CN108865977A - A kind of newborn rat skin end septal cell massive amplification method and application - Google Patents
A kind of newborn rat skin end septal cell massive amplification method and application Download PDFInfo
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Abstract
The invention belongs to animal cell culture technology field, it is related to a kind of newborn rat skin end septal cell massive amplification method and application.Steps are as follows：The pretreatment of newborn rat skin sample；The configuration of enzymic digestion liquid；Enzymic digestion liquid handles sample, obtains whole septal cell suspension；Prepare newborn rat skin end septal cell primary cell；The passage of primary cell expands culture, prepares passage cell；c-kit+/CD34+Cell liquid is resuspended in selected by flow cytometry apoptosis derma end septal cell, expands culture, prepares the higher skin end septal cell of purity.The present invention uses 0.05% clostridiopetidase A II, 0.05% trypsase, simultaneous digestion method is separately cultured skin end septal cell, the primary whole septal cell quantity that this method obtains is big, purity is high, vigor is good, after selected by flow cytometry apoptosis, can rapid, high volume amplification, cellular morphology is stablized after amplification, and vigor is high, can be widely used for subsequent relevant final septal cell Morphology observation and functional study.
Description
Technical field
The present invention relates to animal cell culture technology field, particularly relates to a kind of newborn rat skin end septal cell and largely expand
Increasing method and application.
Background technique
Whole septal cell（Telocytes, TCs), it is a kind of interstitial cell that is newly discovered and being named, is distributed widely in skin
In the Various Tissues such as skin, heart, kidney, liver, gastrointestinal tract, bladder, the parotid gland, placenta, lung, bronchus and brain.Whole septal cell tool
There is typical morphological feature：Cell space is smaller, stretches out one or more elongated protrusions different in size from cell space（Telopodes,
TPs）, there is narrowing portion (podomer) and bulb (podom) to be alternately arranged in protrusion, show and read pearlitic texture, between surrounding
Cell plastid forms complicated three-dimensional network.
Identification for whole septal cell, transmission electron microscope observing tissue horizontal configuration feature is still unique gold mark at present
It is quasi-.And for the TCs of Isolation and culture different organ and tissue, still lack efficiently separate cultural method at present, greatly limits
Further investigation to TCs.As a kind of interstitial cell of specific form, the specific antigen of its own is not found at present, therefore
Researchers cannot still obtain the whole septal cell of purifying for scientific research by fluidic cell sorting technology.As people give birth to it
The development of the deep understanding and primitive cell culture technology of object feature, researchers are also gradually exploring whole septal cell point
From with the preferable method of originally culture, further investigation to obtain higher degree TCs, for form and function assessment.
Currently, the primary TCs that is separately cultured has been reported in heart, oesophagus and kidney.The ingredient of tissue digestion liquid is not to the utmost
It is identical.Such as in oesophagus, it is configured to digestive juice for II Collagenase Type and deoxyribonuclease, cell centrifugation rate is
2000rpm, cell culture differential velocity adherent time are 60-90 min, and cell culture fluid is DMEM in high glucose culture medium；For skin
TCs is primary to be separately cultured, and tissue digestion liquid is 0.05% collagenase P and 0.1% trypsase；But it is separately cultured by these methods
Each tissue T Cs quantity it is few, purity is lower, is unfavorable for carrying out subsequent related experiment.Currently, being directed to newborn rat skin TCs
It is separately cultured, proposes a kind of new efficient primary separation and culture method.
Summary of the invention
The present invention is the technical issues of solving rapid expansion culture and purify newborn rat skin end septal cell, to disclose one
Kind newborn rat skin end septal cell massive amplification method and application.
The technical proposal of the invention is realized in this way：
A kind of newborn rat skin end septal cell primary separation and culture method, steps are as follows：
（1）The pretreatment of newborn rat skin sample；
（2）The configuration of enzymic digestion liquid；
（3）Enzymic digestion liquid handles sample, obtains whole septal cell suspension；
（4）The cell inoculation of whole septal cell suspension, prepares primary cell；
（5）The passage of primary cell expands culture, prepares passage cell；
（6）Fixed passage cell, carries out Electronic Speculum and cellular immunofluorescence Testing and appraisal respectively；
（7）Secondary culture 2-3 generation whole septal cell, through c-kit+/CD34+Selected by flow cytometry apoptosis purifying, expands culture.
The step（1）In pretreated step be that newborn rat skin sample is taken under aseptic condition, it is slow with ice-cold PBS
After fliud flushing repeated flushing, subcutaneous tissue is carefully rejected, by historrhexis is 0.5-1.0mm with eye scissors3Organize fritter.
The step（2）The configuration of middle enzymic digestion liquid：0.05g clostridiopetidase A II and 0.05g trypsase is weighed respectively, is filled
Divide and be dissolved in the sterile PBS of 100mL, filter membrane aseptic filtration, tubule packing, -20 DEG C save backup.
The step（3）Middle enzymic digestion liquid handles the step of sample as the mixing with skin histology fritter same volume is added
Enzyme handles 10min at 37 DEG C, is blown and beaten 80-120 times, 37 DEG C of standing 5min with suction pipe, Aspirate supernatant, repeats enzymic digestion and blows
It beats step 3-5 times.
