CN101339104B - Mechanical damaging device for cell damage processing - Google Patents
Mechanical damaging device for cell damage processing Download PDFInfo
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- CN101339104B CN101339104B CN200710127846.1A CN200710127846A CN101339104B CN 101339104 B CN101339104 B CN 101339104B CN 200710127846 A CN200710127846 A CN 200710127846A CN 101339104 B CN101339104 B CN 101339104B
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Abstract
The invention relates to a multi-channel mechanical damaging device which is used for damaging cells and which comprises a pin support body 1, a plurality of pins 2 which are arranged in parallel and connected with the pin support body, and a handle which is connected with the pin support body; the length the pins extending out of the pin support body can be adjusted. The pin support body is provided with a plurality of holes 5; the pins can be inserted into the holes and are suitable for the one-time suction head which can be replaced. The multi-channel mechanical damaging device can also be provided with guide components 4 which are arranged on two sides of the pin support body 1; and the angle between the guide components can be regulated. In particular, the invention provides an 8-channel mechanical damaging device which can be used in the cell injury experiment of cells which are cultured by 96-hole microplate. The invention also provides a method which uses the multi-channel mechanical damaging device to damage the cell culture and an application thereof in cell migration and the potential biological phenomena thereof.
Description
Invention field
The present invention relates to a kind of multiple channel mechanical damage device for cell is damaged.Concrete, the invention provides a kind of 8 passage mechanical damage devices of the damage test for 96 hole micro plate form cultured cells.
Background technology
Cell migration plays an important role in multiple normal physiological processes.These processes comprise embryo's formation, vascularization, wound healing, intestinal mucosal injury reparation and immune defense.In some pathologic conditions such as artery sclerosis or gastrointestinal ulceration, often find that large-area cell strips off, therefore need to rebuild immediately intact cell monolayer and be repaired.
Cell migration is a complex process, needs in various kinds of cell and the coordination of extracellular event, and for example cytoskeleton is organized again, matrix rebuilding, the adjusting of cell-cell adhesion and inducing of chemical inhibitor.
Damage test is a kind of common method in research cell migration and potential biological phenomena thereof, as matrix rebuilding or polarization.In addition, damage test also is used to study Angiogenesis, Malignant tumor of bonal metastasis and other physiology and pathologic process.
But, in many situations, the research of cell migration usually relates to the reaction of confluent monolayer cells for mechanical wounding (mechanical damage).Usually by remove a certain proportion of cell that is paved be grown on each cover glass or in porous plate with imbibition head, blade, injection needle, mechanical friction device or scraper, to cause cellular damage (referring to Obeso JL, Auerbach R:A newmicrotechnique for quantitating cell movement in vitro using polystyrenebead monolayers.J Immunol Methods 1984,70 (2): 141-52; With Sholley MM, Gimbrone MA Jr, Cotran RS:Cellular migration and replication inendothelial regeneration:a study using irradiated endothelial cultures.LabInvest 1977,36 (1): 18-25).Then can observe, measure and quantize by digital camera or the computer generated image system of microscope preparation and strip off regional closed situation in a period of time, namely alleged epithelium regenerates, endothelium regenerates or the situation of wound healing.
But most damage methods need to be processed respectively each sample well.Do not have simple method to produce a plurality of and consistent mechanical damage to the cell be paved with, for screening several samples.Therefore, need a kind of high-throughout damage method to reach this purpose.
Summary of the invention
The invention provides a kind of multiple channel mechanical damage device for cell is damaged, be applicable to the mode cultured cells of porous cell culture plate is carried out to mechanical damage.It can produce in the same position in each hole big or small homogeneous, shape consistent, be easy to quantitative damage.
Multiple channel mechanical damage device of the present invention comprises:
In one aspect of the invention, wherein sell support and be provided with a plurality of holes 5, pin can insert in described hole, and is fixed and regulates by 6 pairs of pins of the screw on the pin support length that it stretches out the pin support.
In another aspect of this invention, described pin is suitable for removable disposable tip.
