CN109810895B - Open type three-dimensional cell culture chip based on contour microcolumn and preparation technology thereof - Google Patents
Open type three-dimensional cell culture chip based on contour microcolumn and preparation technology thereof Download PDFInfo
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- CN109810895B CN109810895B CN201910147892.0A CN201910147892A CN109810895B CN 109810895 B CN109810895 B CN 109810895B CN 201910147892 A CN201910147892 A CN 201910147892A CN 109810895 B CN109810895 B CN 109810895B
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- 238000005516 engineering process Methods 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 44
- 239000000758 substrate Substances 0.000 claims abstract description 41
- 230000008569 process Effects 0.000 claims abstract description 23
- 238000009616 inductively coupled plasma Methods 0.000 claims abstract description 10
- 239000000463 material Substances 0.000 claims abstract description 9
- 238000000465 moulding Methods 0.000 claims abstract description 8
- 230000010076 replication Effects 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims abstract description 7
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 44
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 44
- 238000005530 etching Methods 0.000 claims description 8
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- 238000001291 vacuum drying Methods 0.000 claims description 3
- 239000000560 biocompatible material Substances 0.000 claims description 2
- 239000003431 cross linking reagent Substances 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 230000007480 spreading Effects 0.000 abstract description 5
- 238000003892 spreading Methods 0.000 abstract description 5
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Abstract
本发明公开了一种可以用于三维细胞培养的芯片及其制备技术。该芯片主要由等高微柱阵列2和凹陷4组成，等高微柱阵列2的顶端面形成凹陷4。该芯片采用MEMS(Micro‑Electro‑Mechanical System)技术和复制模塑技术制备。本发明的有益效果：1)芯片包含的三维结构可以为细胞提供功能化的三维生长条件，同时由于等高微柱阵列2的存在，该芯片可以为细胞的黏附和铺展提供硬度均一的基底；2)芯片所包含的三维结构是一种开放式的三维结构，与常见显微观测技术兼容；3)芯片制备时以高硬度材料作为模板基底6，克服了软性材料容易损坏的问题；4)经各向同性微加工技术在模板基底上制备凹坑，工艺可控性高；4)经电感耦合等离子体反应(ICP)制备微柱阵列，微柱高度容易控制，避免了微柱深宽比过大时倒伏等缺陷。
The invention discloses a chip that can be used for three-dimensional cell culture and its preparation technology. The chip is mainly composed of an equal-height micro-pillar array 2 and a recess 4 , and a recess 4 is formed on the top surface of the equal-height micro-pillar array 2 . The chip is fabricated by MEMS (Micro-Electro-Mechanical System) technology and replication molding technology. The beneficial effects of the present invention: 1) the three-dimensional structure contained in the chip can provide functionalized three-dimensional growth conditions for cells, and at the same time, due to the existence of the equal-height micro-pillar array 2, the chip can provide a substrate with uniform hardness for cell adhesion and spreading; 2) The three-dimensional structure contained in the chip is an open three-dimensional structure, which is compatible with common microscopic observation techniques; 3) When the chip is prepared, a high-hardness material is used as the template substrate 6, which overcomes the problem of easy damage of soft materials; The isotropic micromachining technology prepares pits on the template substrate, and the process is highly controllable; 4) The micropillar array is prepared by inductively coupled plasma reaction (ICP), the height of the micropillar is easy to control, and the aspect ratio of the micropillar is avoided. Defects such as lodging when large.
Description
Technical Field
The invention relates to a cell culture chip with an open three-dimensional structure and a preparation technology thereof.
Background
The traditional cell culture technology can only provide a flat two-dimensional surface for spreading and growing cells, but the two-dimensional surface is far from the growth environment of the cells in vivo, which causes the cells to change in aspects of morphology, functions and the like, such as: osteoblasts have no proliferative capacity in vivo, but have proliferative capacity if isolated and cultured in a conventional two-dimensional method.
