CN104017768A - Cell in-vitro collection method and application thereof as well as in-vitro cell material and application thereof - Google Patents
Cell in-vitro collection method and application thereof as well as in-vitro cell material and application thereof Download PDFInfo
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Abstract
The invention discloses a cell collection method and a cell material. Aiming at the defect that scientific content for prompting experiment result in the conventional Transwell migration research is limitedly utilized, the invention provides an in-vitro collection method for the first time. According to the method, after the Transwell cell migration culture is completed and culture liquids inside an upper chamber and a lower chamber of Transwell are eliminated, cells in the upper chamber and the lower chamber after the Transwell cell migration experiment are digested and collected by using a method with the combination of trypsinization and mechanical oscillation; in pancreatin cell digestion operation, a phosphate buffer liquid of which the pH value is 7.35-7.45 is used as a washing liquid, and a pancreatin cell digestion liquid contains 0.25% of pancreatin and 0.01 EDTA (Ethylene Diamine Tetraacetic Acid). The invention further provides a high migration living cell material and a low migration living cell material generated from identical chemotactic factor induction through separation. The invention further provides a collection method and an application of the cell material in a cell biological behavior analysis experiment. The Transwell in-vitro migration experiment is expanded to cell biological behavior and molecular biology study on cell migration.
Description
Technical field
The present invention relates to a kind of cell collection method, cell material, particularly relate to a kind of cells in vitro collection method, and cell in vitro material.Belong to biological, medical cell experiment in vitro technical field.
Background technology
Cell migration is the Chemotaxis that cell produces under exogenous token stimulus, and normally cell moves from lower concentration king area with high mercury after experiencing the concentration gradient of Cucumber.Under physiological status, in the processes such as cell migration is looked for food at small cell, wound healing, embryo's generation, tissue repair, immune response, play an important role; Under pathological state, cell migration has also promoted generation and the development of the diseases such as cancer metastasis, atherosclerosis and sacroiliitis.Thereby cell migration is an important research direction of current life science.
Transwell migrating technology is the experimental technique of current most widely used research cell in vitro migration.Testing required Transwell migration system adopts Transwell cell (Transwell insert) and common porous cell culture plate jointly to build.Transwell cell profile is a cup-formed device that can be placed in ordinary cells culture plate orifice plate, bottom band permeability film, on film with the micropore of pore size 0.1-12.0 μ m.In research, according to different experiment purposes, select different micro-pore diameters.Cell migration research is selected the Transwell system with 8.0 μ m or 12.0 μ m micropores conventionally.In experiment, Transwell cell is put into ordinary cells culture plate, in Transwell cell, deserve to be called Huo Nei chamber, chamber, in Tissue Culture Plate, claim lower chamber or mistress.In experiment, by adding various chemokines in lower chamber, inducing cell is down chamber migration from upper chamber.According to production firm's difference, Transwell cell claims again Boyden Chamber or Thincert cell.Due to the general Transwell cell that adopts Corning company of the U.S. to produce in field, thereby this class cell in vitro migration research method extensively adopts " Transwell method " as its conventional title at present.Transwell migrating technology reliability is strong, reproducible, is cell migration research method the most widely at present.But the analytical procedure in Transwell migration research, experimental result being adopted is at present all fairly simple, normally simple analytical calculation cell migration rate, utilizes limited to the scientific contents of experimental result prompting.
In cell paste, in research, disclosed the various cell behaviors of the active high and low cell of cell migration, comprised that cell adhesion, propagation, differentiation, apoptosis etc. are evenly significantly different, Chinese scholars conduct in-depth research this.But the restriction of research in acceptor, investigator is difficult to obtain the cell with different migratory activities and further studies.From the principle of design analysis of Transwell migration experiment, the external migration experiment of Transwell can be divided into cell material the two group cells of transfer ability by force and a little less than transfer ability, has created precondition for completing above-mentioned cells in vitro behavioral study.But, the migration of cell in Transwell cell is a very complicated process: the protein of cell interior and ion rearrange after the concentration gradient of the upper and lower cell of perception, and utmost point shape foot is stretched out in sex change, form adhesion plaque, tractive cell forward, thereby through narrow and small Transwell micropore, from Transwell film, chamber moves to lower chamber gradually.Because eukaryotic diameter is generally between 10 μ m～100 μ m, much larger than the micro-pore diameter of Transwell migration experiment 8.0 μ m～12.0 μ m.Thereby in Transwell cell migration experiment, cell, except sticking on two bottom surfaces up and down of Transwell film, also has a large amount of cell meetings and " falls into " in the micropore on Transwell film, be difficult to effectively collect for follow-up research.Especially Transwell cell area is less, adopts conventional chemical digestion mode cannot carry out batched operation, and cell harvesting amount is lower, is not enough to for follow-up Celluar and Molecular Biology research.In addition, in conventional cell dissociation method, cell is to cultivate plane from it to come off, and only need to destroy the connection between cell when cell dissociation; And in Transwell migration system, cell will be less than through aperture the narrow and small micropore of its diameter, when digestion collecting cell, need cell to depart from fast from the attached micropore of its embedding, very easy damaged cells of this process, even causes lysis, fragmentation.Therefore, on the whole, although it is simple that cell in vitro investigative technique has intervention factor, experiment condition is easily controlled, fast economical, reproducible, and can carry out the advantages such as deep Mechanism Study, but be subject to the restriction of prior art condition, make cell in vitro migration experiment conventionally can only understand simply the transfer ability of cell by analysis of cells mobility, and the desirable result cell by migration experiment of investigator further utilizes, for example obtaining the cell of high migratory activity further studies, the research of comparing the biological behaviour of migrating cell and non-migrating cell under cells in vitro experiment condition is difficult to carry out all the time.
