CN110157617A - Cervical cancer cell three-dimensional cultivation device and its application - Google Patents
Cervical cancer cell three-dimensional cultivation device and its application Download PDFInfo
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- CN110157617A CN110157617A CN201910354787.4A CN201910354787A CN110157617A CN 110157617 A CN110157617 A CN 110157617A CN 201910354787 A CN201910354787 A CN 201910354787A CN 110157617 A CN110157617 A CN 110157617A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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Abstract
The invention belongs to bioengineered tissues and biomedicine field, and in particular to a kind of cervical cancer cell three-dimensional cultivation device and its application.Described device is made of outer cylinder 2, inner cylinder 4, deceleration device 8 and motor 9, the inner cylinder 4 is made of polycarbonate membrane 11, inner cylinder top cover 6, inner cylinder bottom cover 5 and culture substrate layer 10, and the culture substrate layer 10 is made of the following components by weight: I 10-20 parts of rat tail collagen protein, 50-100 parts of agarose, 5-10 parts of fibronectin and 1-3 parts of fibroin albumen.The cervical cancer cell three-dimensional cultivation device and cultural method are practical, to cytotoxic side effect, to human and environment close friend, and price is lower, greatly reduce the cost of three-dimensional cell cultivation, it can be applied in external medicament for resisting cervical cancer screening and cytotoxicity detection, there is important scientific meaning and commercial value to the inspection of cervical carcinoma, diagnosing and treating.
Description
Technical field
The invention belongs to bioengineered tissues and biomedicine field, and in particular to a kind of cervical cancer cell dimensional culture dress
Set the preparation method and applications with a kind of culture substrate layer 10, a kind of method and one kind of external dimensional culture cervical cancer cell
The method of in vitro study cervical cancer cell invasion.
Background technique
Cervical carcinoma is the pernicious carcinoma that a kind of disease incidence is only second to breast cancer for the mankind especially women, it
Second is in the female cancer death rate.Its pathogenic factor includes that sexual life is unhealthy, the childbearing age is too early, thought is despised, sought
Support unbalanced element, smoking, virus infection (such as HPV) etc..
It include at present cytodiagnosis, viral diagnosis and Specific marker for the inspection of cervical carcinoma and diagnostic method
Detection.It clinically mostly uses the methods of radiotherapy, chemotherapy, operation, drug to be treated, the research of cervical carcinoma is be unable to do without thin
The culture of born of the same parents and tumour cell research.
Multiple studies have shown that cell is in three-dimensional environment, form and function and single layer two in people's tumor growth
Difference is very big when dimension cell is cultivated in vitro.Constantly there is document to confirm that cell has significance difference under two dimension and dimensional culture mode
It is different.
Common two dimension cell culture and human body or the intracorporal cell growth pattern of animal be not identical, usually cannot be correct
The structure or function under the conditions of normal or pathological tissue is embodied, thus being carried out using this method may when such as drug screening is tested
As a result it is inaccurate.
Compared with two-dimentional cell culture, three-dimensional cell cultivation is the best culture side for being more nearly cell and growing in vivo
Formula, because this training method has fully considered between cell and critical interaction between cell and environment.It is multinomial to grind
Study carefully confirm three-dimensional cell cultivation mode can the growing state of more preferable simulation cell in vivo, make cell assay in vitro institute
The result of acquisition is more accurate, thus toxicity detection and drug screening etc. play a significant role and huge applications valence in vitro
Value.
Current dimensional culture technology includes spontaneous cell aggregation, matrix covering culture, rotary flask culture, microcarrier
Culture, preset bracket culture and Rotary cell culture system etc..Above-mentioned dimensional culture technology respectively has advantage and disadvantage, present inventor
There is experience very rich in cervical cancer cell dimensional culture field, for the problem in cervical cancer cell incubation
Have developed a kind of cervical cancer cell three-dimensional cultivation device and corresponding cultural method.
Summary of the invention
In view of this, described device can it is an object of the present invention to provide a kind of cervical cancer cell three-dimensional cultivation device
Applied to the research to cervical carcinoma, can the growing state of more preferable simulation cervical cancer cell cell in vivo, make cell in vitro
It is more accurate to test result obtained.