In view of the limitations of the two-dimensional cell culture techniques described above, three-dimensional cell culture techniques have been proposed: the biological scaffold material with a three-dimensional structure is used for culturing cells, so that the cells can grow, proliferate and migrate in a three-dimensional space to form a three-dimensional cell-cell or cell-carrier compound. Compared with two-dimensional culture, three-dimensional culture can ensure that cells have a certain spatial arrangement form, and the interaction between the cells and the scaffold material is closer to the real environment of the cells growing in vivo.
Most of the current three-dimensional cell culture studies adopt a closed structure similar to a date cake, and then cells are implanted into the structure, which is expected to simulate the in-vivo microenvironment of the cells. However, such closed structures are not well compatible with conventional microscopic observation techniques. Thus, three-dimensional cell culture chips with an open structure (Zhang Ying, bright and bright, King Kong. cell culture chips with variable surface shapes: CN 2011.) have been produced. The open three-dimensional structure of the chip can be well compatible with the conventional microscopy technology, and the defects are that: 1) the chip is composed of micro-columns with different heights, which can cause that the height of the micro-columns at the edge of the three-dimensional structure is higher and the rigidity is lower, so that sufficient support can not be provided for cells, and the adhesion and the spreading of the cells are not facilitated; 2) the three-dimensional structure in the chip is prepared by adopting a light resistance reflux process, and the method is influenced by factors such as temperature, material surface tension and the like, so that the controllability is poor, and the preparation of the three-dimensional structure with good consistency is not facilitated; 3) the microcolumn of the chip is directly prepared on the photoresist material through photoetching, the height and the line width of the microcolumn are influenced by the coupling of photoresist and a photoresist photoetching process and cannot be well controlled, and the chip is prepared by a photoetching process every time, which brings great negative influence on the complexity of the process and the difference between batches; 4) although the template for large-scale preparation can be obtained by an electroplating process, the template is made of a soft material, and the structure of the microcolumn is easily damaged after multiple uses;
the invention content is as follows:
the invention provides an open type three-dimensional cell culture chip based on contour columns and a preparation technology thereof, aiming at the problems of uneven surface hardness, complex preparation method, poor controllability and the like of the existing three-dimensional cell culture chip based on an open type structure.
The three-dimensional cell culture chip is made of a casting-molded biocompatible material, for example, Polydimethylsiloxane (PDMS), and comprises a substrate 1, an array of equal-height micro-pillars 2, micro-pits 3 and depressions 4 formed on the upper surface of the array of equal-height micro-pillars 2. The cavity 4 is a semi-ellipsoid for three-dimensional cell culture, and the major axis diameter R and the depth R of the semi-ellipsoid should be in a suitable range so that the cell can sense the change of the surface shape, and in the present invention, the major axis diameter R should be in the range of 20-200 μm and the depth R should be in the range of 5-50 μm; the arrangement of the micro-pits 3 can be in a regular quadrangle, a regular hexagon or a circumference shape so as to reduce the area of the area 5 between the micro-pits 3, so that the cells can enter the pits 4 as much as possible, and the cells can be promoted to grow in a three-dimensional environment. In FIG. 1, a plurality of different parameters of the well 4 are shown, but in practice, the diameter R of the major axis and the depth R of the well 4 should have the same values within a single cell culture chip. The surface of the substrate 1 is provided with a plurality of micro pits 3, and the micro pits 3 are identical to the depressions 4, which will not be described in detail. In fig. 2, for convenience of explanation, the regions 5 and the depressions 4 between the respective micro pits 3 are subjected to a gradation process, which does not exist in an actual chip.
The upper surface of the equal-height micro-column array 2 is provided with a recess 4, the diameter L of the micro-column is between 1 and 10 mu m, the height H of the micro-column is between 1 and 50 mu m, and the height H of each micro-column is the same, so that a substrate with uniform hardness and suitable adhesion and spreading is provided for cell growth. The distance d between adjacent microcolumns should be between 1 and 10 μm so that the cell culture solution can enter without allowing the cells to fall into the gap between the microcolumns. The height H of the microcolumn should be in the range of 1-50 μm to prevent the structure from being deformed due to the height H of the microcolumn being too high or the three-dimensional cell culture from being impossible due to the height H of the microcolumn being too low.