Summary of the invention
Object of the present invention is exactly for the deficiencies in the prior art, and a kind of cells in vitro collection method is provided, and the method specific aim combines with cell migration experiment, and the cell after migration experiment is finished is effectively collected; The cell of collecting is the cell under the induction of identical chemokine with different migratory activities, can be applicable to the comparative studies of migrating cell and non-migrating cell under cells in vitro experiment condition; This cells in vitro collection method can be applicable to cell clone, propagation, the tests such as apoptosis, gene and protein expression, morphologic detection.
For achieving the above object, first the present invention provides a kind of cells in vitro collection method, and its technical scheme is as follows:
A kind of cells in vitro collection method, is characterized in that: be applied to Transwell cell migration experiment; Transwell cell migration is cultivated and is finished and remove after the upper chamber of Transwell, lower indoor cultivation liquid, adopts trysinization and respectively the cell in upper chamber, lower chamber after Transwell cell migration experiment is digested, collected in conjunction with the method for mechanical oscillation; In the operation of pancreatin cell dissociation, washings is pH 7.35～7.45 phosphate buffered saline buffers, and pancreatin cell dissociation buffer is containing 0.25% pancreatin and 0.01%EDTA.
Cells in vitro collection method technology basic technique principle of the present invention is that external Transwell migration experiment is combined with external cell culture method.Transwell cell migration is cultivated and is finished and remove Transwell and go up chamber, after lower indoor cultivation liquid, cell residual and that be attached to Transwell permeability film upper surface is non-migrating cell (transfer ability is low), migrating cell (transfer ability is high) and be attached to the cell that Transwell permeability film lower floor surface and embedding be attached in Transwell permeability membrane micropore, can will be attached to permeability film surface and be embedded in the cell dissociation in micropore and collect by trysinization and in conjunction with the method for mechanical oscillation, thereby the cell that obtains non-migratory behavior under Transwell cell in vitro migration experiment condition carries out next step research with there being the cell of migratory behaviour.
Under optimal way, above-mentioned cells in vitro collection method can be implemented according to following steps:
Step S1, washing for the first time
Transwell cell migration is cultivated and is finished and remove after the upper chamber of Transwell, lower indoor cultivation liquid, in upper chamber, lower chamber, add respectively phosphate buffered saline buffer washing culturing room, in collection, in chamber Yu Xia chamber, washings deposits in respectively in two centrifuge tubes, is labeled as respectively centrifuge tube A, centrifuge tube B;
Step S2, digestion are collected
In the upper chamber of Transwell, lower chamber, add respectively pancreatin cell dissociation buffer, treat cell dispersion suspension, collect respectively Digestive system correspondence in upper chamber, lower chamber and add in centrifuge tube A, centrifuge tube B, in centrifuge tube A, centrifuge tube B, add bovine serum to stop cell dissociation respectively;
Step S3, washing collection again
In the upper chamber of Transwell, lower chamber, add phosphate buffered saline buffer respectively, level vibration, collects respectively washings correspondence in upper chamber, lower chamber and adds in centrifuge tube A, centrifuge tube B;
Step S4, centrifugal collecting cell
By centrifugal to centrifuge tube A, centrifuge tube B, remove supernatant liquor, in centrifuge tube A, centrifuge tube B, add phosphate buffered saline buffer respectively, resuspended and collect centrifuge tube bottom cell mass, obtain collecting cell.
Above-mentioned cells in vitro collection method, need where necessary repeating step S3, again washing operation once, digestion collect operation after carry out twice washing collection.
Above-mentioned cells in vitro collection method is carried out the collection of 2～3 attached cells altogether, only have the 1st time, be in step S2, in trysinization liquid, carry out, all the other all adopt phosphate buffered saline buffer washing to collect in conjunction with the method for horizontal shaking table mechanical oscillation, such operation can reduce cell and be exposed to the time in pancreatin, can improve again the collection effciency of cell.
