CN109810895A - Open three dimensional cell cultivation chip and its technology of preparing based on contour microtrabeculae - Google Patents
Open three dimensional cell cultivation chip and its technology of preparing based on contour microtrabeculae Download PDFInfo
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- CN109810895A CN109810895A CN201910147892.0A CN201910147892A CN109810895A CN 109810895 A CN109810895 A CN 109810895A CN 201910147892 A CN201910147892 A CN 201910147892A CN 109810895 A CN109810895 A CN 109810895A
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- 238000004113 cell culture Methods 0.000 title claims abstract description 39
- 238000005516 engineering process Methods 0.000 title claims abstract description 23
- 239000000758 substrate Substances 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 31
- 230000008569 process Effects 0.000 claims abstract description 17
- 239000000463 material Substances 0.000 claims abstract description 14
- 238000009616 inductively coupled plasma Methods 0.000 claims abstract description 10
- 238000001259 photo etching Methods 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 238000001039 wet etching Methods 0.000 claims description 6
- 238000005530 etching Methods 0.000 claims description 5
- 239000003431 cross linking reagent Substances 0.000 claims description 3
- 238000006116 polymerization reaction Methods 0.000 claims description 3
- 238000005266 casting Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000004205 dimethyl polysiloxane Substances 0.000 claims 14
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims 14
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims 14
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims 14
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims 14
- 238000007872 degassing Methods 0.000 claims 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims 1
- 229920005573 silicon-containing polymer Polymers 0.000 claims 1
- 238000003756 stirring Methods 0.000 claims 1
- 238000012545 processing Methods 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000007547 defect Effects 0.000 abstract description 2
- 230000012010 growth Effects 0.000 abstract description 2
- 238000007306 functionalization reaction Methods 0.000 abstract 1
- 239000002243 precursor Substances 0.000 abstract 1
- 239000000376 reactant Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 17
- 229920002120 photoresistant polymer Polymers 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 210000000963 osteoblast Anatomy 0.000 description 2
- 241000209202 Bromus secalinus Species 0.000 description 1
- 229920001730 Moisture cure polyurethane Polymers 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 240000008866 Ziziphus nummularia Species 0.000 description 1
- 238000004026 adhesive bonding Methods 0.000 description 1
- 239000000560 biocompatible material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000008447 perception Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- -1 poly dimethyl silicon Oxygen alkane Chemical class 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
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Abstract
The invention discloses a kind of chip that can be used for Three-dimensional cell culture and its technologies of preparing.The chip is mainly made of contour micro-pillar array 2 and recess 4, and the top end face of contour micro-pillar array 2 forms recess 4.The chip is prepared using MEMS (Micro-Electro-Mechanical System) technology and duplicating molded technology.Beneficial effects of the present invention: 1) three-dimensional structure that chip includes can provide the three dimensional growth condition of functionalization for cell, simultaneously because the presence of contour micro-pillar array 2, which can sticking and sprawling and provide hardness uniform substrate for cell；2) three-dimensional structure that chip is included is a kind of open three-dimensional structure, compatible with common micro-observation technology；3) using high hardness material as template substrate 6 when prepared by chip, the problem of flexible material is easily damaged is overcome；4) pit is prepared on template substrate through isotropism micro-processing technology, process controllability is high；4) prepare micro-pillar array through inductively coupled plasma precursor reactant (ICP), microtrabeculae height is easy to control, avoid microtrabeculae depth-to-width ratio it is excessive when the defects of lodging.
Description
Technical field
The present invention relates to a kind of cell cultivation chip and technology of preparing with open three dimensional structure.
Background technique
Traditional cell culture technology can only provide a flat two-dimensional surface and sprawl and grow for cell, but this two
Dimension table face greatly differs from each other with the growing environment of cell in vivo, cause cell form and in terms of change, such as:
Osteoblast is in vivo without proliferative capacity, but if osteoblast is separated, using conventional two-dimensional method culture, then has and increases
Grow ability.