The invention adopts MEMS technology to prepare the cell culture chip template, and then adopts twice replication molding technology to prepare the cell culture chip. The template preparation comprises the following specific steps:
the method comprises the following steps: a first mask 7 for preparing template micro-pits 9 is formed on the template substrate 6 by a photolithography process.
Step two: with the aid of the first mask 7, template micro-pits 9 are produced on the template substrate 6 using a wet etching technique.
Step three: a second mask 10 for preparing a template micropillar array 11 is formed on the template substrate 6 containing the template micropits 9 by a photolithography process.
Step four: with the aid of a second mask 10, template micro-pits 9 are transferred to the surface of a template micro-pillar array 11 by using an inductively coupled plasma etching technique (ICP), forming template recesses 12.
After the template is obtained, the PDMS three-dimensional cell culture chip can be prepared in batch by means of a copy molding technology, and only needs to be copied each time. The two-shot replication molding technique comprises the following specific steps:
the method comprises the following steps: mixing PDMS prepolymer and cross-linking agent according to a proper proportion, stirring uniformly, placing in a vacuum drying oven for degassing until bubbles generated in the stirring process are completely removed.
Step two: and pouring the PDMS prepolymer on the template substrate 6, standing for a period of time, and heating to enable the PDMS prepolymer to generate a crosslinking reaction so as to be cured. After the PDMS is cooled, the PDMS template 13 is peeled off from the template substrate 6, and the PDMS template 13 has a structure completely opposite to the template substrate 6.
Step three: the PDMS prepolymer is poured onto the PDMS template 13, left for a while, and then heated to cause crosslinking reaction of the PDMS prepolymer and cure. And after cooling the PDMS, stripping the PDMS template 13 to obtain the PDMS three-dimensional cell culture chip of the invention, wherein the chip has the structure completely the same as that of the template substrate 6.
The invention has the beneficial effects that:
the open three-dimensional structure provided by the invention is compatible with the conventional microscopic observation technology; the three-dimensional structure of the invention is composed of the equal-height micro-column array 2, the rigidity of the micro-column is uniform and is convenient to control, and the cell adhesion and spreading can be better promoted.
The invention takes the high-hardness material as the template substrate, overcomes the problem that the soft material is easy to damage, and does not damage the template when preparing chips in large scale; the micro-pit structure is prepared by adopting a wet etching technology, the process steps are simple, the controllability is high, and the consistency of the prepared micro-pit structure is good; the ICP etching technology is adopted to prepare the micro-column array, the height of the micro-column is irrelevant to the photoetching process, the height of the micro-column is easy to control, and the defects of lodging, fracture and the like caused by the overlarge depth-to-width ratio of the micro-column are avoided.
Drawings
The accompanying drawings, which are described below, illustrate exemplary embodiments of the present invention and are not intended to limit the scope of the invention, since the invention may admit to other equally effective embodiments. The drawings are not necessarily to scale and certain features may be particularly exaggerated or simplified in scale from the drawings in the interest of clarity and conciseness.
FIG. 1 is a schematic diagram of an open three-dimensional cell culture chip based on contour microcolumns.
FIG. 2 is a top view of a three-dimensional cell culture chip.
FIG. 3 is a cross-sectional view of a three-dimensional cell culture chip.
FIG. 4 is a schematic diagram of the process of obtaining the template micro-pits by the first photolithography in the template fabrication process in the embodiment.
Fig. 4A depicts a process of forming a first mask 7 on the surface of the template substrate 6 by photolithography.
Fig. 4B is the result of a first lithography on the template substrate 6.
Fig. 4C depicts the results of a wet etch technique to prepare template micropits 9.
FIG. 5 is a schematic diagram illustrating a second photolithography process performed during the template fabrication process according to an embodiment of the present invention.
Fig. 5A depicts a process of forming a second mask 10 on the surface of the template substrate 6 by photolithography.
Fig. 5B is the result of a second lithography on the template substrate 6.
Fig. 5C depicts the result of the ICP etching technique for preparing the micropillar array 11.
FIG. 6 is a schematic diagram of the process of preparing a cell culture chip by the two-shot replication technique in the examples.
Fig. 6A depicts the process of casting PDMS prepolymer on the surface of the template substrate 6.