To achieve the above object, the present invention uses following scheme:
A kind of cervical cancer cell three-dimensional cultivation device, which is characterized in that described device is by outer cylinder 2, inner cylinder 4, deceleration device 8
It is constituted with motor 9, the inner cylinder 4 is made of polycarbonate membrane 11, inner cylinder top cover 6, inner cylinder bottom cover 5 and culture substrate layer 10, institute
It states culture substrate layer 10 to be made of the following components by weight: I 10-20 parts of rat tail collagen protein, 50-100 parts of agarose, fibre
5-10 parts and fibroin albumen 1-3 parts of Fibronectin.
Preferably, the culture substrate layer 10 is made of the following components by weight: I 12 parts of rat tail collagen protein, fine jade
55 parts of lipolysaccharide, 7 parts of fibronectin and 1 part of fibroin albumen.
Collagen type-I is a kind of natural medium, it, which has, promotes cultured cell in vitro (especially epithelial cell) adherent
Effect, while it is also a kind of natural binder, when we need to place small slide in culture bottle or culture dish, preparation is thin
When born of the same parents' creep plate, one layer of collagen can be applied in bottom of bottle, then put small slide, small slide is just fixed among culture bottle after natural drying.
Agarose is linear polymer, and basic structure is in the β-D- galactolipin of 1,3 connections and 3,6- of Isosorbide-5-Nitrae connection
The long-chain [1-2] that ether-L- galactolipin alternately connects.Agar pectin is by the heterogeneous mixing of many smaller molecular compositions
Object.
Fibronectin is a kind of glycoprotein of large size, is present in all vertebrates, molecule 4.5-9.5% containing sugar, sugar
Chain structure is different according to histocyte source and differentiation state.Cell can be connected on extracellular matrix by FN.
Fibroin albumen is the natural polymer fibrin extracted from silk, and content accounts for about the 70%~80% of silk,
Containing 18 kinds of amino acid, wherein glycine (Gly), alanine (Ala) and serine (Ser) account for about 80% of total composition or more.
Fibroin itself has good mechanical performance and physicochemical property, and such as good flexibility and tensile strength, are delayed breathable moisture permeability
Property released etc., and pass through the available different form of different disposal, such as fiber, solution, powder, film and gel.
Result caused by selection to culture substrate composition of layer and proportion itself has Unpredictability, needs to research and develop people
The continuous exploration of member is adjustable, and present inventor is obtaining the culture substrate finally after largely testing, so that
The dimensional culture of the entirely appropriate cervical cancer cell of the culture substrate and the vegetative activity of cervical cancer cell.
Further, as shown in Figure 1, the top cover 1 of the outer cylinder 2 is openable and cell culture medium is added, the bottom of the outer cylinder 2
Lid is connect by shaft 12 with the deceleration device 8, and the deceleration device 8 is connect with the motor 9.
Further, the inner cylinder top cover 6 and inner cylinder bottom cover 5 are connect with shaft 12, and the stack shell of the inner cylinder 4 is poly- carbonic acid
Ester film 11,11 inner face of polycarbonate membrane are covered with culture substrate layer 10, and the polycarbonate membrane 11 is detachable.
Further, the inner cylinder 4 is outer when use is full of cell culture medium between cylinder 2.
Further, the volume of the cell culture medium is 5-10ml.
Further, the revolving speed of the shaft 12 is 5r/min-15r/min.
The second object of the present invention is to provide a kind of preparation method of culture substrate layer 10.
To achieve the above object, the present invention uses following scheme:
The culture substrate layer 10 is made of the following components by weight: I 10-20 parts of rat tail collagen protein, agarose
50-100 parts, 5-10 parts of fibronectin and 1-3 parts of fibroin albumen, preparation method the following steps are included:
1) agarose of formula ratio is dissolved in 90-100 DEG C with distilled water；
2) acetic acid of the 0.006mol/L of rat tail collagen protein I, culture of the fibronectin without serum, sodium magnesium
Base dissolution, fibroin albumen calcium chloride water alcohol system dissolve；
3) it is dissolved to agarose, when temperature is down to 60-70 DEG C, by rat tail collagen protein I, fibronectin and the silk of dissolution
Fibroin, which is added, to be stirred rapidly in agarose solution and is evenly applied on polycarbonate membrane 11, is cooled to room temperature to get described
Culture substrate layer 10.