In view of limitation existing for above-mentioned two-dimentional cell culture technology, there has been proposed Three-dimensional cell culture technologies: utilizing
The biologic bracket material of three-dimensional structure cultivates cell, enables cell in three-dimensional space growth, proliferation and migration, constitutes three-dimensional
Cell-ECM or cell-vector compound.Compared with two dimension culture, dimensional culture can make iuntercellular have certain space
Spread pattern, the interaction between cell and timbering material are more nearly the true environment that cell is grown in vivo.
The research of majority Three-dimensional cell culture all uses class in the enclosed construction of " jujube cake " at present, is then implanted into cell
Inside configuration, it is expected that simulating cell in body microenvironment.However this enclosed construction cannot be compatible with conventional microscopic observation
Technology.Then the Three-dimensional cell culture chip with Open architecture comes into being, and [Zhang Ying, Fang Ye, Wang Jianguo have variable
The cell cultivation chip of surface shape: CN 2011.].The open three dimensional structure of this chip can be compatible with conventional aobvious
Microtechnology is disadvantageous in that: 1) chip is made of not contour microtrabeculae, the microtrabeculae height that will lead to three-dimensional structure edge is higher,
Rigidity is lower, and enough supports can not be provided for cell, is unfavorable for sticking and sprawling for cell；2) three-dimensional structure in chip is adopted
It is prepared with photoresist reflux technique, this method is led to poor controllability by the factors coupling influence such as temperature, material surface tension, is unfavorable for
Prepare the good three-dimensional structure of consistency；3) microtrabeculae of the chip standby on photoresist, the microtrabeculae height that directly passes through optical graving
With line width by photoresist and the coupling influence of gluing photoetching process, cannot control well, and prepare chip every time all and need through
Photoetching process is gone through, this all brings biggish negative effect to the complexity and differences between batches of technique；4) although plating can be passed through
Technique obtains the template for large scale preparation, but the template is made of flexible material, and rear micro-column structure is used for multiple times and is easy damage
It is bad；
Summary of the invention:
Inhomogenous, the preparation method for surface hardness existing for the existing Three-dimensional cell culture chip based on Open architecture
The problems such as complicated and poor controllability, the invention proposes a kind of open three dimensional cell cultivation chip based on contour microtrabeculae and its
Technology of preparing.
Three-dimensional cell culture chip is made of the biocompatible materials of moulded by casting, such as material is poly dimethyl silicon
Oxygen alkane (PDMS), chip include substrate 1, contour micro-pillar array 2, the recess 4 in micro- hole 3 and the formation of 2 upper surface of contour micro-pillar array.
Recess 4 is semielliptical shape, and for realizing Three-dimensional cell culture, the major diameter R and depth r of semielliptical should be suitable at one
In range, so as to the variation of cells perception to surface shape, in the present invention, major diameter R should in the range of 20-200 μm,
Depth r should be in the range of 5-50 μm；The arrangement in micro- hole 3 can be in regular quadrangle, regular hexagon or circumferential arrangement, to reduce
The area in region 5, enters cell in recess 4 as much as possible, cell is promoted to grow in three-dimensional environment between each micro- hole 3.Fig. 1
In show the recess 4 of multiple and different parameters, but in practical preparation in a single cell cultivation chip, be recessed 4 length
Shaft diameter R and depth r should numerical value having the same.There are multiple micro- holes 3 on 1 surface of substrate, this cheats 3 and 4 complete phases of recess slightly
Together, it repeats no more.In Fig. 2 for convenience of description, region 5 and recess 4 between each micro- hole 3 shade of gray processing has been subjected to, simultaneously
It is not present in actual chips.
The upper surface of contour micro-pillar array 2 forms recess 4, micro post diameter L between 1-10 μm, height H 1-50 μm it
Between, the height H of each microtrabeculae is identical, and to provide for cell growth, a hardness is uniform, is suitable for sticking the substrate sprawled.Phase
The distance d of adjacent microtrabeculae so as to cell culture fluid entrance, while should be unlikely to that cell is made to fall into microtrabeculae gap between 1-10 μm
It is interior.Microtrabeculae height H should be in the range of 1-50 μm, to prevent microtrabeculae height H is excessively high from leading to malformation or microtrabeculae height H mistake
It is low to cannot achieve Three-dimensional cell culture.
