CN105695328A - Co-culture device for cell culture and cell-cell interaction and using method of co-culture device - Google Patents
Co-culture device for cell culture and cell-cell interaction and using method of co-culture device Download PDFInfo
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- CN105695328A CN105695328A CN201610222671.1A CN201610222671A CN105695328A CN 105695328 A CN105695328 A CN 105695328A CN 201610222671 A CN201610222671 A CN 201610222671A CN 105695328 A CN105695328 A CN 105695328A
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Abstract
The invention discloses a co-culture device for cell culture and cell-cell interaction and a using method of the co-culture device. The device comprises a culture plate, wherein the culture plate is provided with at least one culture hole, at least one detachable semipermeable membrane spacing block and detachable spacing blocks; each detachable semipermeable membrane spacing block divides the corresponding culture hole into a plurality of cell culture rooms; and the detachable spacing blocks are positioned on two sides of each detachable semipermeable membrane spacing block. The using method of the device comprises the following steps: inserting the detachable semipermeable membrane spacing blocks and the detachable spacing blocks in the culture holes after sterilization; culturing tumor cells in a segmented cell culture room; culturing adherent cells in another cell culture room; detaching the detachable spacing blocks, and enabling the tumor cells and the adherent cells to be in co-culture; and continuing co-culture, observing a tumor cell balling condition and a migration condition of the balled tumor cells from one cell culture room to the other cell culture room. The co-culture device for cell culture and cell-cell interaction is simple in structure, simple and practical in operation, and visual and clear in effect.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of cell and cultivate the co-culture device with cell-cell interaction and using method thereof。
Background technology
Utilize the cultivation of ordinary two dimensional cell to study the machine-processed and truly internal environment of tumor development to be very different, such as cell and cell, cell and substrate interphase interaction, cell differentiation etc.。Animal model, owing to there is species variation with human body, can not well repeat characteristics of human body, such as human tumor growth's transfer, Drug therapy reaction, immunoreation and tumor stem cell (TSCs) differentiation etc.。
Three-dimensional cell culture technology is simple to operation, it is possible to simulated in vivo environment feature。In dimensional culture, it is possible to comprise tumor cell or the TSCs of single cell type, it is also possible to comprise many cells system such as tumor cell, substrate and immunocyte, such model is cultivated than common two dimension more can simulate tumor true growing state in vivo。In the incubation of cell, dimensional culture can promote the connection between cell and iuntercellular and cell and extracellular matrix, Three-dimensional cell culture is cultivated to be more easy to than attached cell and is supplied to one, cell close to internal environment, therefore, the application in Cultural Course of Tumor Cells of the dimensional culture technology receives people and more and more payes attention to, but Three-dimensional cell culture is a kind of new technique, also has some incomplete place, as lacked the three-dimensional nodule cell culture studies with other cell-cell interactions at present。
In dimensional culture technology, cell balling-up technology is subject to increasing attention。Tumor cell ball sample aggregation is cultivated, have another name called multicellular spheriods to cultivate, cultivate referred to as balling-up, its objective is scattered oncocyte is carried out In vitro culture, make it again form the solid tumor sample tuberosity of morphologically similar tumor growth, do some tumor experiments with this tuberosity (spheroid) and study closer to tumor growth。Dittmer etc. find in dimensional culture model breast cancer cell (MCF-7) can attractive interstital stem cell (hMSCs) strengthen they transfer abilities, and hMSCs also can enter mammary gland ball that breast carcinoma formed to affect growth development and the immunoreation of the latter。But its experiment simply simple breast cancer cell that hMSCs is added balling-up is observed the haptoreaction of the two, owing to two kinds of cells have been thoroughly mixed in together, this is difficult to follow-up be kept completely separate two kinds of cells, observe the characteristic of two kinds of cells respectively, and the hMSCs observation to breast cancer cell balling-up capacity cannot be accomplished。The limitation of this research and the co-culture device lacking cell balling-up cultivation and other cell-cell interactions have some relations。The lymphocyte that research is more at present there is also Similar Problems with tumor cell Co-culture。