Preferably, the solution temperature of step 1) is 95 DEG C.
The third object of the present invention is to provide a kind of method of external dimensional culture cervical cancer cell.
To achieve the above object, the present invention uses following scheme:
Poly- carbonic acid is first taken when carrying out cervical cancer cell dimensional culture using the cervical cancer cell three-dimensional cultivation device
Ester film 11, and culture substrate layer 10 will be prepared and be covered on polycarbonate membrane 11, then cervical cancer cell is inoculated in the training
It supports on hypothallus 10, then the inner cylinder 4 equipped with the polycarbonate membrane 11 is placed in outer cylinder 2, and open top cover 1 and inject in right amount
Device is placed in cell incubator and cultivates after adjusting shaft 12 by cell culture medium.
Specifically, the cervical cancer cell include but is not limited to Hela, siha, caski, c4-1, DoTc2-4510 and
C33A。
The fourth object of the present invention is to provide a kind of method of in vitro study cervical cancer cell invasion.
To achieve the above object, the present invention uses following scheme:
After method using the external dimensional culture cervical cancer cell, cell culture medium is removed, culture substrate is wiped
Cervical cancer cell on layer 10 recrystallizes purple dyeing, is incubated at room temperature and passes through the number of micro- sem observation cell.
The fifth object of the present invention is to provide the cervical cancer cell three-dimensional cultivation device and the culture substrate layer
A kind of application of 10 preparation method.
Specifically, the preparation method of the cervical cancer cell three-dimensional cultivation device and the culture substrate layer 10 is in body
Application in outer medicament for resisting cervical cancer screening and cytotoxicity detection.
The beneficial effects of the present invention are:
1) the cervical cancer cell three-dimensional cultivation device and cultural method provided by the invention are practical, are more nearly palace
The best training method that neck cancer cell is grown in vivo, the growing state of more preferable simulation cell in vivo, tries cell in vitro
It is more accurate to test result obtained；
2) culture substrate provided by the invention is to cytotoxic side effect, and to human and environment close friend, and price is lower, greatly
The cost of three-dimensional cell cultivation is reduced greatly；
3) cervical cancer cell three-dimensional cultivation device of the present invention can be applied to external medicament for resisting cervical cancer screening and thin
In cellular toxicity detection, there is important scientific meaning and commercial value to the inspection of cervical carcinoma, diagnosing and treating.
Detailed description of the invention
Fig. 1 is the schematic diagram of cervical cancer cell three-dimensional cultivation device；
In figure, 1 is the top cover of outer cylinder, and 2 be outer cylinder, and 3 be cell culture medium, and 4 be inner cylinder, and 5 be inner cylinder bottom cover, and 6 be inner cylinder
Top cover, 7 be the bottom cover of outer cylinder, and 8 be deceleration device, and 9 be motor, and 10 be culture substrate layer, and 11 be polycarbonate membrane, and 12 be to turn
Axis.
Fig. 2 is the longitudal section sectional view of cervical cancer cell three-dimensional cultivation device inner cylinder；
In figure, 10 be culture substrate layer, and 11 be polycarbonate membrane, and 13 be hela cell.
Fig. 3 is cervical cancer cell Matrigel violet staining figure.
Specific embodiment
Illustrated embodiment is in order to which preferably the present invention will be described, but is not that the contents of the present invention are limited only to institute
For embodiment.So those skilled in the art according to foregoing invention content to embodiment carry out it is nonessential improvement and
Adjustment, still falls within protection scope of the present invention.