The present invention prepares above-mentioned cell cultivation chip template using MEMS technology, then using duplicating molded technology system twice
Standby cell cultivation chip.Specific step is as follows for template preparation:
Step 1: the first exposure mask 7 for being used to prepare the micro- hole 9 of template is formed on template substrate 6 by photoetching process.
Step 2: under the auxiliary of the first exposure mask 7, the micro- hole of template is prepared on template substrate 6 using wet etching technique
9。
Step 3: it is formed on the template substrate 6 in hole 9 micro- comprising template by photoetching process and is used to prepare template microtrabeculae battle array
Second exposure mask 10 of column 11.
Step 4: under the auxiliary of the second exposure mask 10, using inductively coupled plasma etching technology (ICP) by the micro- hole of template
9 are transferred to 11 surface of template micro-pillar array, form template recess 12.
After obtaining template, PDMS Three-dimensional cell culture chip can be prepared in batches by duplicating molded technology, every time only
It need to carry out replica.Specific step is as follows for duplicating molded technology twice:
Step 1: PDMS prepolymer and crosslinking agent are mixed in accordance with the appropriate ratio, are put into vacuum drying after mixing evenly
It is deaerated in case until the bubble generated in whipping process excludes completely.
Step 2: PDMS prepolymer is cast on template substrate 6, is heated after standing a period of time, keeps PDMS pre-
Polymers crosslinks reaction and solidifies.After PDMS is cooling, removing obtains PDMS template 13, PDMS template from template substrate 6
13 have and the antipodal structure of template substrate 6.
Step 3: PDMS prepolymer is cast in PDMS template 13, is heated after standing a period of time, makes PDMS
Prepolymer crosslinks reaction and solidifies.After PDMS is cooling, removing obtains PDMS tri- of the present invention from PDMS template 13
Cell cultivation chip is tieed up, chip has and the identical structure of template substrate 6.
The beneficial effects of the present invention are:
Open three dimensional structure proposed by the present invention is compatible with conventional micro-observation technology；In the present invention three-dimensional structure by etc.
High micro-pillar array 2 forms, and microtrabeculae rigidity is uniform and convenient for control, can advantageously promote cell adhesion and sprawl.
The present invention overcomes the problem of flexible material is easily damaged using the material of high rigidity as template substrate, high-volume
Template will not be damaged when preparing chip；Dimple structure is prepared using wet etching technique, processing step is simple and controllability is high, system
Standby obtained dimple structure consistency is good；Micro-pillar array is prepared using ICP lithographic technique, the height and photoetching process of microtrabeculae without
Close, microtrabeculae height be easy to control, avoid microtrabeculae depth-to-width ratio it is excessive when generate lodging and fracture the defects of.
Detailed description of the invention
Figures described below illustrates exemplary embodiment of the invention, but this is not the limit to the scope of the invention
System, because the present invention allows other equally valid embodiments.Attached drawing is not necessarily proportional to material object, and for clarity
With it is succinct, the scale of view of specific feature and attached drawing particularly may be amplified or be simplified signal.
Fig. 1 is the open three dimensional cell cultivation chip schematic diagram based on contour microtrabeculae.
Fig. 2 is the top view of Three-dimensional cell culture chip.
Fig. 3 is the sectional view of Three-dimensional cell culture chip.
Fig. 4 is to pass through the process schematic for being lithographically derived the micro- hole of template for the first time in embodiment during template construct.
Fig. 4 A, which is described, forms the process of the first exposure mask 7 on 6 surface of template substrate by photoetching.
Fig. 4 B is the result in template substrate 6 upper first time photoetching.
Fig. 4 C describes the result in the micro- hole 9 of wet etching technique preparation template.
Fig. 5 is the process schematic for passing through second of photoetching in embodiment during template construct.
Fig. 5 A, which is described, forms the process of the second exposure mask 10 on 6 surface of template substrate by photoetching.