Utilize what traditional Transwell cell realized tumor cell balling-up and other cell to co-culture the particular/special requirement that there is problems in that (1) cultivates due to tumour 3 D balling-up, tumor cell can only be confined to the lower floor of Transwell cell, therefore limits the research to some function effects of tumor cell of balling-up of other cell；(2) Transwell cell is that levels cell cultivates structure, it is impossible to get rid of the defect of gravity interference。
Summary of the invention
Present invention aims to the defect of above-mentioned prior art, a kind of cell is provided to cultivate the co-culture device with cell-cell interaction and using method thereof, this co-culture device simple in construction, with low cost, simple to operation, overcome above-mentioned conventional vertical co-culture device cannot get rid of the defect of the balling-up tumor cell gravity interference that volume is bigger, and solve tumor cell balling-up in prior art and cultivate the problem being difficult to other co-culture of cells, it is simple to observe tumor and become glomus cell and other intercellular interaction。
For achieving the above object, the technical solution used in the present invention is:
A kind of cell cultivates the co-culture device with cell-cell interaction, including culture plate, on described culture plate at least provided with a culture hole, culture hole is divided into the detachable semipermeable membrane shaping piece of several cell culture chambers and is positioned at the detachable shaping piece of detachable semipermeable membrane partition both sides by least one。
As the improvement to technique scheme, the hole wall of described culture hole is arranged with the draw-in groove setting detachable semipermeable membrane shaping piece, detachable shaping piece for card, the longitudinally opposed distribution of described draw-in groove vertically in parallel；The height of described draw-in groove is identical with the degree of depth of culture hole, in the insertion draw-in groove that described detachable semipermeable membrane shaping piece, detachable shaping piece are detachable。
As the improvement to technique scheme, the junction of described culture hole and detachable semipermeable membrane shaping piece, the junction of detachable semipermeable membrane shaping piece, draw-in groove and detachable semipermeable membrane shaping piece, detachable semipermeable membrane shaping piece is provided with the sealing device preventing liquid between cell culture chamber from flowing mutually。This sealing device be can fill in draw-in groove with detachable shaping piece or form the diaphragm seal, the sealing gasket etc. that seal with detachable semipermeable membrane shaping piece。
As the improvement to technique scheme, described detachable shaping piece is symmetrically distributed in the both sides of detachable semipermeable membrane shaping piece, closely adjacent with detachable semipermeable membrane shaping piece, at a distance of width≤0.5mm。
As the improvement to technique scheme, described detachable semipermeable membrane shaping piece is dismountable semipermeable membrane shaping piece being suitable to culture medium or cell traverse。
As the improvement to technique scheme, described detachable semipermeable membrane shaping piece is polycarbonate membrane shaping piece。
As the improvement to technique scheme, the pore size of described detachable semipermeable membrane shaping piece is 3-8 μm。
As the improvement to technique scheme, described detachable shaping piece is the plastic sheet of dismountable acellular adhesion, and height is identical with the culture hole degree of depth。
As the improvement to technique scheme, described culture plate is Tissue Culture Plate or the Tissue Culture Dish of 6 culture hole or 12 culture hole or 24 culture hole。
Invention additionally provides the using method of above-mentioned co-culture device, the step of this using method is:
S1, co-culture device is carried out autoclaving and ultra-vioket radiation sterilizing；
S2, insert detachable semipermeable membrane shaping piece, detachable shaping piece；
Tumor cell suspension adding under S3, sterile working the cell culture chamber being coated matrigel, makes tumor cell be sunken to matrigel, in cell culture chamber, stable growth is cultivated 3 days；
S4, the 4th day time, be coated another cell culture chamber with poly-D-lysine, another kind of attached cell is planted in the cell culture chamber being coated poly-D-lysine。37 DEG C of incubated overnight, so as in cell culture chamber adherent growth；