The preparation of 1 culture substrate layer 10 of embodiment
The culture substrate layer 10 is made of the following components by weight: I 10 parts of rat tail collagen protein, agarose 50
Part, 5 parts of fibronectin and 1 part of fibroin albumen, preparation method the following steps are included:
1) agarose of formula ratio is dissolved in 90 DEG C with distilled water；
2) acetic acid of the 0.006mol/L of rat tail collagen protein I, culture of the fibronectin without serum, sodium magnesium
Base dissolution, fibroin albumen calcium chloride water alcohol system dissolve；
3) it is dissolved to agarose, when temperature is down to 60 DEG C, by rat tail collagen protein I, fibronectin and the fibroin of dissolution
Albumen, which is added, to be stirred rapidly in agarose solution and is evenly applied on polycarbonate membrane 11, is cooled to room temperature to get the training
Support hypothallus 10.
The preparation of 2 culture substrate layer 10 of embodiment
The culture substrate layer 10 is made of the following components by weight: I 15 parts of rat tail collagen protein, agarose 75
Part, 8 parts of fibronectin and 2 parts of fibroin albumen, preparation method the following steps are included:
1) agarose of formula ratio is dissolved in 95 DEG C with distilled water；
2) acetic acid of the 0.006mol/L of rat tail collagen protein I, culture of the fibronectin without serum, sodium magnesium
Base dissolution, fibroin albumen calcium chloride water alcohol system dissolve；
3) it is dissolved to agarose, when temperature is down to 65 DEG C, by rat tail collagen protein I, fibronectin and the fibroin of dissolution
Albumen, which is added, to be stirred rapidly in agarose solution and is evenly applied on polycarbonate membrane 11, is cooled to room temperature to get the training
Support hypothallus 10.
The preparation of 3 culture substrate layer 10 of embodiment
The culture substrate layer 10 is made of the following components by weight: I 20 parts of rat tail collagen protein, agarose 100
Part, 10 parts of fibronectin and 3 parts of fibroin albumen, preparation method the following steps are included:
1) agarose of formula ratio is dissolved in 100 DEG C with distilled water；
2) acetic acid of the 0.006mol/L of rat tail collagen protein I, culture of the fibronectin without serum, sodium magnesium
Base dissolution, fibroin albumen calcium chloride water alcohol system dissolve；
3) it is dissolved to agarose, when temperature is down to 70 DEG C, by rat tail collagen protein I, fibronectin and the fibroin of dissolution
Albumen, which is added, to be stirred rapidly in agarose solution and is evenly applied on polycarbonate membrane 11, is cooled to room temperature to get the training
Support hypothallus 10.
4 cervical cancer cell three-dimensional cultivation device of embodiment
A kind of cervical cancer cell three-dimensional cultivation device as shown in Figure 1, described device is by outer cylinder 2, inner cylinder 4, deceleration device 8
With motor 9 constitute, the inner cylinder 4 by polycarbonate membrane 11, inner cylinder top cover 6, inner cylinder bottom cover 5 and embodiment 1 culture substrate layer
10 are constituted, and the culture substrate layer 10 is made of the following components by weight: I 10-20 parts of rat tail collagen protein, agarose
50-100 parts, 5-10 parts of fibronectin and 1-3 parts of fibroin albumen.
The top cover 1 of the outer cylinder 2 is openable and cell culture medium 3 is added, the bottom cover 7 of the outer cylinder 2 by shaft 12 with
The deceleration device 8 connects, and the deceleration device 8 is connect with the motor 9.
The inner cylinder top cover 6 and inner cylinder bottom cover 5 are connect with shaft 12, and the stack shell of the inner cylinder 4 is polycarbonate membrane 11,
11 inner face of polycarbonate membrane is covered with culture substrate layer 10, and the polycarbonate membrane 11 is detachable.
The inner cylinder 4 is outer when use is full of cell culture medium 3 between cylinder 2.Described device can fill the body of cell culture medium
Product is 5ml, and the revolving speed of the shaft 12 is 5r/min.
5 cervical cancer cell three-dimensional cultivation device of embodiment
A kind of cervical cancer cell three-dimensional cultivation device as shown in Figure 1, described device is by outer cylinder 2, inner cylinder 4, deceleration device 8
With motor 9 constitute, the inner cylinder 4 by polycarbonate membrane 11, inner cylinder top cover 6, inner cylinder bottom cover 5 and embodiment 2 culture substrate layer
10 are constituted, and the culture substrate layer 10 is made of the following components by weight: I 10-20 parts of rat tail collagen protein, agarose
50-100 parts, 5-10 parts of fibronectin and 1-3 parts of fibroin albumen.