In another aspect of this invention, described multiple channel mechanical damage device also has the guide 4 that is positioned at pin support 1 both sides, and its angle can be regulated.For example can be fixed and adjusting angle by 7 pairs of guides 4 of the screw on pin support 1.
In a preferred version of the present invention, multiple channel mechanical damage device is 8 passage mechanical damage devices, comprising:
8 pins that are arranged in parallel 2, it inserts in the hole 5 of pin support, the length of by the fixing described pin of screw 6 on the pin support and the described pin of adjusting, stretching out the pin support;
Handle 3, it vertically is fixed on the pin support; With
Guide 4, it is positioned at pin support both sides, by 7 pairs of guides 4 of the screw on pin support 1, is fixed and adjusting angle.
In one aspect of the invention, the diameter of selling in 8 passage mechanical damage devices of the present invention is 3.5 ± 0.1mm, and each pin is at a distance of 9 ± 0.5mm, and the distance between two guides is 8.2cm.In an example, wherein handle is the long hollow stainless-steel tube of 12cm.
In one aspect of the invention, described multiple channel mechanical damage device is made by stainless steel.
The present invention also provides the method that cell is damaged, and comprising:
A. removable disposable tip is contained on the pin of multiple channel mechanical damage device of the present invention;
B. the length of pin is regulated, the length that makes each suction nozzle stretch out is identical, so that the while contacts with the lower surface in the hole of Tissue Culture Dish;
C. regulate the operating angle of two guides, so that pin and the disposable tip on it are positioned at the Central Line of each culture hole;
The disposable tip that d. will damage device vertically is placed on the same row's of Tissue Culture Dish the end in each hole, then will damage device and laterally move to its relative end;
E. optionally repeat same process, until the cell in upper all rows' of Tissue Culture Dish hole all passes through, scrape damage.
Aspect of said method, wherein said disposable tip is p-10 imbibition head.
Aspect of said method, wherein said cell is cell monolayer.Described cell can be HUVEC cell or IEC-6 cell.
Multiple channel mechanical damage device of the present invention can be made by stainless steel, and therefore whole damage device can carry out sterilizing by hyperbaric heating.Multiple channel mechanical damage utensil of the present invention has a plurality of pins that are arranged in parallel 2, its length of stretching out pin support 1 can be regulated respectively, this design can be guaranteed each pin, and when being equipped with suction nozzle, the bottom in each hole of all suction nozzles and porous cell culture plate is good contact simultaneously.The pin support 1 of multiple channel mechanical of the present invention damage device can insert pin 2 in each holes containing porose 5, by 6 pairs of pins of the screw on the pin support, be fixed and regulate the length that it stretches out the pin support.Pin support 1 both sides can have two guides 4.Guide 4 operating angles can be regulated, and to guarantee pin, are positioned at the Central Line of each culture hole.Can be fixed and adjusting angle by 7 pairs of guides 4 of the screw on pin support 1.Can insert removable disposable tip on pin, the bottom good contact of itself and porous cell culture plate, produce consistent damage to cell.Disposable tip can prevent cross pollution, and avoids suction nozzle because being recycled and reused for the damaging cells rust.Described suction nozzle can be plastics imbibition head, it has several advantages: 1) this suction nozzle has enough elasticity and hardness, can guarantee suction nozzle and hole surface good contact, 2) smoothness of suction nozzle can guarantee hole surface not to be caused to serious physical damage, 3) suction nozzle is cheap and can buy.Multiple channel mechanical damage utensil of the present invention has handle 3, vertically is fixed on the pin support.
In one aspect of the invention, provide a kind of 8 passage mechanical damage devices, be applicable to the mode cultured cells of 96 hole micro plates is carried out to mechanical damage.The pin 2 of this damage device is complementary with disposable p-10 imbibition head.This utensil is made by stainless steel, and whole damage device can carry out sterilizing by hyperbaric heating.Handle 3 permanently vertically is fixed on pin support 1.The pin support is provided with 8 holes 5, will sell and insert in each hole, by 6 pairs of pins 2 of the hex(agonal)screw on the pin support, is fixed and regulates the length that it stretches out the pin support.Use 8 pins that can regulate individually 2, this design can guarantee that, when the mechanical damage device is equipped with suction nozzle, the bottom in each hole of each suction nozzle and 96 hole micro plates is good contact simultaneously.This damage device is applicable to 96 orifice plates, and the operating angle of two guides 4 also can be regulated, and can guarantee that pin is positioned at the Central Line of each culture hole.These pins 2 are complementary with disposable p-10 imbibition head.
