CN101250500A - Method for cultivating adult distal arteria pulmonalis smooth muscle cells - Google Patents
Method for cultivating adult distal arteria pulmonalis smooth muscle cells Download PDFInfo
- Publication number
- CN101250500A CN101250500A CNA2007100330162A CN200710033016A CN101250500A CN 101250500 A CN101250500 A CN 101250500A CN A2007100330162 A CNA2007100330162 A CN A2007100330162A CN 200710033016 A CN200710033016 A CN 200710033016A CN 101250500 A CN101250500 A CN 101250500A
- Authority
- CN
- China
- Prior art keywords
- smooth muscle
- cell
- muscle cells
- solution
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for culturing adult distal end pulmonary artery smooth muscle cells, which comprises following steps: firstly, separating pulmonary artery smooth muscle cells from an adult distal end pulmonary artery smooth muscle layer which is obtained and culturing primary cells, secondly, obtaining the adult distal end pulmonary artery smooth muscle cells which are subcultured, taking out the primary cells and cleaning with PBS solution after the primary cells in the first step grow to the logarithmic growth phase, adding trypsin solution 0.3-0.5ml 0.1%-0.2%, digesting for 2-3 minutes under the temperature of 35-37 DEG C, gettering the trypsin solution when cells appear retraction and the intercellular space is enlarged, adding complete culture medium 3-5ml to enable the trypsin solution to form cell suspension, inoculating in a new culture bottle, changing medium each three days, then, finishing culturing subculture cells, and obtaining the culturing adult distal end pulmonary artery smooth muscle cells which are subcultured. The method for culturing has stable technique and good repeatability, and the cells which are cultured have uniform forms with good growth and have the forms and characteristics of typical smooth muscle cells.
Description
Technical field
The present invention relates to a kind of cell culture processes, especially a kind of cultural method of adult distal arteria pulmonalis smooth muscle cells belongs to biological technical field.
Background technology
Adult distal arteria pulmonalis smooth muscle cells is the important cells material of disease incidence mechanism such as research pulmonary circulation relative disease, especially pulmonary hypertension.The method of cultivating arteria pulmonalis smooth muscle cells at present comprises enzyme digestion and tissue block paster method.Animal-origin mostly is rat, and rat differs greatly with people's the different iuntercellulars of kind, so carry out the meaning of pulmonary hypertension pathogenesis and correlative study obviously greater than the cell with animal with adult's arteria pulmonalis smooth muscle cells.But because the difficulty of drawing materials, and mankind are that its cell of organism of high evolution is difficult to unicellular lower eukaryote far away external successfully the cultivation, thus simultaneously because obviously be longer than the latent period of adult's cell in-vitro growth animal such as rat external successfully cultivate difficult more.In view of above reason, it is material that the at present domestic arteria pulmonalis smooth muscle cells that is used for the experimental study cultivation adopts rat more; Part scholar adopts the main pulmonary artery of induced labor foetus (in 18 weeks) to experimentize.Because the fetal cell multiplication capacity is easy to by force cultivate, and main pulmonary artery unstriated muscle bed thickness cell is easy to separate, but fetus its cell of undeveloped mature and adult's cell difference to some extent still, and the main pathological change position of pulmonary hypertension is positioned at the lung distal artery.So it is unsatisfactory that employing fetus main pulmonary artery carries out the experiment of pulmonary hypertension Mechanism Study.And the foreign scholar is the material (most of is rat) except that adopting animal; The arteria pulmonalis smooth muscle cells strain that part scholar also buys the adult experimentizes but cell strain costs an arm and a leg and non-former generation cultured cells, the cytobiology feature changes to some extent influences experimental result; And the small part scholar distal arteria pulmonalis smooth muscle cells of also bringing out costs an arm and a leg but its substratum that adopts is a this substratum of smooth muscle cell basic medium (SMBM), and domestic general laboratory is difficult to accept.In sum, seek the key that a kind of adult distal arteria pulmonalis smooth muscle cells cultured method of former generation efficient, easy, that cost is low becomes Success in Experiment.
Summary of the invention
The cultural method that the purpose of this invention is to provide a kind of adult distal arteria pulmonalis smooth muscle cells.This cultural method is consistent, good reproducibility, and cultured cells form homogeneous, well-grown, and have the form and the characteristics of typical smooth muscle cell.
The objective of the invention is to be achieved through the following technical solutions: a kind of cultural method of adult distal arteria pulmonalis smooth muscle cells, it may further comprise the steps composition:
(1) obtains the former foster adult distal arteria pulmonalis smooth muscle cells of being commissioned to train: from the adult distal arteria pulmonalis smooth muscle layer of obtaining, separate distal arteria pulmonalis smooth muscle cells, and carry out primary cell culture.To obtain the former foster adult distal arteria pulmonalis smooth muscle cells of being commissioned to train;
(2) obtain the adult distal arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate: the primary cell in step (1) grows to the logarithmic growth after date, after the taking-up primary cell cleans with PBS (phosphate buffered saline buffer) solution, add 35～37 ℃ of digestion of 0.3～0.5ml, 0.1%～0.2% trypsin solution 2～3 minutes, when retraction appears in cell, when the intercellular substance increases, absorb trypsin solution, add 3～5ml complete culture solution, make it to form cell suspending liquid, be inoculated in the new culturing bottle, changed liquid once in per 3 days, promptly finish passage cell and cultivate, obtain the adult distal arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate.