The top cover 1 of the outer cylinder 2 is openable and cell culture medium 3 is added, the bottom cover 7 of the outer cylinder 2 by shaft 12 with
The deceleration device 8 connects, and the deceleration device 8 is connect with the motor 9.
The inner cylinder top cover 6 and inner cylinder bottom cover 5 are connect with shaft 12, and the stack shell of the inner cylinder 4 is polycarbonate membrane 11,
11 inner face of polycarbonate membrane is covered with culture substrate layer 10, and the polycarbonate membrane 11 is detachable.
The inner cylinder 4 is outer when use is full of cell culture medium 3 between cylinder 2.Described device can fill the body of cell culture medium
Product is 10ml, and the revolving speed of the shaft 12 is 15r/min.
6 cervical cancer cell three-dimensional cultivation device of embodiment
A kind of cervical cancer cell three-dimensional cultivation device as shown in Figure 1, described device is by outer cylinder 2, inner cylinder 4, deceleration device 8
It is constituted with motor 9, the inner cylinder 4 is by polycarbonate membrane 11,3 culture substrate layer 10 of inner cylinder top cover 6, inner cylinder bottom cover 5 and embodiment
It constitutes, the culture substrate layer 10 is made of the following components by weight: I 10-20 parts of rat tail collagen protein, agarose 50-
100 parts, 5-10 parts of fibronectin and 1-3 parts of fibroin albumen.
The top cover 1 of the outer cylinder 2 is openable and cell culture medium 3 is added, the bottom cover 7 of the outer cylinder 2 by shaft 12 with
The deceleration device 8 connects, and the deceleration device 8 is connect with the motor 9.
The inner cylinder top cover 6 and inner cylinder bottom cover 5 are connect with shaft 12, and the stack shell of the inner cylinder 4 is polycarbonate membrane 11,
11 inner face of polycarbonate membrane is covered with culture substrate layer 10, and the polycarbonate membrane 11 is detachable.
The inner cylinder 4 is outer when use is full of cell culture medium 3 between cylinder 2.Described device can fill the body of cell culture medium
Product is 7.5ml, and the revolving speed of the shaft 12 is 10r/min.
7 cervical cancer cell Matrigel of embodiment
Using cervical cancer cell three-dimensional cultivation device described in embodiment 4-6, (this embodiment is with device as described in example 4
For), when carrying out cervical cancer cell dimensional culture, polycarbonate membrane 11 is first taken, and culture substrate layer 10 will be prepared and be covered in
On polycarbonate membrane 11, then by 1*103A cervical cancer cell (Hela, siha, caski, c4-1, DoTc2-4510 and C33A
Tested, this embodiment is by taking Hela as an example) it is inoculated on the culture substrate layer 10 that (same culture substrate layer 10 exists
Crystal violet and then and micro- sem observation are carried out after equivalent inoculating cell), then the inner cylinder 4 equipped with the polycarbonate membrane 11 is set
It in outer cylinder 2, and opens top cover 1 and injects appropriate cell culture medium, adjust device to be placed in cell incubator after shaft 12 and train
It supports.
After cultivating 48h, cell culture medium is removed, the cervical cancer cell on culture substrate layer 10, violet staining, room are wiped
Temperature is incubated for and the number for micro mirror observation cell of holding high office.As shown in figure 3, being compared with cell number when rigid inoculation hela cell, training
Hela cell after having supported 48h is because invasion reason, cell number are reduced, and the cell of reduction enters cell culture medium 3, therefore
Illustrate that the cervical cancer cell three-dimensional cultivation device provided by the invention can the growing state of fine simulation cell in vivo
And correctly reflect the invasive ability of tumour cell, it can be applied in external medicament for resisting cervical cancer screening and cytotoxicity detection.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
Claims (10)
1. a kind of cervical cancer cell three-dimensional cultivation device, which is characterized in that described device is filled by outer cylinder (2), inner cylinder (4), deceleration
It sets (8) and motor (9) is constituted, the inner cylinder (4) is by polycarbonate membrane (11), inner cylinder top cover (6), inner cylinder bottom cover (5) and culture
Hypothallus (10) is constituted, and the culture substrate layer (10) is made of the following components by weight: I 10-20 of rat tail collagen protein
Part, 50-100 parts of agarose, 5-10 parts of fibronectin and 1-3 parts of fibroin albumen.