The present invention also provides the method for cell culture being damaged with above-mentioned 8 passage mechanical damage devices, comprising:
A. p-10 imbibition head is contained on pin;
The length of b. each pin being stretched out by hex(agonal)screw 6 with hex wrench is regulated, and the length that makes each disposable tip stretch out is identical, in order to contact with the lower surface in the hole of Tissue Culture Dish simultaneously;
The operating angle of c. by hex(agonal)screw 7, regulating two guides 4, so that pin and the p-10 imbibition head on it are positioned at the Central Line of each culture hole;
The p-10 imbibition head that d. will damage device vertically is placed on the same row's of Tissue Culture Dish the high order end in each hole, then will damage device and laterally move to low order end;
E. optionally repeat same process, until the cell in upper all rows' of Tissue Culture Dish hole all passes through, scrape damage.
In one aspect of the invention, described method is for damaging processing to cell monolayer.Wherein said cell can be HUVEC cell or IEC-6 cell.
The present invention also provides and has used the application of above-mentioned multiple channel mechanical damage device in research cell migration and potential biological phenomena thereof.
Above-mentioned general introduction of the present invention does not also mean that and has represented each embodiment of the present invention or various aspects.By the following detailed description of listing and accompanying drawing, other characteristics of the present invention and advantage will become apparent.
The accompanying drawing explanation
Fig. 1. 8 hole mechanical damage devices of the present invention
Handle 3 vertically is fixed on pin support 1.Pin support 1 is provided with 8 holes 5, and the pin 2 be arranged in parallel inserts in each hole, by the fixing length of stretching out with adjustment pin 2 of hex(agonal)screw 6.On pin support both sides, there is the operating angle of 4, two guides of two guides to regulate by hex(agonal)screw 7.
Fig. 2 A plugs the mechanical damage device photo of suction nozzle
The adjusting of the pin of Fig. 2 B. damage device.
The length that each pin stretches out is regulated by hex(agonal)screw.By trickle like this adjusting, can guarantee that the length that each suction nozzle stretches out is identical, in order to contact with the lower surface in hole is coherent.
Fig. 3. the damage device of duty.
The mechanical damage device vertically is placed on to the high order end in each hole in same row, then level is shifted to low order end.Each round is repeated to identical process.
Fig. 4 A and Fig. 4 be the effect of passage mechanical damage device B.8.
The image of whole 96 orifice plates after Fig. 4 A damage.The IEC-6 cell is seeded on 96 orifice plates and spends the night, and inoculum density is 4 * 10
4.Scraped damage with 8 passage damage devices, thereby produced damage.After damage, with Hemacolour 3, to these cell dyeings, examine whole plate.
The statistical study of damage in each hole of Fig. 4 B.After damage, for checking the consistance of damage, measure immediately the width of the damage in each hole.In 96 orifice plates, the mean breadth of each damage is 600 μ m, and SD is 36.6.
The migration of IEC-6 cell after Fig. 5 A and Fig. 5 B. damage.
The IEC-6 cell of Fig. 5 A damage epidermal growth factor (EGF) (20ng/ml) or DL-alpha-difluoromethyl ornithine (DFMO) (5mM) exist or non-existent situation under, process 24 hours.At 0 time point, damage 16 hours after and damage take the microphotogram of healing after 24 hours.
Fig. 5 B EGF and DFMO reflect in order to damage make percent for the work of IEC-6 cell migration.Result means with the standard deviation of mean value ± mean value.With respect to medium contrast, p<0.001.
The migration of HUVEG after Fig. 6 A and Fig. 6 B. damage.
The microphotogram of impaired HUVEC before and after Fig. 6 A FBS processes.With the FBS (0%, 1% and 20%) of variable concentrations, impaired HUVEC is processed 24 hours, at 0 time point, damage latter 16 hours and damage and within latter 24 hours, take microphotogram.