Aforesaid method can at utmost improve collection effciency by the cooperation between the main operational steps of front and back.Specifically:
In step S1, in the upper chamber of Transwell, lower chamber, add phosphate buffered saline buffer washing, collect a small amount of floating non-adherent cell.
In step S2, in the upper chamber of Transwell, lower chamber, add respectively pancreatin cell dissociation buffer can digest the cell that is attached to the upper and lower both sides of Transwell film, having to collection in the centrifuge tube of phosphate buffered saline buffer and trypsin solution adds bovine serum to stop digestion reaction, stop digestion reaction but not directly add in chamber, lower chamber containing the cell culture medium of bovine serum on Transwell, can avoid producing bubble in Yu Xia chamber, upper chamber, both reduce the damage to cell, improved again cell harvesting rate.In the process of conventional trypsin digestion cell, in pancreatin, to stop cell dissociation reaction by adding containing the substratum of bovine serum, if and in the method like this operation, due to the existence of serum composition, in piping and druming collecting cell process, be easy to produce bubble, directly produce 3 aspect harm: the first, bubble mechanical stress in the time breaking is easy to damaging cells; The second, once bubble produces the collection that can affect liquid, cause cell harvesting not thorough; The 3rd, the sharpness in the visual field once bubble exerts an influence, is difficult for the digestible degree at observation of cell under inverted microscope.And in the inventive method step S2, owing to not needing to blow and beat cell, can thoroughly avoid Bubble formation.Meanwhile, owing to directly adding in bovine serum and digestion reaction, instead of add the substratum containing a small amount of bovine serum, also can reduce the volume of whole collection liquid, thereby reduce the time of follow-up centrifugal collection.
In step S3, by again adding phosphate buffered saline buffer washing and vibrate in conjunction with mechanical level in the upper chamber of Transwell, lower chamber, be convenient to residual cell detachment, can again collect and residue in the cell of Transwell film both sides and be embedded in the cell in Transwell membrane micropore.In washing, adopt horizontal mechanical vibration make cell from Transwell permeability film, upper surface departs from and unconventional suction pipe piping and druming cell causes it and departs from, and can effectively reduce damage to cell, improve collection rate, and simplified operation, improves operation efficiency.
In step S4, through centrifugal, resuspended operation, collect and obtain target cell respectively.
Under optimum condition, above-mentioned cells in vitro collection method can be done respectively following optimization:
Optimize one: in method, use the phosphate buffer solution of washings for pH 7.4.
Optimize two: in step S2, contain under 0.25% pancreatin and 0.01%EDTA condition at pancreatin cell dissociation buffer, pancreatin cell dissociation buffer add-on is advisable to cover respectively bottom, Yu Xia chamber, the upper chamber of Transwell.The centrifugation of trypsinase to cell and the type of cell and the character of cell are in close relations, and different tissues or clone are different to tryptic action-reaction, and the activity of trypsinase cell dispersion is also relevant with its concentration, temperature and action time.Premenstruum, test was determined, in the inventive method, testing under the condition combining with Transwell cell migration, in the upper chamber of Transwell, lower chamber, the amount of pancreatin cell dissociation buffer is important cell decentralised control condition.For conventional Transwell culturing room, the pancreatin cell dissociation buffer add-on of suggestion is: the upper chamber of 24 hole Transwell culturing room (Transwell cell diameter 6.5mm) adds 0.1ml, lower chamber adds 0.6ml; The upper chamber of 12 hole Transwell culturing room (Transwell cell diameter 12mm) adds 0.5ml, lower chamber adds 1.5ml; The upper chamber of 6 hole Transwell culturing room (Transwell cell diameter 24mm) adds 1.5ml, lower chamber adds 2.6ml.
Optimize three: in step S2, use 100% bovine serum to stop digestion reaction, its objective is and reduce total liquid volume in centrifuge tube, reduce follow-up centrifugation time and improve cell harvesting amount.Add bovine serum to stop digestion reaction owing to adopting in step S2 in collection has the centrifuge tube of phosphate buffered saline buffer and trypsin solution, the phosphate buffered saline buffer of collecting in advance in centrifuge tube plays dissemination to trypsin solution, therefore use 100% bovine serum can stop smoothly digestion reaction, and control on this basis total liquid volume in centrifuge tube, improve collection effciency.