Fig. 5 B is the result of second of photoetching on template substrate 6.
Fig. 5 C describes the result of ICP lithographic technique preparation micro-pillar array 11.
Fig. 6 is to prepare cell cultivation chip process schematic using duplicating molded technology twice in embodiment.
Fig. 6 A describes the process that PDMS prepolymer is cast in 6 surface of template substrate.
Fig. 6 B is the result in 6 upper PDMS prepolymer of template substrate.
Fig. 6 C is the PDMS template 13 stripped down from template substrate 6.
Fig. 6 D is the open three dimensional cell cultivation chip with contour microtrabeculae stripped down from PDMS template 13.
In attached drawing: 1- substrate, the high micro-pillar array such as 2-, the micro- hole 3-, 4- recess, 5- two dimension void area, 6- template substrate,
The first exposure mask of 7-, 8- etching window, the micro- hole of 9- template, the second exposure mask of 10-, 11- template micro-pillar array, 12- template recess, 13-
PDMS template
Specific embodiment
In order to which cell cultivation chip described in this patent is explained in more detail, cell training is described with an example below
Support the manufacturing method of chip.
Chip in the present embodiment is for cultivating SD rat bone marrow mesenchymal stem cells (rBMSCs).
As shown in Figure 1 to Figure 3, Three-dimensional cell culture chip is made of PDMS material, includes substrate 1, contour micro-pillar array
2, the recess 4 that micro- hole 3 and micro-pillar array upper surface are formed.Micro post diameter L is 3 μm, and micro- hole 3 and 4 diameter R of recess are 20 μm, deep
Spending r is 5 μm, and adjacent recessed distance D is 12 μm, and microtrabeculae spacing d is 4 μm.Recess 4 be in arranged in regular hexagon shape, i.e., it is any recess 4 with
The centre distance of adjacent recessed 4 is same fixed value D, and the center of circle of the recess 4 of any row or column should be in adjacent row or column recess 4
On the perpendicular bisector of circle center line connecting.
As shown in Figures 4 to 6, this example prepares cell cultivation chip template using wet etching and ICP lithographic technique, with
Cell cultivation chip is prepared using duplicating molded technology twice afterwards.
The step of preparing template is as follows:
Step 1: as illustrated in figures 4 a and 4b, photoresist is formed into 4 μ on 6 surface of template substrate by way of rotary coating
The thin layer of m, subsequent photoresist form the first mask 7 comprising etching window 8 on 6 surface of template substrate by exposure and imaging；
Template substrate 6 is the single-sided polishing silicon wafer of 500 μ m-thicks, and 8 diameter of etching window is 10 μm.
Step 2: as shown in Figure 4 C, the micro- hole 9 of template is obtained by wet etching (HNA solution) template substrate 6.
Step 3: as shown in Figure 5 A and 5B, photoresist is formed 4 μm on 6 surface of template substrate by way of spraying
Thin layer, subsequent photoresist form the second exposure mask 10 on 6 surface of template substrate by exposure and imaging.
Step 4: the micro- hole 9 of template is transferred to by 11 surface of template micro-pillar array by ICP lithographic technique, it is recessed to form template
Fall into 12.
It is as follows using the step of duplicating molded technology prepares cell cultivation chip twice:
Step 1: as shown in Figure 6 A and 6B, PDMS prepolymer and crosslinking agent is mixed according to the ratio of 10:1, stirred evenly
It is put into vacuum oven and is deaerated until the bubble generated in whipping process is excluded completely afterwards.PDMS is then cast in template
In substrate 6, standing heats 1h at 90 DEG C after five minutes, and PDMS prepolymer is made to crosslink reaction and solidify.It is cooling to PDMS
Afterwards, removing obtains PDMS template 13 from template substrate 6, as shown in Figure 6 C.
Step 2: PDMS is cast in PDMS template 13, and standing heats 1h at 90 DEG C after five minutes, makes PDMS pre-polymerization
Object crosslinks reaction and solidifies.After PDMS is cooling, removing obtains three-dimensional cell core of the present invention from PDMS template 13
Piece, as shown in Figure 6 D.