Fig. 6 B FBS reflects by the damage make percent for the effect of HUVEC migration.Result means with the standard deviation of mean value ± mean value.With respect to medium contrast, p<0.001.
Embodiment
Although the present invention easily is subject to the impact of various changes and replaceable form, we by the example in accompanying drawing concrete embodiment and being explained in detail at this.Yet it is to be understood that, the present invention is not intended to be limited to particular forms disclosed.But, present invention resides in marrow of the present invention that appending claims limits and all deformations, equivalent and the substitute within scope.
As provided 8 passage mechanical damage devices of one of them embodiment of the present invention in Fig. 1, be applicable to the mode cultured cells of 96 hole micro plates is carried out to mechanical damage.
Whole utensil is made by stainless steel, can carry out sterilizing by hyperbaric heating.Handle 3, for the long hollow stainless-steel tube of 12cm, vertically is fixed on pin support 1.Pin support 1 is provided with 8 holes 5, and the pin 2 be arranged in parallel inserts in each hole, and the corresponding pin in each hole, by the fixing length of stretching out with adjustment pin 2 of hex(agonal)screw 6.Use 8 pins 2, the diameter of these pins is 3.5 ± 0.1mm, is suitable for disposable p-10 imbibition head.Each pin is arranged in parallel within on the pin support, at a distance of 9 ± 0.5mm (center distance).Pin has two guides 4 on the support both sides.Distance between two guides is made as 8.2cm, equals 96 well culture plates (Iwaki, code: width 3860-096).The operating angle of two guides can be fixed and regulate by hex(agonal)screw 7, guarantees that pin is positioned at the Central Line of each culture hole.
During use, with hex wrench, regulate hex(agonal)screw, thereby regulate the length that each pin stretches out.By trickle like this adjusting, can guarantee that the length that each suction nozzle stretches out is identical so that with the lower surface in hole coherent contact the simultaneously.
Before cell is damaged, p-10 imbibition head is contained on pin, the extension elongation of each pin can be regulated by hex(agonal)screw with hex wrench, makes the p-10 imbibition head be arranged in parallel can contact with the culture hole surface simultaneously.This design, add the plastics suction nozzle and have suitable consistency and elasticity, can greatly strengthen the good contact on suction nozzle and culture hole surface, guarantees cell monolayer is produced to coherent, consistent damage.
When cellular damage is damaged to operation, the imbibition head is contained on the damage device, vertically is placed on the high order end in each hole in same row, then will damage device and laterally move to low order end.Repeat same process, until each row's cellular layer all passes through, scrape damage.
The effect that this mechanical damage device has been damaged for the cell monolayer of estimating human umbilical vein endothelial cell (HUVEC) to cultivating and intestine in rats epithelial cell (IEC) in 96 orifice plates and for studying the damage migration of HUVEC cell and IEC cell afterwards.
Experimental technique and material
Clone and cell culture
Intestine in rats epithelial cell strain (IEC-6 cell; ATCC CRL 1592).The IEC-6 cell line comes from the intestines crypt cell, presses morphology and the judgement of immunology standard.The reserve cell culture remains on 75cm
2in Dulbecco improvement Eagle medium (DMEM) in culture flask, supplement 10% hyclone (FBS), 10 μ g/ml insulin and 50 μ g/ml gentamicin sulphates.These cells are cultivated under 37 degrees centigrade in the carbon dioxide culture apparatus (5% carbon dioxide) of humidification.The 14th generation in generation to 20 cell for experimental study.
Human umbilical vein endothelial cell (HUVEC) is purchased from Clonetics (TCS Biologicals, UK).HUVEC cultivates in the M199 medium, supplement 20 μ g/ml endothelial cell growth replenishers (ECGS), 20% thermal inactivation hyclone (FCS), 1% penicillin-streptomysin (PS), 50ng/ml amphotericin-B and 90 μ g/ml heparin are cultivated on the culture flask that scribbles 0.1% gelatin.These cells are under 37 degrees centigrade in the carbon dioxide culture apparatus (5% carbon dioxide) of humidification, cultivate.2nd generation to 7 generation cell is for experimental study.