Complete culture solution in the described step (2) is; Contain 0.1%EDTA (ethylenediamine tetraacetic acid (EDTA)) in the trypsin solution, described cell inoculation density is 2～3 * 10
5Individual/ml, to keep the cell growth enough spaces, nutrition are arranged.
Perfect medium described in the step (2) is: contain the low sugar DMEM (Dulbcco ' s Modifed Eagle Medium) of 90～105U/ml penicillin, 90～105mg/ml Streptomycin sulphate and the mixing solutions of foetal calf serum, described foetal calf serum accounts for 10～20% of substratum cumulative volume.
Described HBSS (Hank ' the s balanced salt solution): contain the aqueous solution of 125～135mol/L sodium-chlor, 3～6mol/L Repone K, 1～1.5mol/L magnesium chloride, 5～15mol/LHEPES (hydroxyethyl piperazine second thiosulfonic acid), 15～25 μ mol/L calcium chloride, 5～15mol/L dextrose anhydrous, 90～105U/ml penicillin and 90～105mg/ml Streptomycin sulphate, the pH value of this solution is 7～7.5.
The concrete steps that described step (1) is obtained former one-tenth human pulmonary artery smooth muscle cells of being commissioned to train foster are:
1〉adopts the gradation enzyme digestion, the tunica media tissue that separates and peeled off interior adventitia is cut into small pieces, put into HBSS and leave standstill 20～30min for 4 ℃, put into no calcareous HBSS room temperature then and leave standstill 10～20min; Tissue block is put into centrifuge tube again and added 35～37 ℃ of digestion of Digestive system, 35～45min, the visible tissue piece is soft, the edge is scared.
2〉sucking-off Digestive system adds perfect medium 2～3ml in centrifuge tube, room temperature is placed 3～5min; Wide-mouth pasteur pipet piping and druming 10～15 times is left standstill and is made the tissue block precipitation a moment, and the sucking-off cell suspending liquid is collected in another centrifuge tube, former Digestive system is added to digest once more in the former centrifuge tube and by above collection step cell again; The cell suspending liquid of collecting is centrifugal, abandon supernatant liquor, the adding perfect medium is adjusted cell density and is planted in culture plate with this density, puts 37 ℃, 5%CO
2Leave standstill cultivation in the incubator, the later half amount of 3d is changed substratum; When cell grows to logarithmic phase, just obtained the former foster adult distal arteria pulmonalis smooth muscle cells of being commissioned to train.
Described step 2〉in according to the situation of tissue digestion, repeated collection cell manipulation 2～4 times.
Described step 2〉in cell density be 2～3 * 10
5Cell/ml.
The HBSS solution of described no calcium is: contain the aqueous solution of 125～135mol/L sodium-chlor, 3～6mol/L Repone K, 1～1.5mol/L magnesium chloride, 5～15mol/L hydroxyethyl piperazine second thiosulfonic acid (HEPES), 5～15mol/L dextrose anhydrous, 90～105U/ml penicillin and 90～105mg/ml Streptomycin sulphate, the pH value of this solution is 7～7.5.
Described Digestive system: contain 125～135mol/L sodium-chlor, 3～6mol/L Repone K, 1～1.5mol/L magnesium chloride, 5～15mol/LHEPES, 5～15mol/L dextrose anhydrous, 2.0～3.0mg/mlI Collagen Type VI enzyme, 9～10U/ml papoid, 1～3mg/ml bovine serum albumin, 0.5～1.51mmol/L DTT (1, the 4-dithiothreitol dithio), the aqueous solution of 90～105U/ml penicillin and 90～105mg/ml Streptomycin sulphate, the pH value of this solution is 7～7.5.
Step 2〉described in perfect medium be: contain the low sugar DMEM (Dulbcco ' s Modifed Eagle Medium) of 90～105U/ml penicillin, 90～105mg/ml Streptomycin sulphate and the mixing solutions of foetal calf serum, described foetal calf serum accounts for 10～20% of substratum cumulative volume.
Reagent used in the present invention is all used 0.2～0.22 μ m membrane filtration degerming.
The inventive method compared with prior art has following beneficial effect:
1, breaks through the technical barrier that the utilization enzyme digestion is turned out highly purified adult distal arteria pulmonalis smooth muscle cells by meticulous instruments such as microscope, microforceps, microscissorses, filled up this field blank.
2, cultural method is consistent, good reproducibility, cultured cells form homogeneous, well-grown.
3, the cell that obtains has typical distal arteria pulmonalis smooth muscle cells form and characteristics through microscopic examination and cellular immunization chemical identification, and the position of drawing materials of arteria pulmonalis smooth muscle cells is positioned at distal arteria pulmonalis, and this part cell is more meaningful to research pulmonary hypertension pathogenesis.Established condition for the pathogenesis of further furtheing investigate pulmonary circulation relative disease, diseases such as little relevant vascular diseases, especially pulmonary hypertension, abundant material source is provided.