Claims (4)
1. the open three dimensional cell cultivation chip based on contour microtrabeculae, which is characterized in that by the bio-compatible of moulded by casting
Property material be made, include substrate 1, contour micro-pillar array 2；There are multiple micro- holes 3 in the substrate 1, meanwhile, it is more in the substrate 1
The form in a micro- hole 3 is transferred to 2 upper surface of contour micro-pillar array and forms multiple recess 4；The recess 4 is semielliptical shape,
For realizing Three-dimensional cell culture, the major diameter R of semielliptical is in the range of 20-200 μm, range of the depth r at 5-50 μm
It is interior；The micro post diameter L of the contour micro-pillar array 2 is between 1-10 μm, and height H is between 1-50 μm, the distance d of adjacent microtrabeculae
Between 1-10 μm.
2. as described in claim 1 based on the open three dimensional cell cultivation chip of contour microtrabeculae, which is characterized in that described micro-
The arrangement in hole 3 is in regular quadrangle, regular hexagon or circumferential arrangement.
3. as described in claim 1 based on the open three dimensional cell cultivation chip of contour microtrabeculae, which is characterized in that described to open
Putting formula Three-dimensional cell culture chip material is dimethyl silicone polymer (PDMS).
4. the preparation method of the open three dimensional cell cultivation chip based on contour microtrabeculae as described in one of claim 1 or 3,
It is characterized in that, first cell cultivation chip template is prepared using MEMS technology when chip material is PDMS, then using twice
Duplicating molded technology prepares cell cultivation chip, the specific steps are as follows:
Step 1: preparation template, including following sub-step:
Sub-step one: the first exposure mask 7 for being used to prepare the micro- hole 9 of template is formed on template substrate 6 by photoetching process；
Sub-step two: under the auxiliary of the first exposure mask 7, the micro- hole 9 of template is prepared on template substrate 6 using wet etching technique；
Sub-step three: it is formed on the template substrate 6 in hole 9 micro- comprising template by photoetching process and is used to prepare template micro-pillar array
11 the second exposure mask 10；
Sub-step four: under the auxiliary of the second exposure mask 10, using inductively coupled plasma etching technology (ICP) by template it is micro- hole 9
It is transferred to 11 surface of template micro-pillar array, forms template recess 12；
Step 2: cell cultivation chip, including following sub-step are prepared by duplicating molded technology twice:
Sub-step one: PDMS prepolymer and crosslinking agent are mixed, and are put into degassing in vacuum oven after mixing evenly until stirring
The bubble generated in the process excludes completely；
Sub-step two: PDMS prepolymer is cast on template substrate 6, is heated after standing a period of time, makes PDMS pre-polymerization
Object crosslinks reaction and solidifies；After PDMS is cooling, removing obtains PDMS template 13, PDMS template 13 from template substrate 6
With with the antipodal structure of template substrate 6；
Sub-step three: PDMS prepolymer is cast in PDMS template 13, is heated after standing a period of time, makes PDMS pre-polymerization
Object crosslinks reaction and solidifies；After PDMS is cooling, it is three-dimensional thin to obtain PDMS of the present invention for removing from PDMS template 13
Born of the same parents cultivate chip, and chip has and the identical structure of template substrate 6.
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| CN110862905A (en) * | 2020-01-09 | 2020-03-06 | 北京航空航天大学合肥创新研究院 | Chip device for cell migration experiment, preparation method and experiment method |
| CN113776722A (en) * | 2021-07-27 | 2021-12-10 | 西北工业大学 | Preparation process of micro-cylinder sensor measurement array |
| CN114058502A (en) * | 2020-08-04 | 2022-02-18 | 中国科学院大连化学物理研究所 | Biological analysis chip capable of realizing three-dimensional co-culture and multi-index detection of cells |
| CN115491307A (en) * | 2022-10-27 | 2022-12-20 | 同腾新创(苏州)科技有限公司 | PET (polyethylene terephthalate) film for cell culture in cell and gene therapy and application |
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