Optimize four: in step S3, level vibration adopts horizontal shaking table vibration, condition is 100r/min～150r/min, vibration 5min.In cell dissociation operation, adopting suction pipe piping and druming cell is to make the attached cell usual manner disperseing that comes off, but test premenstruum of the present invention is found, due to the singularity of Transwell cell bottom permeability membrane structure, adopting suction pipe to blow and beat the cell that is difficult to embedding is attached in permeability membrane micropore splits away off completely, and easily produce bubble, large to cell damage, cell harvesting efficiency is not high.And the slight mechanical oscillation the effective oscillation amplitude of controlling that adopt horizontal shaking table to realize, cell unbalance stress while avoiding blowing and beating cell avoids producing bubble simultaneously, is beneficial to be attached in poromerics evenly to come off with the cell on permeability film surface.Can less cell injury, significantly improve again collection rate.
Optimize five: in step S4, upper Transwell chamber, lower chamber centrifuge tube is centrifugal, and centrifugal condition is: rotating speed 200RCF (xg)～250RCF (xg), time 10min.
Optimize six: in step S4, in centrifuge tube, add pH 7.35～7.45 phosphate buffered saline buffer 0.5ml～1ml.
Cells in vitro collection method of the present invention can be applicable to cell behaviors analytical test, includes but not limited to cell clone, cell proliferation, and expression test, the morphocytology of apoptosis, cytogene and albumen detect test etc.
The cell that adopts cell in vitro collection method of the present invention to collect is the cell that shows high and low two kinds of migratory activities under identical chemokine induction, can act on cell behaviors and molecular biology research that desirable cell material is applied to cell migration, comprise cell clone, propagation, the expression of apoptosis, gene and albumen, and morphologic detection etc.Based on this, the invention provides a kind of cell in vitro material, its technical scheme is as follows:
A kind of cell in vitro material, is characterized in that: comprise by the lower high transport property viable cell material and the low migratory activity cell material that produce of separating of identical chemokine induction; First the preparation of described cell in vitro material adopts Transwell migration experiment that the migration under chemokine induction of initiating cell material is cultivated, the above-mentioned cell in vitro collection method of employing collecting cell after Transwell cell migration cultivation finishes, in the upper chamber of Transwell, collecting the cell material obtaining is described low migration viable cell material, and under Transwell, in chamber, collecting the cell material obtaining is described high transport property viable cell material.
Above-mentioned cell in vitro material can be applicable to cell behaviors analytical test, includes but not limited to cell clone, cell proliferation, and apoptosis, cytogene and protein expression, morphocytology detect test etc.
Somatocyte collection method of the present invention can combine with various kinds of cell biological behaviour experimental technique, comprises cell clone experiment, cell proliferation experiment, the expression experiment of cell apoptosis assay, gene and albumen, and morphocytology detection etc.Its technical scheme is as follows:
A kind of cell behaviors analytical procedure, it is a kind of morphocytology detection method, it is characterized in that: first adopt Transwell migration experiment that the migration under chemokine induction of initiating cell material is cultivated, the above-mentioned cell in vitro collection method of employing collecting cell after Transwell cell migration cultivation finishes, obtains respectively high transport property viable cell material and low migratory activity cell material; Secondly respectively described high transport property viable cell material and low migratory activity cell material are seeded to cell culture medium and carry out cells in vitro cultivation; Cultivate and finish to detect respectively high transport property viable cell material and low migratory activity cell material form when cell in vitro; The substratum that described cells in vitro is selected in cultivating and culture condition are determined according to initiating cell material behavior.
A kind of cell behaviors analytical procedure, that a kind of cytoskeleton is learned detection method, it is characterized in that: first adopt Transwell migration experiment that the migration under chemokine induction of initiating cell material is cultivated, the above-mentioned cell in vitro collection method of employing collecting cell after Transwell cell migration cultivation finishes, obtains respectively high transport property viable cell material and low migratory activity cell material; Secondly the cytoskeleton dyeing to described high transport property viable cell material and low migratory activity cell material respectively; Dyeing finishes rear observation and detects cytoskeleton; Described cytoskeleton dyeing process is determined according to initiating cell material behavior.
A kind of cell behaviors analytical procedure, it is a kind of cell self-renewal ability detection method, it is characterized in that: first adopt Transwell migration experiment that the migration under chemokine induction of initiating cell material is cultivated, the above-mentioned cell in vitro collection method of employing collecting cell after Transwell cell migration cultivation finishes, obtains respectively high transport property viable cell material and low migratory activity cell material; Secondly respectively high transport property viable cell material and low migratory activity cell material are carried out to cell clone culture; Clone cultivates the poststaining detection cell self-renewal ability that finishes; Described cell clone culture condition is determined according to initiating cell material behavior.
A kind of cell behaviors analytical procedure, it is a kind of ability of cell proliferation detection method, it is characterized in that: first adopt Transwell migration experiment that the migration under chemokine induction of initiating cell material is cultivated, the above-mentioned cell in vitro collection method of employing collecting cell after Transwell cell migration cultivation finishes, obtains respectively high transport property viable cell material and low migratory activity cell material; Secondly the ability of cell proliferation that detects respectively high transport property viable cell material and low migratory activity cell material, detection method is determined according to initiating cell material behavior.