Description of drawings
Fig. 1 is that former being commissioned to train supported 20 days adult distal arteria pulmonalis smooth muscle cells photos under the inverted phase contrast microscope (100 *);
Fig. 2 is that former being commissioned to train supported 35 days adult distal arteria pulmonalis smooth muscle cells photos under the inverted phase contrast microscope (100 *);
Fig. 3 goes down to posterity under the inverted phase contrast microscope (100 *) to cultivate 3 days adult distal arteria pulmonalis smooth muscle cells photos;
Fig. 4 goes down to posterity under the inverted phase contrast microscope (100 *) to cultivate 7 days adult distal arteria pulmonalis smooth muscle cells photos;
Fig. 5 is that former being commissioned to train formed people's distal arteria pulmonalis smooth muscle cells through the cellular immunofluorescence photo that dyes under the laser scanning co-focusing microscope (200 *);
The rubescent look fluorescence of nucleus of the cytoplasm α of FITC mark-Actin antigen green-emitting fluorescence, DAPI mark;
Fig. 6 is that former being commissioned to train formed people's distal arteria pulmonalis smooth muscle cells through the cellular immunofluorescence photo that dyes under the laser scanning co-focusing microscope (600 *);
Fig. 7 is that former being commissioned to train formed people's distal arteria pulmonalis smooth muscle cells through the cellular immunofluorescence photo that dyes under the laser scanning co-focusing microscope (200 *);
Negative control does not have the α of adding-Actin monoclonal antibody, and only with DAPI labeled cell nuclear, visible cell is authorized red fluorescence;
Fig. 8 is that former being commissioned to train formed people's distal arteria pulmonalis smooth muscle cells through the cellular immunofluorescence photo that dyes under the laser scanning co-focusing microscope (100 *);
Fig. 9 is that former being commissioned to train formed people's distal arteria pulmonalis smooth muscle cells under the transmission electron microscope (28000 *), myofilament (↑) in the visible endochylema, dense body (△), dense patch (
).
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto, according to foregoing of the present invention, ordinary skill knowledge and customary means according to this area, not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways.
Embodiment one:
Concrete steps:
1, main experiment material
(1) reagent preparation:
1, HBSS: contain 130mol/L sodium-chlor, 5mol/L Repone K, 1.2mol/L magnesium chloride, 10mol/L hydroxyethyl piperazine second thiosulfonic acid (HEPES), 20 μ mol/L calcium chloride, 10mol/L dextrose anhydrous, 100U/ml penicillin and 100mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
2, the HBSS of no calcium: contain 130mol/L sodium-chlor, 5mol/L Repone K, 1.2mol/L magnesium chloride, 10mol/L hydroxyethyl piperazine second thiosulfonic acid (HEPES), 10mol/L dextrose anhydrous, 100U/ml penicillin and 100mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
3, predigestion liquid: contain 130mol/L sodium-chlor, 5mol/L Repone K, 1.2mol/L magnesium chloride, 10mol/L hydroxyethyl piperazine second thiosulfonic acid (HEPES), 10mol/L dextrose anhydrous, 1.5mg/mlI Collagen Type VI enzyme, 100U/ml penicillin and 100mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
4, Digestive system: contain 130mol/L sodium-chlor, 5mol/L Repone K, 1.2mol/L magnesium chloride, 10mol/L hydroxyethyl piperazine second thiosulfonic acid (HEPES), 10mol/L dextrose anhydrous, 2.5mg/mlI Collagen Type VI enzyme, 9.5U/ml papoid, 2mg/ml bovine serum albumin, 1mmol/L DTT, 100U/ml penicillin and 100mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
5, trypsin solution: the EDTA that contains 0.2% (m/v) trypsinase and 0.1% (m/v).
6, perfect medium is: contain the low sugar DMEM of 100/ml penicillin, 100mg/ml Streptomycin sulphate and the mixing solutions of foetal calf serum, described foetal calf serum accounts for 10% of substratum cumulative volume
Above reagent is through 0.22 μ m membrane filtration degerming.
(2) tissue-derived: the patient of no pulmonary hypertension gets the far-end lung tissue of the excision lobe of the lung because of disease row pulmonay lobectomys such as lung cancer.
2, operation steps
(1) obtain adult's arteria pulmonalis smooth muscle:
1) under aseptic condition, cut lobe of the lung peripheral tissues with scissors, tweezers, put into cold HBSS, insert in the ice chest cryopreservation and in 4 hours, send into the laboratory super clean bench as early as possible and experimentize.
2) lung tissue is sent in the super clean bench operated, clean with cold HBSS and remove blood stains; Place under the Stereo microscope, with microforceps and micro-scissors careful separation distal arteria pulmonalis under microscopic examination; In sepn process, need constantly to change cold HBSS; Separating the pulmonary artery tissue that obtains places cold HBSS to clean removal blood.