S5, separate detachable shaping piece, make tumor cell and attached cell co-culture；
S6, continue to co-culture, observe tumor cell balling-up situation, and the tumor cell of balling-up is from a cell culture chamber to the migration situation of another cell culture chamber。
Compared with prior art, the present invention has the advantage that and has the benefit effect that
The cell of the present invention is cultivated and the co-culture device of cell-cell interaction, and 1, simple and practical, it is to avoid the loaded down with trivial details separating step after co-culturing is cultivated in conventional tumor cell balling-up with other cell direct contact type, and separates halfway interference；2, owing to becoming spheroid mass relatively big, the factor that cannot get rid of gravity interference during Transwell co-cultures is overcome；3, the suitability is wide, is not only suitable for tumor balling-up and cultivates and the co-culturing of attached cell, and is also applied for co-culturing of tumor balling-up cultivation and suspension cell；4, can Reusability, with low cost, simple to operation。The using method of the present invention can utilize this co-culture device to carry out tumor cell balling-up cultivation and cell-cell interaction effect observation, and practicality simple to operate, effect are simple and clear。
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, the accompanying drawing used required in embodiment or description of the prior art will be briefly described below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the premise not paying creative work, it is also possible to obtain other accompanying drawing according to these accompanying drawings。
Fig. 1 is the overall structure schematic diagram of the present invention；
Fig. 2 is the structural representation of the single culture hole of the present invention；
Fig. 3 is the assembling structural representation of the single culture hole of the present invention。
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments。Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain under not making creative work premise, any amendment made, equivalent replacement, improvement etc., should be included within protection scope of the present invention。
As shown in Figure 1, 2, 3, the cell of the present invention cultivates the co-culture device with cell-cell interaction, including culture plate 1, on described culture plate 1 at least provided with a culture hole 2, culture hole 2 is divided into the detachable semipermeable membrane shaping piece 6 of several cell culture chambers 3,4 and is positioned at detachable shaping piece 7a, 7b of detachable semipermeable membrane partition 6 both sides by least one；Be arranged with draw-in groove 5a, 5b, 5c, 5a', 5b of setting detachable semipermeable membrane shaping piece 6, detachable shaping piece 7a, 7b for card on the hole wall of described culture hole 2 vertically in parallel ', 5c ', described draw-in groove 5a, 5b, 5c, 5a', 5b ', the longitudinally opposed distribution of 5c '；Described draw-in groove 5a, 5b, 5c, 5a', 5b ', the height of 5c ' identical with the degree of depth of culture hole 2, in insertion draw-in groove 5c, 5c that described detachable semipermeable membrane shaping piece 6 is detachable ', in detachable detachable for shaping piece 7a insertion draw-in groove 5a, 5a', detachable detachable for shaping piece 7b insertion draw-in groove 5b, 5b ' in；Described culture hole 2 and detachable semipermeable membrane shaping piece 6, detachable semipermeable membrane shaping piece 7a, the junction of 7b is provided with the sealing device preventing liquid between cell culture chamber from flowing mutually, detachable semipermeable membrane shaping piece 6 and draw-in groove 5c, the junction of 5c ', detachable shaping piece 7a and draw-in groove 5a, the junction of 5a', detachable shaping piece 7b and draw-in groove 5b, the junction of 5b ' is provided with the sealing device preventing liquid between cell culture chamber from flowing mutually, this sealing device be can fill in draw-in groove with detachable shaping piece, detachable semipermeable membrane shaping piece forms the diaphragm seal sealed, sealing gasket etc.。
Described detachable shaping piece 7a, 7b are symmetrically distributed in the both sides of detachable semipermeable membrane shaping piece 6, closely adjacent with detachable semipermeable membrane shaping piece 6, at a distance of width≤0.5mm；Described detachable semipermeable membrane shaping piece 6 is dismountable semipermeable membrane shaping piece being suitable to culture medium or cell traverse；Described detachable semipermeable membrane shaping piece 6 is polycarbonate membrane shaping piece；The pore size of described detachable semipermeable membrane shaping piece 6 is 3-8 μm；Described detachable shaping piece 7a, 7b are the plastic sheet of dismountable acellular adhesion, and height is identical with the culture hole degree of depth。