Material
Dulbecco improvement Eagle medium (DMEM), FBS, FCS, phosphate buffer (PBS), penicillin-streptomysin (PS), trypsase, gentamicin sulphate, insulin and Sodium Pyruvate are from Invitrogen (Carlsbad, California, USA) obtain.The M199 medium, endothelial cell growth replenishers (ECGS), heparin, amphotericin-B and epidermal growth factor (EGF) obtain (Saint Louis, Missouri, USA) by Sigma.Matrigel is obtained by BDBioscience (Palo Alto, California, USA).Hemacolour 3 is purchased from Merck, and DL-alpha-difluoromethyl ornithine (DFMO) is purchased from Calbiochem (USA).
The cellular damage test
The IEC-6 cell is seeded in in the 96 flat micro plates in hole, (Iwaki, code: 3860-096), density is every hole 4 * 10
4individual cell, spend the night with Matrigel (1: 7 (the v/v)) covering of PBS dilution in advance.Cultivate in the DMEM that has supplemented 10%FBS after 24 hours, these cells are not being cultivated 24 hours containing in the medium of serum again.For carrying out the damage test of HUVEC, cell is seeded in to the 96 flat micro plates in hole that scribble in advance 0.1% gelatin, and (Iwaki, code: 3860-096), density is every hole 3 * 10
4individual cell.Scrape the damage cell monolayer with the 8 passage mechanical damage devices that p-10 imbibition head (Axygen, T-30) is housed, cause cell to strip off zone.Regulate hex(agonal)screw with hex wrench, the length of pin is proofreaied and correct, with coherent contacting between the bottom that guarantees suction nozzle and hole.Under the help of guide, will damage device and be fixed on 96 orifice plates, make each passage (imbibition head) be positioned at 8 same depth level in hole of same row.During damage, the damage device is moved on to the right side internal edge (Fig. 3) in hole from the left side internal edge in hole, cell monolayer is scraped to damage.Then, the diameter in each Nei Yan hole, hole forms sharp keen damage.Each round to 96 orifice plates repeats this damage process (Fig. 4 A).Then, for control wells, with fresh serum-free medium, replace medium in hole; For the IEC-6 cell, with the fresh serum-free medium that contains 20ng/ml EGF or 5mM DFMO, replace medium in hole; For HUVEC, with the FBS of different weight percentage, replace medium in hole.
Image capture
By catching cell image, can observe the situation of impaired cell monolayer.
Catch the cell migration image by the time interval with different, easily the on cell migration degree is quantitative.Take the image of 0 time point, to record initial damage width, after 16 hours and after 24 hours, utilize quantitatively (Motic InstrumentsInc. of Motic image and 2.0 softwares, Richmond, Canada) for being estimated to the recovery of stripping off the impaired cell monolayer that regional migration causes because of cell.Cell means with the damage make percent to the migration of damage.
Damage make percent=[(Wt
0-Wt
16 or 24)/Wt
0] * 100%
Wherein, Wt
0the=0th, scrape the damage width recorded immediately after damage, Wt
16 or 24to scrape to damage the damage width recorded in latter 16 hours or 24 hours
Statistical study
Every test is at least carried out three times, and each is processed and repeats 8 parts.Data mean with mean value ± standard deviation.Adopt Si Shi t check to carry out statistical study.P<0.05, think significant difference arranged.
With cellular damage device of the present invention, and by enterocyte (IEC-6) and Human umbilical vein endothelial cells (HUVEC) as model, according to above-described cell, cultivate and the damage method of operating, in 96 orifice plates, checking cellular damage device of the present invention damages the effect of operation to cultured cells, particularly estimates the consistance that it produces damage.
By catching cell image, can observe the situation of impaired cell monolayer.Take the image of impaired cell monolayer, measure the width of each damage.Can see that from Fig. 4 A there is consistent injury mark in the central authorities in each hole.In 96 orifice plates, as shown in Figure 4 B, mean breadth is 600 μ m to the Size Distribution of each damage, and standard deviation is 36.6.