3) the pulmonary artery tissue is put under 25 ℃ of temperature of predigestion liquid and carried out predigestion 10min, blood vessel is put into cold HBSS with flush away predigestion liquid, carefully peel off tunica adventitia vasorum with microforceps and micro-scissors down in microscopic examination; The vascular strip of peeling off adventitia is vertically cut off, in face up and strike off inner membrance from top to bottom, film smooth muscle layer in staying with the elbow pincet;
(2) obtain the former foster adult distal arteria pulmonalis smooth muscle cells of being commissioned to train:
1) middle membrane tissue is cut into small pieces about 2 * 3mm puts into HBSS and leaves standstill 30min for 4 ℃, puts into no calcareous HBSS room temperature then and leaves standstill 20min;
2) tissue block is put into centrifuge tube again and added 37 ℃ of digestion of Digestive system 45min, the visible tissue piece is soft, the edge is scared;
3) sucking-off Digestive system gently adds perfect medium 3ml, and room temperature is placed 3min.Wide-mouth pasteur pipet piping and druming 10 times has floss in the visible liquid;
4) leave standstill and make a moment tissue block be deposited in centrifuge tube bottom, the sucking-off cell suspending liquid is collected in another centrifuge tube.Continue digestion 5min in the former Digestive system add-back tissue block, by the above-mentioned steps collecting cell.Repeat 5min digestion operation 2 times again;
5) centrifugal (800rpm 5min), abandons supernatant liquor, adds substratum and adjusts cell density with 3 * 10 with the cell suspending liquid of collecting
5The density of individual/ml is planted in 6 well culture plates (wherein cover glass is put in 2 holes, uses during evaluation).Put 37 ℃, 5%CO
2Leave standstill cultivation in the incubator, change substratum after 10～14 days.When cell grows to logarithmic phase, promptly finish primary cell culture, obtain the former foster adult distal arteria pulmonalis smooth muscle cells of being commissioned to train.
(3) obtain the adult distal arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate:
After primary cell covers with bottle wall, can go down to posterity.When the time comes, clean cell 2 times with 2mlPBS solution, add 37 ℃ of digestion of 0.5ml0.2% (m/v) trypsin solution (containing 0.1%EDTA) 3 minutes, when retraction, intercellular substance increase appear in cell, absorb trypsin solution, add the 5ml nutrient solution, gently the cell on the piping and druming bottle wall, make it to form cell suspending liquid, with 2 * 10
5The cell density of individual/ml is inoculated in the new culturing bottle, places 37 ℃, 5% carbonic acid gas incubator to cultivate, and changes liquid once in per 2 days, grows to logarithmic phase to cell, obtains the adult distal arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate.
3, cultivation results
(1), inverted phase contrast microscope is observed rounded, bright, the single uniform distribution of cell of firm plantation down.Former adult distal arteria pulmonalis smooth muscle cells length in latent period of being commissioned to train foster, 14 days left and right sides of poor growth visible part cell attachment, to launch cellular form various, is prismatic, polygon, star or irregular shape, and size is slightly variant; Endochylema is abundant; It is monokaryon that karyon mostly is the most cells of oval, and a few cell has double-core or three nuclears, and one or more kernels are arranged.The cell quantity showed increased is characteristic of concentration growth (Fig. 1) after 20～25 days, and iuntercellular often has projection to link to each other.Cell growth in 30～35 days reaches the logarithmic phase growth, and subregion cell overlap accumulated growth forms " peak ", and the cell number of plies is gradually few between " peak " and " peak ", forms " paddy ".After cell grows to fusion, the iuntercellular bunchy that is arranged parallel to each other, height rises and falls, and forms typical smooth muscle cell growth feature " peak " " paddy " shape growth (Fig. 2).
(2) inverted phase contrast microscope is observed down, the adult distal arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate.The cell speed of growth is obviously accelerated than primary cultured cell.Go down to posterity back 1 day cell attachment, launch visible cell and be prismatic, polygon, star or irregular shape, the cell quantity showed increased is the characteristic of concentration growth after 3 days, iuntercellular often has projection link to each other (Fig. 3).Cell grows to fusion after 7 days, forms typical smooth muscle cell growth feature " peak " " paddy " shape growth (Fig. 4).In former generation,, cultured cells was consistent with the passage cell form.
(3) the antigenic immunofluorescence of unstriated muscle α-Actin detects: adopt unstriated muscle α-Actin monoclonal antibody that cultured cells is identified, and adopt international redyeing process commonly used to dye, with DAPI all cells nuclear is dyeed, guarantee the cell purity computation's reliability.Under laser scanning co-focusing microscope, observe the cytoplasm unstriated muscle α-Actin antigen green-emitting fluorescence of visible FITC mark, the rubescent look fluorescence of the nucleus of DAPI mark (Fig. 5).High power lens is observed down, and the myofilament in the smooth muscle cell endochylema is parallel to the cell major axis and is filament shape expression (Fig. 6).Negative control does not have and adds one anti-ly, and the nucleus that only detects the DAPI mark does not detect the unstriated muscle α-Actin of FITC mark, so rarely seen rubescent look fluorescence (Fig. 7).Low power lens calculates cell purity, and every sheet is got 5 visuals field at random, and 200 cells are detected in each visual field at least.Calculate α in each visual field-Actin and express the ratio that positive cell accounts for this total cell count in visual field, be smooth muscle cell purity.Average out to is (Fig. 8) more than 98%.
(4) transmission electron microscope detects: observing under the transmission electron microscope has the myofilament parallel with the cell major axis and coupled dense body, dense patch (Fig. 9) is arranged in the distal arteria pulmonalis smooth muscle endochylema, show the typical outline structure feature of shrinking.