Described culture plate 1 is Tissue Culture Plate or the Tissue Culture Dish of 6 culture hole or 12 culture hole or 24 culture hole, is 6 culture hole shown in Fig. 1。
Embodiment 1
S1, co-culture device is carried out autoclaving and ultra-vioket radiation sterilizing；
S2, insert, at middle draw-in groove, the detachable semipermeable membrane shaping piece 6 that pore size is 3 μm, insert detachable shaping piece 7a, 7b at both sides draw-in groove；
Tumor cell suspension adding under S3, sterile working the cell culture chamber 3 being coated matrigel, makes tumor cell be sunken to matrigel, in cell culture chamber 3, stable growth is cultivated 3 days；
S4, the 4th day time, be coated another cell culture chamber 4 with poly-D-lysine, another kind of attached cell is planted in the cell culture chamber 4 being coated poly-D-lysine。37 DEG C of incubated overnight, so as in cell culture chamber 4 adherent growth；
S5, separate detachable shaping piece 7a, 7b, make tumor cell and attached cell co-culture；
S6, continue to co-culture, observe tumor cell balling-up situation, and the tumor cell of balling-up is from a cell culture chamber 3 to the migration situation of another cell culture chamber 4。
Embodiment 2
S1, co-culture device is carried out autoclaving and ultra-vioket radiation sterilizing；
S2, insert, at middle draw-in groove, the detachable semipermeable membrane shaping piece 6 that pore size is 8 μm, insert detachable shaping piece 7a, 7b at both sides draw-in groove；
Tumor cell suspension adding under S3, sterile working the cell culture chamber 3 being coated matrigel, makes tumor cell be sunken to matrigel, in cell culture chamber 3, stable growth is cultivated 3 days；
S4, the 4th day time, be coated another cell culture chamber 4 with poly-D-lysine, another kind of attached cell is planted in the cell culture chamber 4 being coated poly-D-lysine。37 DEG C of incubated overnight, so as in cell culture chamber 4 adherent growth；
S5, separate detachable shaping piece 7a, 7b, make tumor cell and attached cell co-culture；
S6, continue to co-culture, observe tumor cell balling-up situation, and the tumor cell of balling-up is from a cell culture chamber 3 to the migration situation of another cell culture chamber 4；
S7, migration balling-up tumor cell be attached on detachable semipermeable membrane shaping piece 6, it is simple to the migration of balling-up tumor is observed in follow-up dyeing。
The cell of the present invention is cultivated and the co-culture device of cell-cell interaction, and 1, simple and practical, it is to avoid the loaded down with trivial details separating step after co-culturing is cultivated in conventional tumor cell balling-up with other cell direct contact type, and separates halfway interference；2, owing to becoming spheroid mass relatively big, the factor that cannot get rid of gravity interference during Transwell co-cultures is overcome；3, the suitability is wide, is not only suitable for tumor balling-up and cultivates and the co-culturing of attached cell, and is also applied for co-culturing of tumor balling-up cultivation and suspension cell；4, can Reusability, with low cost, simple to operation。The using method of the present invention can utilize this co-culture device to carry out tumor cell balling-up cultivation and cell-cell interaction effect observation, and practicality simple to operate, effect are simple and clear。
Claims (10)
1. a cell cultivates the co-culture device with cell-cell interaction, including culture plate, it is characterised in that: on described culture plate at least provided with a culture hole, culture hole is divided into the detachable semipermeable membrane shaping piece of several cell culture chambers and is positioned at the detachable shaping piece of detachable semipermeable membrane partition both sides by least one。
2. cell according to claim 1 cultivates the co-culture device with cell-cell interaction, it is characterized in that: the hole wall of described culture hole is arranged with in parallel the draw-in groove setting detachable semipermeable membrane shaping piece, detachable shaping piece for card, the longitudinally opposed distribution of described draw-in groove vertically；The height of described draw-in groove is identical with the degree of depth of culture hole, in the insertion draw-in groove that described detachable semipermeable membrane shaping piece, detachable shaping piece are detachable。