Compared with prior art, the invention has the beneficial effects as follows: (1) cell in vitro collection method of the present invention combines external Transwell migration experiment with external cell culture method, the later cell of the external migration of Transwell is digested and collected, collect and obtain the cell of high migratory activity and low migratory activity respectively, make the external migration of Transwell test the cell behaviors and the molecular biology research that are extended to cell migration from the transfer ability of simple analysis of cells and transport efficiency, (2) the outer cell collection method of the present invention is in each operation steps, by design different cell collection methods (collect or collect) in phosphate buffered saline buffer in trysinization liquid, control pancreatin cell dissociation buffer concentration and digestion time, selection adds bovine serum to stop step and the environment of cell dissociation reaction, in conjunction with come off from the culturing room optimization of the six aspect underlying conditions such as method of disperseing of mechanical oscillation helper, ensure on the whole can effectively collect the few cells of migration results under the meticulous condition of Transwell migration experiment, guarantee is collected enough cells for follow-up research, (3) the invention provides by identical chemokine induction is lower and separate the high transport property viable cell material and the low migratory activity cell material that produce, this material is desirable cell migration material, can be applied to cell behaviors and molecular biology research, (4) the invention provides the various kinds of cell biological behaviour experimental technique combining with somatocyte collection method, (5) the inventive method provides new technique means for cell in vitro migration mechanism and behavioral study, and this technique means is based upon on the external migration experiment of ripe Transwell, applied range.
Brief description of the drawings
Fig. 1-1: embodiment 1 cell inoculation-collecting amount curve.The collecting amount of result showed cell becomes positive correlation with inoculum size.
Fig. 1-2 a, Fig. 1-2 b showed cell collection effciency.Fig. 1-2 a shows that in embodiment 1, cell, after Transwell cultivates 16h, migrates to the human marrow mesenchymal stem cell violet staining picture of lower chamber, and visible migrating cell well spreads over the film bottom surface of cell; In Fig. 1-2 b demonstration embodiment 1, take the present invention's cell dissociation method used to collect upper chamber non-migrating cell and lower chamber migrating cell, the purple dyeing of Transwell film surface crystallization picture.On visible Transwell film, almost do not have cell remaining, illustrate that cell harvesting efficiency of the present invention is higher.
Fig. 2 a, Fig. 2 b show that in embodiment 2, cell material is cultivated respectively the inverted microscope photo after 24h.Fig. 2 a represents chamber non-migrating cell, and Fig. 2 b represents lower chamber migrating cell.From picture, different from upper chamber non-migrating cell, in the cell of migration, there are a large amount of cell debriss, visible cell has physical abuse to a certain degree in transition process.
Fig. 3 a, Fig. 3 b show cell material skeleton coloration result in embodiment 3.Fig. 3 a represents chamber non-migrating cell, and Fig. 3 b represents lower chamber migrating cell.Visible cell cellular form in transition process changes, and makes chamber non-migrating cell and lower chamber migrating cell present visibly different cytoskeleton feature.
In Fig. 4 a, Fig. 4 b demonstration embodiment 4, cell material carries out the result of colony formation.Fig. 4 a represents chamber non-migrating cell, and Fig. 4 b represents lower chamber migrating cell.Result demonstration, the upper chamber plastidogenetic cell clone of non-migrating is compared with little and sparse, and the clone that lower chamber migrating cell forms, compared with large and fine and close, illustrates that the self-renewal capacity of the cell of different migratory activities differs.
Fig. 5 shows that CKK-8 method detects the growth curve of cell material in embodiment 5.As can be seen from the figure, the multiplication capacity of the cell of different migratory activities is also obviously different.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are further described.
Embodiment mono-
As shown in Fig. 1-1, Fig. 1-2, by the inventive method, the external migration of Transwell is cultivated to the cell finishing and collect.
The present embodiment adopts mescenchymal stem cell (the human bone marrow mesenchymal stem cells of people's derived from bone marrow, hereinafter to be referred as hMSCs) as research object, adopt the restructuring PDGF-BB (Thr6 PDGF BB) in people source to move as chemokine inducing cell.
1, experiment material and reagent
Transwell cell (being the upper chamber of Transwell): the Transwell cell that is applicable to 6 porocyte culture plates of Coring company, cell area 4.67cm
2, diameter 24mm; Article No.: #3428, #354576; Mould material is polycarbonate (Polycarbonate, PC) or polyethylene terephthalate (Polyethylene Terephthalate, PET), pore size 8.0um.