Result proves that this damage device uses disposable plastics imbibition head, can be in 96 orifice plates the middle body in each hole strip off equably the cell monolayer of certain area in each hole, average lesion size is 600 ± 36.6 μ m.
With cellular damage device of the present invention, research cell migration promoter (EGF or FBS) or cellular migration inhibition agent (DFMO) are for the impact of healing.
1. the restructuring procedure of impaired IEC-6 cell
The IEC-6 cell is often used as the cell model in damage test, because it has transport property, can demonstrate typical wound healing reaction.In order to simulate the recovery of epithelial damage in enteron aisle, remove the part cell in the IEC-6 cell monolayer with the damage device, within after causing damage 16 hours and 24 hours, observe and strip off regional recovery situation due to what cell migration caused.Introducing may be regulated the material of IEC-6 cell migration character, further to estimate the applicability of this damage device.DFMO is a kind of known cellular migration inhibition agent, and DFMO suppresses ornithine decarboxylase and reduces the synthetic of polyamine.Owing to what recover, needing in early days polyamine, so lack polyamine, can affect cell migration.On the other hand, select the derivant of EGF as cell migration.In the damage system of this experiment, the IEC-6 cell has typical reaction [Fig. 5 A and 5B] for the effect of DFMO and EGF.The closed percent of damage that compared with control cells is damaged latter 16 hours is approximately 60%, and after the cellular damage that EGF processes, the closed percent of the damage of 16 hours increases to 90%; After the cellular damage that DFMO processes, the closed percent of the damage of 16 hours only drops to 33%.Damage latter 24 hours, damaging closed percent further increases.The closed percent of damage that compared with control cells is damaged latter 24 hours is 76%, and after the cellular damage that EGF processes, the closed percent of the damage of 24 hours is almost 100%; After the cellular damage that DFMO processes, the closed percent of the damage of 24 hours only about 50%.Damage system of the present invention clearly shows, and processes latter 24 hours, and EGF is the inhibiting effect to the IEC-6 cell migration to the facilitation of IEC-6 cell migration and DFMO.Therefore, this damage system can be used for the Large-scale Screening of cell migration derivant or inhibitor.
2. the restructuring procedure of impaired HUVEC cell
HUVEC is usually used in studying Angiogenesis and wound healing.In latter 16 hours of damage and observation of cell migration [Fig. 6 A] in 24 hours.The cell of processing with different proportion (0%, 1% and 20%) FBS, the migration rate difference, demonstrate the wound healing reaction [Fig. 6 B] of concentration dependent.Only use the cell migration speed of media processes relatively low, damage closure after 24 hours is lower than 40%, and the cell migration of processing with 20%FBS is very fast, after 24 hours, damage is fully closed, the cell migration speed of processing with 1%FBS is placed in the middle, after 16 hours, the damage closure is that after 60%, 24 hour, the damage closure is 72%.
Conclusion
Successfully prove, 8 passage mechanical damage devices of the present invention are to carry out the useful instrument of damage test, can, in 96 hole microtest plates, make cell monolayer that consistent and coherent stripping off occur in the zone limited.This easy assembling and be convenient to device that the user uses be highly suitable in cell migration research or the wound healing test in high-throughout sample screening.
From following table 1 can find out 8 passage mechanical damage devices of the present invention and the method cell damaged with it with respect to the advantage of tradition damage device and method.
Table 1. 8 passage mechanical damage devices of the present invention and use and traditional comparison that damages device and method.