Embodiment two:
Concrete steps:
1, main experiment material
(1) reagent preparation:
1, HBSS: contain 125mol/L sodium-chlor, 6mol/L Repone K, 1mol/L magnesium chloride, 5mol/LHEPES, 15 μ mol/L calcium chloride, 15mol/L dextrose anhydrous, 105U/ml penicillin and 105mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
2, the HBSS of no calcium: contain 125mol/L sodium-chlor, 6mol/L Repone K, 1mol/L magnesium chloride, 5mol/LHEPES, 15mol/L dextrose anhydrous, 105U/ml penicillin and 105mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
3, predigestion liquid: contain 125mol/L sodium-chlor, 6mol/L Repone K, 1mol/L magnesium chloride, 5mol/LHEPES, 15mol/L dextrose anhydrous, 1.3mg/mlI Collagen Type VI enzyme, 105U/ml penicillin and 105mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
4, Digestive system: contain 125mol/L sodium-chlor, 6mol/L Repone K, 1mol/L magnesium chloride, 5mol/LHEPES, 15mol/L dextrose anhydrous, 2.0mg/ml type i collagen enzyme, 10U/ml papoid, 1mg/ml bovine serum albumin, 0.5mmol/L DTT, 105U/ml penicillin and 105mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
5, trypsin solution: contain 0.1% (m/v) trypsinase and 0.1% (m/v) EDTA.
6, substratum:: contain the low sugar DMEM and FBS (foetal calf serum) mixing solutions of 90U/ml penicillin, 90mg/ml Streptomycin sulphate, described foetal calf serum accounts for 20% of substratum cumulative volume.
Above reagent is through 0.2 μ m membrane filtration degerming.
(2) tissue-derived: the patient of no pulmonary hypertension gets the far-end lung tissue of the lobe of the lung of excision because of disease row lobectomy of lungs such as lung cancer.
2, operation steps
(1) obtain adult's arteria pulmonalis smooth muscle:
1) under aseptic condition, cut lobe of the lung peripheral tissues with scissors, tweezers, put into cold HBSS, insert in the ice chest cryopreservation and in 4 hours, send into the laboratory super clean bench as early as possible and experimentize.
2) lung tissue is sent in the super clean bench operated, clean with cold HBSS and remove blood stains; Place under the Stereo microscope, with microforceps and micro-scissors careful separation distal arteria pulmonalis under microscopic examination; In sepn process, need constantly to change cold HBSS; Separating the pulmonary artery tissue that obtains places cold HBSS to clean removal blood.
3) the pulmonary artery tissue is put under 20 ℃ of temperature of predigestion liquid and carried out predigestion 8min, blood vessel is put into cold HBSS with flush away predigestion liquid, carefully peel off tunica adventitia vasorum with microforceps and micro-scissors down in microscopic examination; The vascular strip of peeling off adventitia is vertically cut off, in face up and strike off inner membrance from top to bottom, film smooth muscle layer in staying with the elbow pincet;
(2) obtain the former foster adult distal arteria pulmonalis smooth muscle cells of being commissioned to train:
1) middle membrane tissue is cut into small pieces about 2 * 3mm puts into HBSS and leaves standstill 20min for 4 ℃, does not have that room temperature leaves standstill 15min among the calcareous HBSS;
2) tissue block is put into centrifuge tube again and added 35 ℃ of digestion of Digestive system 35min, the visible tissue piece is soft, the edge is scared;
3) sucking-off Digestive system gently adds substratum 2.5ml, and room temperature is placed 4min.The piping and druming of wide-mouth pasteur pipet has floss in the visible liquid for several times;
4) leave standstill and make a moment tissue block be deposited in centrifuge tube bottom, the sucking-off cell suspending liquid is collected in another centrifuge tube.Continue digestion 5min in the former Digestive system add-back tissue block, by the above-mentioned steps collecting cell.Repeat 5min digestion operation 3 times again;
5) centrifugal (800rpm 5min), abandons supernatant liquor, adds substratum and adjusts cell density with 2 * 10 with the cell suspending liquid of collecting
5The density of individual/ml is planted in 6 well culture plates (wherein cover glass is put in 2 holes, uses during evaluation).Put 37 ℃, 5%CO
2Leave standstill cultivation in the incubator, change substratum after 10～14 days.When cell grows to logarithmic phase, promptly finish primary cell culture, obtain the former foster adult distal arteria pulmonalis smooth muscle cells of being commissioned to train.
(3) obtain the adult distal arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate:
After primary cell covers with bottle wall, can go down to posterity.When the time comes, clean cell 2 times with 2mlPBS solution, add 35 ℃ of digestion of 0.5ml0.1% (m/v) trypsin solution (containing 0.1%EDTA) 3 minutes, when retraction, intercellular substance increase appear in cell, absorb trypsin solution, add the 5ml nutrient solution, gently the cell on the piping and druming bottle wall, make it to form cell suspending liquid, with 2 * 10
5The cell density of individual/ml is inoculated in the new culturing bottle, places 37 ℃, 5% carbonic acid gas incubator to cultivate, and changes liquid once in per 2 days, grows to logarithmic phase to cell, obtains the adult distal arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate.
3, cultivation results
The result is identical with embodiment one.