Tissue Culture Plate (being chamber under Transwell): 6 porocyte culture plates of Corning company, article No. CLS3516-50EA
The low sugar culture-medium of DMEM: Gibco company product, article No. 11885-076
Phosphate buffered saline buffer: pH 7.35～7.45
Preparation chemotactic substratum: add DMEM solution to make with 20ng/ml Thr6 PDGF BB (PDGF-BB), matching while using.
Thr6 PDGF BB (PDGF-BB): R & D company product, article No. 220-BB-050
Pancreatin cell dissociation buffer: containing the phosphate buffered saline buffer of 0.25% pancreatin and 0.01%EDTA, Gibco company product, article No. R-001-100
Bovine serum: Gibco company product, article No. 10082147
Viola crystallina: Sigma company product, article No. HT90132
Whizzer: Eppendorf company product, model 5810
Centrifuge tube: material is polycarbonate, 50ml, Corning company product
Hematocyte Counter: sigma company product, article No. Z359629-1EA
Liquid-transfering gun: Eppendorf company product, specification 1ml
Leica inverted microscope (model DMI3000B)
2, experimental implementation
(experiment) cultivated in the external migration of 2.1Transwell
1) cell pretreatment
Carry out changing the low sugar culture-medium of DMEM by the hungry 24h of cell before cell migration experiment.
2) make cell suspension, preparation cell migration
Hungry cultured cells discards nutrient solution, wash 1 time with PBS, add pancreatin cell dissociation buffer 1ml～2ml, the amount adding is advisable to cover Tissue Culture Plate bottom, adds containing the low sugar DMEM substratum of 10%FBS and stop digestion after cell rounding, employing Hematocyte Counter counting, centrifugal collecting cell, rotating speed 200RCF (xg)～250RCF (xg), centrifugation time 5min, resuspended with the low sugar culture-medium of DMEM, and adjust cell density to 2.5 × 10
5/ ml obtains cell suspension, stand-by.
3) inoculating cell and cultivation
Transwell cell is added in Tissue Culture Plate, form the Yu Xia chamber, upper chamber of Transwell culturing room; In chamber under Transwell, add preparation chemotactic substratum, add-on 2ml/ hole;
Obtained cell suspension 1ml adds the upper chamber of Transwell, and when inoculation, attention action is slow, and cell is evenly inoculated, and avoids producing bubble;
Cell is cultivated to 16h～24h in 37 DEG C of 95% CO2gas incubator.
The external migration culturing cell of 2.2Transwell is collected
Step S1, first washing are collected
Transwell cell migration is cultivated and is finished and remove after the upper chamber of Transwell, lower indoor cultivation liquid, adds respectively phosphate buffered saline buffer washing culturing room in upper chamber, lower chamber, and add-on is upper chamber, the each 1ml in lower chamber;
In collection, in chamber Yu Xia chamber, washings deposits in respectively in two 50ml centrifuge tubes, is labeled as centrifuge tube A, centrifuge tube B.
Step S2, digestion are collected
Go up the pancreatin cell dissociation buffer that adds respectively 37 DEG C of preheatings in chamber, lower chamber to Transwell, add-on is 1.5ml/ hole, upper chamber, 2.6ml/ hole, lower chamber; Transwell culturing room is placed in to 95% CO2gas incubator, cultivates 2min～5min for 37 DEG C; Can take out during this time Transwell culturing room and observe under inverted microscope,, after rounded and dispersion suspension, collect respectively Digestive system correspondence in upper chamber, lower chamber and add in centrifuge tube A, centrifuge tube B until cell; If find cell not rounded and dispersion suspension under inverted microscope, can put into carbon incubator and continue to cultivate until cell is rounded and dispersion suspension.
In centrifuge tube A, B, add 100% bovine serum to stop cell dissociation respectively, add-on is 10%～20% of pancreatin cell dissociation buffer.
Step S3, washing collection again
In the upper chamber of Transwell, lower chamber, add phosphate buffered saline buffer washing culturing room respectively, add-on is with step S1;
Transwell culturing room (the Corning6 porocyte culture plate of Transwell cultivation cell is housed) is placed in to horizontal shaking table, 100r/min～150r/min, time 5min,
Collecting respectively washings correspondence in upper chamber, lower chamber adds in centrifuge tube A, centrifuge tube B;
Repeat above operation.
Step S4, centrifugal collecting cell
By centrifugal to centrifuge tube A, centrifuge tube B, rotating speed 200RCF (xg)～250RCF (xg), time 10min;
Remove respectively supernatant liquor, in centrifuge tube A, centrifuge tube B, add phosphate buffered saline buffer 0.5ml～1ml; Collect respectively hanging core barrel A, the B bottom cell mass of laying equal stress on, collect target cell.Wherein, in centrifuge tube A, be to separate as chemokine induction is lower the low migratory activity cell producing taking the restructuring PDGF-BB in people source, in centrifuge tube B, be to separate taking the restructuring PDGF-BB in people source the high migratory activity cell producing as chemokine induction is lower.