| traditional damage device and method | damage device of the present invention and method |
| the great majority such as imbibition head or strigil damage device is made of plastics, and easily damages. | damage device of the present invention is made by stainless steel, very durable. |
| great majority damage device is non-autoclavable due to the character of plastics. | damage device of the present invention can be through hyperbaric heating. |
| be difficult to the cellular damage effect that reaches coherent | the length that the pin of damage device of the present invention stretches out can be regulated, and guarantees suction nozzle and hole surface good contacting simultaneously, therefore can obtain damage simultaneously, coherent.The design of guide can guarantee that the damage position in each hole is consistent. |
| when being damaged with metallic spatula, often culture hole is caused to serious mechanical damage.Such damage can have a strong impact on cell migration and image analysis. | with plastics imbibition head, damaged, its elasticity can be guaranteed to contact with the coherent of hole lower surface, but can not cause damage to culture hole. |
| same damage device (for example scraper) is used in whole test, and the chance of cross pollution is relatively large. | with disposable imbibition head, can prevent the cross pollution between Kong Yukong, be convenient to large-scale test and high-throughout screening. |
The present invention has been easy to various changes and replacement form, and its concrete embodiment is expressed out and is explained in detail at this by the example in accompanying drawing.Yet, it is to be understood that, and do not mean that the present invention only is confined to disclosed particular form, and but contrary, the objective of the invention is to have comprised the marrow of the present invention that limits and all changes, equivalent and the substitute within scope in appending claims.
Claims (11)
1. one kind is damaged device for the multiple channel mechanical that cell is damaged, and described damage device comprises:
Pin support (1);
A plurality of pins that are arranged in parallel (2), it is connected with pin support (1), and described pin stretches out the adjustable in length of pin support; With
Handle (3), it is connected with the pin support;
Described damage utensil has the guide (4) that is positioned at pin support (1) both sides, and its angle can be regulated;
Wherein sell support and be provided with a plurality of holes (5), pin can insert in described hole, and pin is fixed and regulates by the screw on the pin support (6) length that it stretches out the pin support.
2. the multiple channel mechanical of claim 1 damage device, wherein be fixed and adjusting angle guide (4) by the screw (7) on pin support (1).
3. in claim 1-2, the multiple channel mechanical of any one damages device, and described pin is suitable for inserting removable disposable tip.
4. in claim 1-2, the multiple channel mechanical of any one damages device, and it is 8 passage mechanical damage devices, comprising:
Pin support (1), it is provided with 8 holes (5);
8 pins (2), it inserts respectively the hole (5) of pin support, the length of by the fixing described pin of the screw on the pin support (6) and the described pin of adjusting, stretching out the pin support;
Handle (3), it vertically is fixed on the pin support; With
Guide (4), its position, in pin support both sides, is fixed and adjusting angle guide (4) by the screw (7) on pin support (1).
5. the multiple channel mechanical of claim 4 damage device, wherein the diameter of pin is 3.5 ± 0.1mm, and each pin is at a distance of 9 ± 0.5mm, and the distance between two guides is 8.2cm.
6. in claim 1-2, the multiple channel mechanical of any one damages device, and it is made by stainless steel.
7. the method for cell being damaged comprises:
A. the multiple channel mechanical that removable disposable tip is contained in to any one in claim 1-6 damages on the pin of device;
B. the length of pin is regulated, the length that makes each suction nozzle stretch out is identical, so that the while contacts with the lower surface in the hole of Tissue Culture Dish;
C. regulate the operating angle of two guides, so that pin and the disposable tip on it are positioned at the Central Line of each culture hole;
The disposable tip that d. will damage device vertically is placed on the same row's of Tissue Culture Dish the end in each hole, then will damage device and laterally move to its relative end;
E. optionally repeat same process, until the cell in upper all rows' of Tissue Culture Dish hole all passes through, scrape damage.
8. the method for claim 7, wherein said cell is cell monolayer.
9. the method for claim 7, wherein said cell is Human umbilical vein endothelial cells or enterocyte.
10. the method for claim 7, wherein said disposable tip is p-10 imbibition head.
11. the application of the multiple channel mechanical of any one damage device in research cell migration and matrix rebuilding or polarization in claim 1-6.
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|---|---|---|---|
| CN200710127846.1A CN101339104B (en) | 2007-07-03 | 2007-07-03 | Mechanical damaging device for cell damage processing |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN101339104B true CN101339104B (en) | 2014-01-08 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1602350A (en) * | 2001-12-19 | 2005-03-30 | 天然植入物公司 | Device for active phase cell or tissue sampling and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1602350A (en) * | 2001-12-19 | 2005-03-30 | 天然植入物公司 | Device for active phase cell or tissue sampling and uses thereof |
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