Fig. 6B is the result of casting a PDMS prepolymer on the template substrate 6.
Fig. 6C shows the PDMS template 13 peeled off from the template substrate 6.
FIG. 6D shows an open three-dimensional cell culture chip with contour micro-pillars peeled from the PDMS template 13.
In the drawings: 1-substrate, 2-equal-height micro-column array, 3-micro-pits, 4-pits, 5-two-dimensional gap area, 6-template substrate, 7-first mask, 8-etching window, 9-template micro-pits, 10-second mask, 11-template micro-column array, 12-template pits, 13-PDMS template
Detailed Description
To illustrate the cell culture chip described in this patent in more detail, a method of manufacturing the cell culture chip is described below with an example.
The chip in this example was used to culture SD rat bone marrow mesenchymal stem cells (rBMSCs).
As shown in fig. 1 to 3, the three-dimensional cell culture chip is made of PDMS material, and includes a substrate 1, a contour micro-pillar array 2, micro-pits 3, and depressions 4 formed on the upper surface of the micro-pillar array. The diameter L of the microcolumn is 3 μm, the diameter R of the dimple 3 and the dimple 4 is 20 μm, the depth R is 5 μm, the adjacent dimple distance D is 12 μm, and the microcolumn spacing D is 4 μm. The depressions 4 are arranged in a regular hexagon, that is, the distance between the center of any depression 4 and the center of the adjacent depression 4 is the same fixed value D, and the center of the depression 4 in any row or column should be on the perpendicular bisector of the connecting line of the centers of the depressions 4 in the adjacent row or column.
As shown in fig. 4 to 6, the present example prepared a cell culture chip template using wet etching and ICP etching techniques, followed by preparing a cell culture chip using a two-shot replication molding technique.
The procedure for preparing the template was as follows:
the method comprises the following steps: as shown in fig. 4A and 4B, a photoresist is spin-coated to form a thin layer of 4 μm on the surface of the template substrate 6, and then the photoresist is exposed and developed to form a first mask 7 including an etching window 8 on the surface of the template substrate 6; the template substrate 6 is a single-sided polished silicon wafer 500 μm thick with an etch window 8 of 10 μm diameter.
Step two: as shown in fig. 4C, the template micropits 9 are obtained by wet etching (HNA solution) the template substrate 6.
Step three: as shown in fig. 5A and 5B, a photoresist is sprayed to form a thin layer of 4 μm on the surface of the template substrate 6, and then the photoresist is exposed and developed to form a second mask 10 on the surface of the template substrate 6.
Step four: template micro-pits 9 are transferred to the surface of the template micro-column array 11 by an ICP etching technology to form template pits 12.
The steps of preparing the cell culture chip by adopting the two-time replication molding technology are as follows:
the method comprises the following steps: as shown in fig. 6A and 6B, the PDMS prepolymer and the crosslinker were mixed in a 10: 1, stirring uniformly, placing the mixture into a vacuum drying oven, and degassing until bubbles generated in the stirring process are completely removed. Then, PDMS was poured on the template substrate 6, left to stand for 5 minutes, and then heated at 90 ℃ for 1 hour to cause crosslinking reaction of the PDMS prepolymer to cure. After the PDMS is cooled, the PDMS template 13 is peeled off from the template substrate 6, as shown in fig. 6C.
Step two: PDMS was poured onto the PDMS template 13, left to stand for 5 minutes and then heated at 90 ℃ for 1 hour to cause the crosslinking reaction of the PDMS prepolymer and cure. After the PDMS is cooled, the three-dimensional cell chip of the present invention is obtained by peeling off the PDMS template 13, as shown in fig. 6D.
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| 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 |
| CN113063831B (en) * | 2021-04-30 | 2025-03-28 | 中国矿业大学(北京) | Microfluidic chip and its manufacturing method, method and device for testing biochemical oxygen demand |
| CN113776722A (en) * | 2021-07-27 | 2021-12-10 | 西北工业大学 | Preparation process of micro-cylinder sensor measurement array |
| CN115491307B (en) * | 2022-10-27 | 2024-01-23 | 同腾新创(苏州)科技有限公司 | PET film for cell culture in cell and gene therapy and application |
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