By Hematocyte Counter counting, the cells ratio of chamber, lower chamber, cell migration rate and cell harvesting efficiency (Fig. 1-1) in calculating; By violet staining analysis of cells collection effciency, (Fig. 1-2 a, Fig. 1-2 are b).
In the present embodiment, violet staining concentration 0.1%, 10 minutes time; Adopt Leica inverted microscope DMI3000 B to take pictures.
Embodiment bis-
The high transport property viable cell that the lower separation of identical chemokine induction produces and the morphologic detection experiment of low migratory activity cell.
Cell culture medium: containing the low sugar culture-medium of DMEM of 10% foetal calf serum
The low sugar culture-medium of DMEM: with embodiment mono-
Inverted microscope: with embodiment mono-
Collect in gained centrifuge tube A, centrifuge tube B cell as material taking embodiment mono-, be seeded to respectively cell culture medium, be placed in 37 DEG C, 95% CO2gas incubator, cultivate respectively and under inverted microscope, detect morphocytology feature after 24h (Fig. 2 a, Fig. 2 are b).
Embodiment tri-
The lower high transport property viable cell of generation and the cytoskeleton of the low migratory activity cell test experience of separating of identical chemokine induction.
Phalloidine (Phalloidin – Tetramethylrhodamine B isothiocyanate:Sigma company product, the article No. P1951 of tetramethyl-rhodamine mark
Inverted microscope: with embodiment mono-
Collect cell in gained centrifuge tube A, centrifuge tube B taking embodiment mono-and, as material, adopt respectively the Phalloidine of tetramethyl-rhodamine mark to cell dyeing, (Fig. 3 a, Fig. 3 are b) under inverted microscope, to detect cytoskeleton feature.
In the present embodiment, Phalloidine dyeing process operates with reference to Sigma product description.
Embodiment tetra-
The lower high transport property viable cell of generation and the self-renewal capacity test experience of low migratory activity cell of separating of identical chemokine induction.
Tissue Culture Plate, inverted microscope, Viola crystallina are all with embodiment mono-
Collect cell in gained centrifuge tube A, centrifuge tube B taking embodiment mono-and as material, be seeded to respectively Tissue Culture Plate, 100/hole of inoculum density, incubation time 8d～10d; See after obvious visible cell clone and finish to cultivate, adopt Viola crystallina to observe to the cell clone employing inverted microscope that dye that (Fig. 4 a, Fig. 4 are b).
In the present embodiment, cell culture condition and violet staining method are with embodiment mono-.
Embodiment five
The lower high transport property viable cell of generation and the multiplication capacity test experience of low migratory activity cell of separating of identical chemokine induction.
CCK-8 reagent: Sigma company product, article No. 96992-500TESTS
Collecting cell in gained centrifuge tube A, centrifuge tube B taking embodiment mono-, as material, adopts respectively CCK-8 method to detect ability of cell proliferation (Fig. 5).
In the present embodiment, adopt 96 orifice plates to detect, cell inoculum size is 2000/hole, and detection time, point was 1,3,5,7,9 day.When detection, CCK-8 add-on is 10ul/ hole, and detection absorbancy is 450nm.
Claims (10)
1. a cells in vitro collection method, is characterized in that: be applied to Transwell cell migration experiment; Transwell cell migration is cultivated and is finished and remove after the upper chamber of Transwell, lower indoor cultivation liquid, adopts trysinization and respectively the cell in upper chamber, lower chamber after Transwell cell migration experiment is digested, collected in conjunction with the method for mechanical oscillation; In the operation of pancreatin cell dissociation, washings is pH 7.35～7.45 phosphate buffered saline buffers, and pancreatin cell dissociation buffer is containing 0.25% pancreatin and 0.01%EDTA.
2. method according to claim 1, is characterized in that: implement according to following steps:
Step S1, washing for the first time
Transwell cell migration is cultivated and is finished and remove after the upper chamber of Transwell, lower indoor cultivation liquid, in upper chamber, lower chamber, add respectively phosphate buffered saline buffer washing culturing room, in collection, in chamber Yu Xia chamber, washings deposits in respectively in two centrifuge tubes, is labeled as respectively centrifuge tube A, centrifuge tube B;
Step S2, digestion are collected
In the upper chamber of Transwell, lower chamber, add respectively pancreatin cell dissociation buffer, treat cell dispersion suspension, collect respectively Digestive system correspondence in upper chamber, lower chamber and add in centrifuge tube A, centrifuge tube B, in centrifuge tube A, centrifuge tube B, add bovine serum to stop cell dissociation respectively;
Step S3, washing collection again
In the upper chamber of Transwell, lower chamber, add phosphate buffered saline buffer respectively, by the level vibration of Transwell culturing room, collect respectively washings correspondence in upper chamber, lower chamber and add in centrifuge tube A, centrifuge tube B;
Step S4, centrifugal collecting cell
By centrifugal to centrifuge tube A, centrifuge tube B, remove supernatant liquor, in centrifuge tube A, centrifuge tube B, add phosphate buffered saline buffer respectively, resuspended and collect centrifuge tube bottom cell mass, obtain collecting cell.
