20:1) is got rid of, by structureless PDMS block, dips the PDMS (20:1) on sheet glass, then sealing-in is structured
Around PDMS chip, array micro-column structure is impaled to the open culturing pond to form limitation, is put in heating 60 minutes in 80 degree of baking ovens.
Chip structure is as shown in Fig. 2, 1 be wherein micro-column structure, 2 be microtrabeculae spacing, and 3 be chip periphery dam structure, is used to
Cell and culture medium are limited, 4 be the embryoid body forming region surrounded there are four micro-column structure.Chip structure enlarged drawing 3,5 is micro-
The diameter of rod structure, 800 microns of length, 6 be microtrabeculae spacing, 30 microns of length；7 be embryoid body forming region, with microtrabeculae around
It is spaced identical, is conducive to cultivate the interregional fluid exchange area size and changes with micro-column structure and spacing.
Embodiment 2
Array micro-column structure PDMS polymer chip is formed for embryoid body
Chip fabrication process such as embodiment 1.Embryoid body is formed and growth in situ incubation step is as follows:
(1) chip sterilizes: the micro-array chip oxygen plasma treatment 1~3 with differentiation is formed in situ in above-mentioned embryoid body
Minute, deionized water is added；120~125 DEG C high pressure sterilization 20~30 minutes；Chip is taken out, mTeSR1 culture medium is added, no
It is disconnected to be blown and beaten with pipettor, the bubble of small intercolumniation is removed to promote cell settlement to depressed area；
(2) embryoid body is formed: by cell density 5 × 106-10×106/cm2A hiPSCs is inoculated into core described in (1)
In piece, the mTeSR1 culture medium of 1.5~2ml is added, and 10~20 μM of Y27632 is added, conventional static gas wave refrigerator 24~48 is small
When；The embryoid body size of formation is uniform, in 300 microns, does not connect each other.As shown in figure 4,1 is microtrabeculae, 2 are
Embryoid body, 3 be microtrabeculae spacing；Embryoid body incubation time is respectively 2 hours, 48 hours and 4 days.
Claims (2)
1. a kind of application of the high-throughput micro-array chip for forming embryoid body, it is characterised in that: utilize said chip high throughput shape
At embryoid body and carry out embryoid body growth in situ culture, specific steps are as follows:
(1) chip sterilization: the micro-array chip that above-mentioned embryoid body is formed in situ was added with oxygen plasma treatment 1~3 minute
Deionized water；120~125 DEG C high pressure sterilization 20~30 minutes；Chip is placed in sterile culture dish and saves or be down to room for 4 DEG C
It is directly used after temperature；
(2) embryoid body is formed: taking out chip, mTeSR1 culture medium is added, is constantly blown and beaten with pipettor, removes the gas of small intercolumniation
Bubble makes microtrabeculae gap full of culture medium；By people's inductivity pluripotent stem cell (hiPSCs) with dispase digestion at unicellular, general
Density is 5 × 106-10×106A hiPSCs/cm2The mTeSR1 of 1.5~2ml is added into chip described in (1) in cell inoculation
Culture medium, and 10~20 μM of Y27632 is added；Static gas wave refrigerator 24~48 hours, form embryoid body of the same size；According to thin
Born of the same parents are inoculated with the control that quantity realizes embryoid body size, (5-10) x106/cm2The cell density of range may be implemented 150-300 microns
The cell ball of diameter；
(3) embryoid body growth in situ: the 24-48 hour that embryoid body is formed replaces the fresh mTeSR1 culture medium without Y27632
Continue to cultivate, removes dead cell and cell fragment, the growth of home position observation embryoid body and differentiation；
(4) being formed by embryoid body can be used for being sliced, tissue staining.
2. a kind of application of the high-throughput micro-array chip for forming embryoid body, it is characterised in that: application is not limited to people and lures
The property led pluripotent stem cell is equally applicable to other stem cells, embryonic stem cell (the embryonic stem including humans and animals
Cells, ESCs) and animal origin inductivity pluripotent stem cell.
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| CN111254118A (en) * | 2018-11-30 | 2020-06-09 | 中国科学院大连化学物理研究所 | Brain-like tissue generation method of hiPSCs source |
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