Embodiment three:
Concrete steps:
1, main experiment material
(1) reagent preparation:
1, HBSS: contain 135mol/L sodium-chlor, 3mol/L Repone K, 1.5mol/L magnesium chloride, 15mol/LHEPES, 25 μ mol/L calcium chloride, 5mol/L dextrose anhydrous, 90U/ml penicillin and 90mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
2, the HBSS of no calcium: contain 135mol/L sodium-chlor, 3mol/L Repone K, 1.5mol/L magnesium chloride, 15mol/LHEPES, 5mol/L dextrose anhydrous, 90U/ml penicillin and 90mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
3, predigestion liquid: contain 135mol/L sodium-chlor, 3mol/L Repone K, 1.5mol/L magnesium chloride, 15mol/LHEPES, 5mol/L dextrose anhydrous, 1.0mg/ml type i collagen enzyme, 90U/ml penicillin and 90mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
4, Digestive system: contain 135mol/L sodium-chlor, 3mol/L Repone K, 1.5mol/L magnesium chloride, 15mol/LHEPES, 5mol/L dextrose anhydrous, 3.0mg/mlI Collagen Type VI enzyme, 9U/ml papoid, 3mg/ml bovine serum albumin, 1.5mmol/L DTT, 90U/ml penicillin and 90mg/ml aqueous streptomycin, the pH value of this solution is 7.4.
5, trypsin solution: contain 0.15% (m/v) trypsinase and 0.1% (m/v) EDTA.
6, substratum:: contain the low sugar DMEM and FBS (foetal calf serum) mixing solutions of 105U/ml penicillin, 105mg/ml Streptomycin sulphate, described foetal calf serum accounts for 15% of substratum cumulative volume.
Above reagent is through 0.21 μ m membrane filtration degerming.
(2) tissue-derived: the patient of no pulmonary hypertension gets the far-end lung tissue of the lobe of the lung of excision because of disease row lobectomy of lungs such as lung cancer.
2, operation steps
(1) obtain adult's arteria pulmonalis smooth muscle:
1) under aseptic condition, cut lobe of the lung peripheral tissues with scissors, tweezers, put into cold HBSS, insert in the ice chest cryopreservation and in 4 hours, send into the laboratory super clean bench and experimentize.
2) lung tissue is sent in the super clean bench operated, clean with cold HBSS and remove blood stains; Place under the Stereo microscope, with microforceps and micro-scissors careful separation distal arteria pulmonalis under microscopic examination; In sepn process, need constantly to change cold HBSS; Separating the pulmonary artery tissue that obtains places cold HBSS to clean removal blood.
3) the pulmonary artery tissue is put under 23 ℃ of temperature of predigestion liquid and carried out predigestion 5min, blood vessel is put into cold HBSS with flush away predigestion liquid, carefully peel off tunica adventitia vasorum with microforceps and micro-scissors down in microscopic examination; The vascular strip of peeling off adventitia is vertically cut off, in face up and strike off inner membrance from top to bottom, film smooth muscle layer in staying with the elbow pincet;
(2) obtain the former foster adult distal arteria pulmonalis smooth muscle cells of being commissioned to train:
1) middle membrane tissue is cut into small pieces about 2 * 3mm puts into HBSS and leaves standstill 25min for 4 ℃, does not have that room temperature leaves standstill 10min among the calcareous HBSS;
2) tissue block is put into centrifuge tube again and added 36 ℃ of digestion of Digestive system 38min, the visible tissue piece is soft, the edge is scared;
3) sucking-off Digestive system gently adds substratum 2ml, and room temperature is placed 5min.Wide-mouth pasteur pipet piping and druming 20 times has floss in the visible liquid;
4) leave standstill and make a moment tissue block be deposited in centrifuge tube bottom, the sucking-off cell suspending liquid is collected in another centrifuge tube.Continue digestion 5min in the former Digestive system add-back tissue block, by the above-mentioned steps collecting cell.Repeat 5min digestion operation 4 times again;
5) centrifugal (800rpm 5min), abandons supernatant liquor, adds substratum and adjusts cell density with 2.5 * 10 with the cell suspending liquid of collecting
5The density of cell/ml is planted in 6 well culture plates (wherein cover glass is put in 2 holes, uses during evaluation).Put 37 ℃, 5%CO
2Leave standstill cultivation in the incubator, change substratum after 10～14 days.When cell grows to logarithmic phase, promptly finish primary cell culture, obtain the former foster adult distal arteria pulmonalis smooth muscle cells of being commissioned to train.
(3) obtain the adult distal arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate:
After primary cell covers with bottle wall, can go down to posterity.When the time comes, clean cell 2 times with 2mlPBS solution, add 36 ℃ of digestion of 0.4ml0.15% (m/v) trypsin solution 3 minutes, when retraction, intercellular substance increase appear in cell, absorb trypsin solution, add the 5ml nutrient solution, gently the cell on the piping and druming bottle wall, make it to form cell suspending liquid, with 2.5 * 10
5The cell density of individual/ml is inoculated in the new culturing bottle, places 37 ℃, 5% carbonic acid gas incubator to cultivate, and changes liquid once in per 2 days, grows to logarithmic phase to cell, obtains the adult distal arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate.
3, cultivation results
The result is identical with embodiment one.
Claims (10)
1, a kind of cultural method of adult distal arteria pulmonalis smooth muscle cells is characterized in that: may further comprise the steps:
(1) obtains former one-tenth human pulmonary artery smooth muscle cells of being commissioned to train foster: from the adult distal arteria pulmonalis smooth muscle layer that obtains, separate arteria pulmonalis smooth muscle cells, and carry out primary cell culture, obtain the former foster adult distal arteria pulmonalis smooth muscle cells of being commissioned to train;
(2) obtain the adult distal arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate: the primary cell in step (1) grows to the logarithmic growth after date, after the taking-up primary cell cleans with PBS solution, add 35～37 ℃ of digestion of 0.3～0.5ml, 0.1%～0.2% trypsin solution 2～3 minutes, when retraction appears in cell, when the intercellular substance increases, absorb trypsin solution, add 3～5ml complete culture solution, make it to form cell suspension, be inoculated in the new culturing bottle, changed liquid once in per 3 days, promptly finish passage cell and cultivate, obtain the adult distal arteria pulmonalis smooth muscle cells that goes down to posterity and cultivate.