3. method according to claim 2, is characterized in that: in described step S2, pancreatin cell dissociation buffer add-on is for covering respectively bottom, Yu Xia chamber, the upper chamber of Transwell.
4. method according to claim 2, is characterized in that: in described step S2, pancreatin cell dissociation buffer add-on is one of following:
24 hole Transwell culturing room, upper chamber adds 0.1ml, lower chamber adds 0.6ml;
12 hole cell Transwell culturing room, upper chamber adds 0.5ml, lower chamber adds 1.5ml;
6 hole cell Transwell culturing room, upper chamber adds 1.5ml, lower chamber adds 2.6ml.
5. method according to claim 2, is characterized in that: in described step S2, add 100% bovine serum to stop cell dissociation reaction respectively in centrifuge tube, add-on is 10%～20% of pancreatin cell dissociation buffer add-on.
6. method according to claim 2, is characterized in that: in described step S3, described level vibration is horizontal shaking table vibration, 100r/min～150r/min, time 5min.
7. according to the arbitrary described cells in vitro collection method of claim 1～6, it is characterized in that: be applied to cell behaviors analytical test.
8. a cell behaviors analytical procedure of utilizing the arbitrary described cells in vitro collection method of claim 1～6 to realize, is a kind of cell detection method, it is characterized in that: implement according to following steps:
Step S1, first adopt Transwell migration experiment that the migration under chemokine induction of initiating cell material is cultivated, the above-mentioned cell in vitro collection method of employing collecting cell after Transwell cell migration cultivation finishes, obtains respectively high transport property viable cell material and low migratory activity cell material;
Step S2, be implemented as follows one of detection method
One of, morphocytology detects: respectively described high transport property viable cell material and low migratory activity cell material are seeded to cell culture medium and carry out cells in vitro cultivation; Cultivate and finish to detect respectively high transport property viable cell material and low migratory activity cell material form when cell in vitro; The substratum that described cells in vitro is selected in cultivating and culture condition are determined according to initiating cell material behavior;
Two, cytoskeleton learns and detects: respectively the cytoskeleton of described high transport property viable cell material and low migratory activity cell material is dyeed; Dyeing finishes rear observation and detects cytoskeleton; Described cytoskeleton dyeing process is determined according to initiating cell material behavior;
Three, cell self-renewal ability detect: respectively high transport property viable cell material and low migratory activity cell material are carried out to cell clone culture; Clone cultivates the poststaining detection cell self-renewal ability that finishes; Described cell clone culture condition is determined according to initiating cell material behavior;
Four, ability of cell proliferation detects: detect respectively the ability of cell proliferation of high transport property viable cell material and low migratory activity cell material, detection method is definite according to initiating cell material behavior.
9. a cell in vitro material, is characterized in that: comprise by the lower high transport property viable cell material and the low migratory activity cell material that produce of separating of identical chemokine induction; First the preparation of described cell in vitro material adopts Transwell migration experiment that the migration under chemokine induction of initiating cell material is cultivated, the above-mentioned cell in vitro collection method of employing collecting cell after Transwell cell migration cultivation finishes, in the upper chamber of Transwell, collecting the cell material obtaining is described low migration viable cell material, and under Transwell, in chamber, collecting the cell material obtaining is described high transport property viable cell material.
10. the application of cell in vitro material according to claim 9 in cell behaviors analytical test.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320494A (en) * | 2013-07-08 | 2013-09-25 | 连云港脂立方生物医药研究所有限公司 | Method for measuring invasiveness of multiple cells to cell matrix embedded with endothelial cells by utilizing Transwell |
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Non-Patent Citations (3)
| Title |
|---|
| YANLING CHEN: "Transwell cell migration assay using human breast epithelial cancer cell", 《BIOPROTOCOL》, 5 March 2011 (2011-03-05), pages 1 - 3 * |
| 李燕，张健: "《细胞与分子生物学常用实验技术》", 31 December 2009, article "Transwell", pages: 17-23 - 18-20 * |
| 黄子明等: "肝癌细胞内 Sox17 基因表达水平与细胞迁移、侵袭力的相关性研究", 《现代肿瘤医学》, 31 December 2012 (2012-12-31), pages 2457 - 2460 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107748130A (en) * | 2017-10-16 | 2018-03-02 | 上海市普陀区中心医院 | A kind of preparation of animal hearts single cell suspension and detection method |
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