2. cervical cancer cell three-dimensional cultivation device according to claim 1, which is characterized in that the top cover of the outer cylinder (2)
(1) openable and be added cell culture medium (3), the bottom cover (7) of the outer cylinder (2) passes through shaft (12) and the deceleration device
(8) it connects, the deceleration device (8) connect with the motor (9).
3. cervical cancer cell three-dimensional cultivation device according to claim 1, which is characterized in that the inner cylinder top cover (6) and
Inner cylinder bottom cover (5) is connect with shaft (12), and the stack shell of the inner cylinder (4) is polycarbonate membrane (11), the polycarbonate membrane
(11) inner face is covered with culture substrate layer (10), and the polycarbonate membrane (11) is detachable.
4. cervical cancer cell three-dimensional cultivation device according to claim 1, which is characterized in that the inner cylinder (4) when use
Cell culture medium 3 is full of between cylinder (2) outside.
5. cervical cancer cell three-dimensional cultivation device according to claim 4, which is characterized in that the body of the cell culture medium
Product is 5-10ml.
6. cervical cancer cell three-dimensional cultivation device according to claim 1, which is characterized in that the revolving speed of the shaft (12)
For 5r/min-15r/min.
7. the preparation method of the culture substrate layer (10), which is characterized in that the culture substrate layer (10) by weight by
Following component composition: I 10-20 parts of rat tail collagen protein, 50-100 parts of agarose, 5-10 parts of fibronectin and fibroin albumen 1-
3 parts, preparation method the following steps are included:
1) agarose of formula ratio is dissolved in 90-100 DEG C with distilled water；
2) acetic acid of the 0.006mol/L of rat tail collagen protein I, fibronectin are used molten without serum, the culture medium of sodium magnesium
Solution, fibroin albumen calcium chloride water alcohol system dissolve；
3) it is dissolved to agarose, when temperature is down to 60-70 DEG C, by the rat tail collagen protein I, fibronectin and fibroin egg of dissolution
It stirs rapidly and is evenly applied on polycarbonate membrane (11) in white addition agarose solution, is cooled to room temperature to get the training
It supports hypothallus (10).
8. a kind of method of external dimensional culture cervical cancer cell, which is characterized in that thin using cervical carcinoma described in claim 1
Born of the same parents' three-dimensional cultivation device first takes polycarbonate membrane (11), and will prepare culture substrate when carrying out cervical cancer cell dimensional culture
Layer (10) is covered on polycarbonate membrane (11), then cervical cancer cell is inoculated on the culture substrate layer (10), then will
Inner cylinder (4) equipped with the polycarbonate membrane (11) is placed in outer cylinder (2), and is opened top cover (1) and injected appropriate cell culture medium,
Device is placed in cell incubator after adjusting shaft (12) and is cultivated.
9. a kind of method of in vitro study cervical cancer cell invasion, which is characterized in that use external three-dimensional according to any one of claims 8
After the method for cultivating cervical cancer cell, cell culture medium is removed, the cervical cancer cell on culture substrate layer (10), crystal violet are wiped
Dyeing, be incubated at room temperature and hold high office micro mirror observation cell number.
10. cervical cancer cell three-dimensional cultivation device described in any one of claims 1-6 and culture substrate as claimed in claim 7
Medicament for resisting cervical cancer screening and the application in cytotoxicity detection in vitro of the preparation method of layer (10).
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| CN1252437A (en) * | 1999-09-23 | 2000-05-10 | 中国科学院力学研究所 | Three-dimensional cell and tissue culturing device with two rotating shafts |
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