2, cultural method according to claim 1 is characterized in that: contain 0.1% ethylenediamine tetraacetic acid (EDTA) in the trypsin solution in the described step (2), described cell inoculation density is 2～3 * 10
5Individual/ml.
3, cultural method according to claim 1, it is characterized in that: described perfect medium is: contain the low sugar DMEM of 90～105U/ml penicillin, 90～105mg/ml Streptomycin sulphate and the mixing solutions of foetal calf serum, described foetal calf serum accounts for 10～20% of substratum cumulative volume.
4, cultural method according to claim 1 is characterized in that: the concrete steps that described step (1) is obtained former one-tenth human pulmonary artery smooth muscle cells of being commissioned to train foster are:
1〉adopts the gradation enzyme digestion, the tunica media tissue that separates and peeled off interior adventitia is cut into small pieces, put into HBSS and leave standstill 20～30min for 4 ℃, put into no calcareous HBSS room temperature then and leave standstill 10～20min; Again tissue block is put into centrifuge tube and added 35～37 ℃ of digestion of Digestive system, 35～45min;
2〉sucking-off Digestive system adds perfect medium 2～3ml in centrifuge tube, room temperature is placed 3～5min; Wide-mouth pasteur pipet piping and druming 10～15 times is left standstill and is made the tissue block precipitation a moment, and the sucking-off cell suspending liquid is collected in another centrifuge tube, former Digestive system is added to digest once more in the former centrifuge tube and by above collection step cell again; The cell suspending liquid of collecting is centrifugal, abandon supernatant liquor, the adding perfect medium is adjusted cell density and is planted in culture plate with this density, puts 37 ℃, 5%CO
2Leave standstill cultivation in the incubator, the later half amount of 3d is changed substratum; When cell grows to logarithmic phase, just obtained the former foster adult distal arteria pulmonalis smooth muscle cells of being commissioned to train.
5, cultural method according to claim 4 is characterized in that: described step 2〉in cell density be 2～3 * 10
5Individual/ml.
6, cultural method according to claim 4, it is characterized in that: described HBSS solution: contain the aqueous solution of 125～135mol/L sodium-chlor, 3～6mol/L Repone K, 1～1.5mol/L magnesium chloride, 5～15mol/L hydroxyethyl piperazine second thiosulfonic acid, 15～25 μ mol/L calcium chloride, 5～15mol/L dextrose anhydrous, 90～105U/ml penicillin and 90～105mg/ml Streptomycin sulphate, the pH value of this solution is 7～7.5.
7, cultural method according to claim 4, it is characterized in that: the HBSS solution of described no calcium is: contain the aqueous solution of 125～135mol/L sodium-chlor, 3～6mol/L Repone K, 1～1.5mol/L magnesium chloride, 5～15mol/L hydroxyethyl piperazine second thiosulfonic acid, 5～15mol/L dextrose anhydrous, 90～105U/ml penicillin and 90～105mg/ml Streptomycin sulphate, the pH value of this solution is 7～7.5.
8, cultural method according to claim 4, it is characterized in that: described Digestive system: contain 125～135mol/L sodium-chlor, 3～6mol/L Repone K, 1～1.5mol/L magnesium chloride, 5～15mol/L hydroxyethyl piperazine second thiosulfonic acid, 5～15mol/L dextrose anhydrous, 2.0～3.0mg/mU Collagen Type VI enzyme, 9～10U/ml papoid, 1～3mg/ml bovine serum albumin, 0.5～1.51mmol/L 1, the aqueous solution of 4-dithiothreitol dithio, 90～105U/ml penicillin and 90～105mg/ml Streptomycin sulphate, the pH value of this solution are 7～7.5.
9, cultural method according to claim 4, it is characterized in that: described perfect medium is: contain the low sugar DMEM of 90～105U/ml penicillin, 90～105mg/ml Streptomycin sulphate and the mixing solutions of foetal calf serum, described foetal calf serum accounts for 10～20% of substratum cumulative volume.