3. cell according to claim 2 cultivates the co-culture device with cell-cell interaction, it is characterised in that: the junction of described culture hole and detachable semipermeable membrane shaping piece, the junction of detachable semipermeable membrane shaping piece, described draw-in groove and detachable semipermeable membrane shaping piece, detachable semipermeable membrane shaping piece is provided with the sealing device preventing liquid between cell culture chamber from flowing mutually。
4. cell according to claim 1 cultivates the co-culture device with cell-cell interaction, it is characterized in that: described detachable shaping piece is symmetrically distributed in the both sides of detachable semipermeable membrane shaping piece, closely adjacent with detachable semipermeable membrane shaping piece, at a distance of width≤0.5mm。
5. cell according to claim 1 cultivates the co-culture device with cell-cell interaction, it is characterised in that: described detachable semipermeable membrane shaping piece is dismountable semipermeable membrane shaping piece being suitable to culture medium or cell traverse。
6. cell according to claim 5 cultivates the co-culture device with cell-cell interaction, it is characterised in that: described detachable semipermeable membrane shaping piece is polycarbonate membrane shaping piece。
7. cell according to claim 6 cultivates the co-culture device with cell-cell interaction, it is characterised in that: the pore size of described detachable semipermeable membrane shaping piece is 3-8 μm。
8. cell according to claim 1 cultivates the co-culture device with cell-cell interaction, it is characterised in that: described detachable shaping piece is the plastic sheet of dismountable acellular adhesion, and height is identical with the culture hole degree of depth。
9. cell according to claim 1 cultivates the co-culture device with cell-cell interaction, it is characterised in that: described culture plate is Tissue Culture Plate or the Tissue Culture Dish of 6 culture hole or 12 culture hole or 24 culture hole。
10. the using method of co-culture device as described in claim 1-9 any one claim, it is characterised in that: the step of this co-culture device using method is:
S1, co-culture device is carried out autoclaving and ultra-vioket radiation sterilizing；
S2, insert detachable semipermeable membrane shaping piece, detachable shaping piece；
Tumor cell suspension adding under S3, sterile working the cell culture chamber being coated matrigel, makes tumor cell be sunken to matrigel, in cell culture chamber, stable growth is cultivated 3 days；
S4, the 4th day time, be coated another cell culture chamber with poly-D-lysine, another kind of attached cell is planted in the cell culture chamber being coated poly-D-lysine。37 DEG C of incubated overnight, so as in cell culture chamber adherent growth；
S5, separate detachable shaping piece, make tumor cell and attached cell co-culture；
S6, continue to co-culture, observe tumor cell balling-up situation, and the tumor cell of balling-up is from a cell culture chamber to the migration situation of another cell culture chamber。
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Cited By (6)
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| CN107488699A (en) * | 2017-09-18 | 2017-12-19 | 江苏大学 | The device and method that a kind of adhesion property to cell culture adhesion material compares |
| CN107523544A (en) * | 2017-10-16 | 2017-12-29 | 中国人民解放军第三军医大学第附属医院 | A kind of preparation method and application of tumour cell ball |
| CN109082379A (en) * | 2018-09-29 | 2018-12-25 | 五邑大学 | It is a kind of for highly emulating the in vitro culture device of Nasopharyngeal neoplasms |
| CN112063525A (en) * | 2019-06-10 | 2020-12-11 | 芬欧汇川集团 | Cell culture plate, method for producing the same, and method for detecting substance |
| CN112852616A (en) * | 2021-01-22 | 2021-05-28 | 福建省微生物研究所 | Fluid bioreactor and use method thereof |
| WO2022134159A1 (en) * | 2020-12-25 | 2022-06-30 | 中国科学院广州生物医药与健康研究院 | Chip for integrated tumor cell behavior experiments |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107488699A (en) * | 2017-09-18 | 2017-12-19 | 江苏大学 | The device and method that a kind of adhesion property to cell culture adhesion material compares |
| CN107523544A (en) * | 2017-10-16 | 2017-12-29 | 中国人民解放军第三军医大学第附属医院 | A kind of preparation method and application of tumour cell ball |
| CN109082379A (en) * | 2018-09-29 | 2018-12-25 | 五邑大学 | It is a kind of for highly emulating the in vitro culture device of Nasopharyngeal neoplasms |
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| CN112852616A (en) * | 2021-01-22 | 2021-05-28 | 福建省微生物研究所 | Fluid bioreactor and use method thereof |
| CN112852616B (en) * | 2021-01-22 | 2024-06-25 | 福建省微生物研究所 | Fluid bioreactor and application method thereof |
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Application publication date: 20160622 |