10, cultural method according to claim 4 is characterized in that: described step 2〉in according to the situation of tissue digestion, repeated collection cell manipulation 2～4 times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100330162A CN101250500B (en) | 2007-12-29 | 2007-12-29 | Method for cultivating adult distal arteria pulmonalis smooth muscle cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100330162A CN101250500B (en) | 2007-12-29 | 2007-12-29 | Method for cultivating adult distal arteria pulmonalis smooth muscle cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101250500A true CN101250500A (en) | 2008-08-27 |
| CN101250500B CN101250500B (en) | 2010-10-06 |
Family
ID=39954100
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007100330162A Expired - Fee Related CN101250500B (en) | 2007-12-29 | 2007-12-29 | Method for cultivating adult distal arteria pulmonalis smooth muscle cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101250500B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168062A (en) * | 2010-12-24 | 2011-08-31 | 首都医科大学附属北京朝阳医院 | Human pulmonary artery smooth muscle cell separation and culturing method and application of same |
| CN102787095A (en) * | 2012-07-06 | 2012-11-21 | 广州医学院第一附属医院 | Method for cultivating rat primary airway epithelial cells |
| CN105039243A (en) * | 2015-07-23 | 2015-11-11 | 中国农业大学 | Isolated culture method for chicken embryo pulmonary arterial smooth muscle cells |
| CN108373994A (en) * | 2018-01-26 | 2018-08-07 | 河北医科大学第四医院 | The tissue block method's original cuiture and identification method of a kind of gastric and esophageal engaging portion smooth muscle cell |
| CN110447633A (en) * | 2018-05-07 | 2019-11-15 | 北京吉尚立德生物科技有限公司 | A kind of lung cancer solid tumor mass Sample preservation liquid |
| CN115322949A (en) * | 2022-07-26 | 2022-11-11 | 唐颐控股(深圳)有限公司 | Isolated culture method of human umbilical vein smooth muscle cells and application thereof |
-
2007
- 2007-12-29 CN CN2007100330162A patent/CN101250500B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168062A (en) * | 2010-12-24 | 2011-08-31 | 首都医科大学附属北京朝阳医院 | Human pulmonary artery smooth muscle cell separation and culturing method and application of same |
| CN102168062B (en) * | 2010-12-24 | 2013-02-13 | 首都医科大学附属北京朝阳医院 | Human pulmonary artery smooth muscle cell separation and culturing method and application of same |
| CN102787095A (en) * | 2012-07-06 | 2012-11-21 | 广州医学院第一附属医院 | Method for cultivating rat primary airway epithelial cells |
| CN105039243A (en) * | 2015-07-23 | 2015-11-11 | 中国农业大学 | Isolated culture method for chicken embryo pulmonary arterial smooth muscle cells |
| CN105039243B (en) * | 2015-07-23 | 2018-01-09 | 中国农业大学 | A kind of isolated culture method of chicken embryo arteria pulmonalis smooth muscle cells |
| CN108373994A (en) * | 2018-01-26 | 2018-08-07 | 河北医科大学第四医院 | The tissue block method's original cuiture and identification method of a kind of gastric and esophageal engaging portion smooth muscle cell |
| CN108373994B (en) * | 2018-01-26 | 2020-10-09 | 河北医科大学第四医院 | Tissue block method primary culture and identification method for smooth muscle cells of esophageal-gastric junction |
| CN110447633A (en) * | 2018-05-07 | 2019-11-15 | 北京吉尚立德生物科技有限公司 | A kind of lung cancer solid tumor mass Sample preservation liquid |
| CN115322949A (en) * | 2022-07-26 | 2022-11-11 | 唐颐控股(深圳)有限公司 | Isolated culture method of human umbilical vein smooth muscle cells and application thereof |
| CN115322949B (en) * | 2022-07-26 | 2024-03-15 | 唐颐控股(深圳)有限公司 | Isolated culture method of human umbilical vein smooth muscle cells and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101250500B (en) | 2010-10-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101250500B (en) | Method for cultivating adult distal arteria pulmonalis smooth muscle cells | |
| CN101803581B (en) | Method for cultivating large granular nuclear pearls | |
| CN106801032B (en) | Construction method of human amniotic epithelial stem cell bank | |
| CN106754674A (en) | Method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN1630715A (en) | Methods of preparing a transplantable product for treatment of skin defects | |
| CN108220229A (en) | A kind of preparation method for improving umbilical cord derived mesenchymal stem cell primary cell yield | |
| CN101306207A (en) | Tissue engineered cornea epithelial transplantation membrane and preparation method and use thereof | |
| CN102807965A (en) | Method for preparing tissue engineered cornea and device of method | |
| CN1321177C (en) | Fish embryo cell separation and culturing method | |
| CN105238738A (en) | Isolated culture method of piglet myocardial fibroblasts | |
| CN104988112A (en) | SD rat thoracic artery smooth muscle cell separation and culture method | |
| CN102697581B (en) | Method for constructing tissue engineering blood vessel | |
| CN109161515A (en) | The isolated culture method of megalobrama amblycephala primary hepatocyte | |
| CN102168062B (en) | Human pulmonary artery smooth muscle cell separation and culturing method and application of same | |
| CN101492653B (en) | Cultivation method for rat venae cavae smooth muscle cell | |
| CN101812427A (en) | Method for culturing rat airway smooth muscle cells | |
| CN1749393A (en) | Constructing method for human corneal endothelium cell system | |
| CN105664258A (en) | Method for preparing smooth muscle cell carrier based on small intestine submucosa acellular matrix | |
| CN110229783A (en) | A method of stem cell diaphragm is prepared based on warm ware | |
| CN102552997A (en) | Pericardium with stem cell, preparation method and application thereof | |
| CN102120983B (en) | Separation and culture method of epidermal stem cells of Beijing ducks | |
| CN106962274A (en) | A kind of organization engineering skin repairs the experiment research of nude mice full thickness dermal | |
| CN109771697B (en) | Dermal fibroblast skin sheet and construction method and application thereof | |
| CN101368171A (en) | Cultivation method for far-end venae pulmonales smooth muscle cell of rat | |
| CN111793568B (en) | Pichia pastoris and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20101006 Termination date: 20141229 |
|
| EXPY | Termination of patent right or utility model |