The step（4）The inoculation step of middle end septal cell suspension is to be added into supernatant in equal volume containing the complete of serum
Full culture medium terminates digestion, obtains cell suspension, with 200 mesh filter screen filtration cell suspensions, filter out the tissue block not digested；
1200 rpm are centrifuged 5 min, are resuspended and are precipitated with complete medium, cell count, with 1 × 105The concentration of a/mL is seeded to culture
In bottle.
The step（4）The step of middle primitive cell culture, removes adherent at fibre on culture bottle for 30 min differential velocity adherents
Cell is tieed up, culture bottle is overturn, complete medium is added and continues to cultivate；Culture medium is replaced for the first time after 24 h, it is not adherent to remove
Cell and cell fragment, later every 48 h change liquid, when cell collects for 83%-87%, with thin containing 0.25% pancreatin and 0.02%EDTA
Born of the same parents' digestive juice digests attached cell, carries out cell secondary culture.
The step（5）The step of middle passage cell is with 1:3 inoculation passages expand culture, and 2-3 is for cell inoculation in hole
In plate, cell climbing sheet is carried out, when cell collects for 68%-72%, the fixation of 4% paraformaldehyde is spare.
The step（6）In the fixed passage cell of 2.5% glutaraldehyde, alcohol serial dehydration is dry through low temperature drying instrument, sweeps
It retouches Electronic Speculum and captures image.
The step（6）In the fixed passage cell of 4% paraformaldehyde, the common molecular labeling skelemin vimentin of TCs,
The double marks of cellular immunofluorescence are carried out with stem cell factor c-kit and transmembrane glycoprotein CD34 respectively.
The step（7）Middle secondary culture 2-3 generation whole septal cell, through c-kit+/CD34+Selected by flow cytometry apoptosis purifying, expands
Big culture.
The newborn rat skin end septal cell of massive amplification, it is right in unicellular sequencing and protein expression pedigree research to meet
The requirement of cell concentration can also meet research of the cell transplantation to injury tissue reparation, mention for clear whole septal cell biological function
Cytological Basis has been supplied, provides a kind of new seed cell for clinical development application.
The beneficial effects of the present invention are：
1. the present invention is directed to the design feature of newborn rat skin tissue, enzyme-linked using 0.05% clostridiopetidase A II, 0.05% tryptose
It closes digestion method and is separately cultured the TCs in newborn rat skin, the cell quantity that this method obtains is big, is conducive to fluidic cell point
Choosing, and then cell purity is high, vigor is good, and cellular morphology is stablized after passage, and vigor is high, can be widely used for subsequent correlation TCs shape
Effective development of state and functional experiment.
2. being separately cultured for skin TCs is primary, tissue digestion liquid is 0.05% collagenase P and 0.1% tryptose at present
Enzyme, cell culture fluid are DMEM in high glucose culture medium.But few by the TCs quantity that these methods are separately cultured, purity is lower, unfavorable
In development TCs related experiment.
Detailed description of the invention
Fig. 1 is the whole septal cell figure that two kinds of different cultural methods obtain under phase contrast microscope, and wherein A is primary is separately cultured
3 days skin TCs（0.05% clostridiopetidase A II, 0.05% trypsase simultaneous digestion method）；B is separately cultured 3 days skin TCs to be primary
（0.05% collagenase P and 0.1% trypsase）.
Fig. 2 be under phase contrast microscope janus green B dyeing liquor to originally culture TCs visitain figure, wherein Fig. 2A be at
The TCs of cluster distribution, Fig. 2 B are that a cell space, the TCs of four protrusions, and protrusion are grown in two branch samples；↑ indicate cell space, be in more
Shuttle row, it is smaller, ▲ indicate that stretch out on cell space 1 arrives multiple elongated protrusions.
Fig. 3 is the skin end septal cell figure that is separately cultured under scanning electron microscope, and Fig. 3 A is two TCs arranged in parallel, 3B and
3C is that the protrusion of whole septal cell and flanking cell are connected with each other, and protrusion has miniliform expand；Wherein ↑ indicate cell space, ▲ table
Show the protrusion stretched out from cell space.
Fig. 4 is to be separately cultured skin end septal cell Double immunofluorescence figure using simultaneous digestion method, and wherein A, E are cell
Core（DAPI dyeing, it is blue）；B, F are respectively c-kit and CD34 dyeing（Cy3 fluorescent marker, takes on a red color）；C, G are waveform egg
White dyeing（FITC fluorescent marker, in green）；D, H are respectively first three columns picture stacking chart, scale：50μm；Cell space is smaller, from
Cell space stretches out one or more TPs different in size, has in protrusion（Podomer) and（Podom it) is alternately arranged, shows beads
Shape characteristic structural.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described, it is clear that institute
The embodiment of description is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention,
Those of ordinary skill in the art's every other embodiment obtained under that premise of not paying creative labor, belongs to this hair
The range of bright protection.
One, experimental material
Rat skin sample（1-3d newborn SD rat）, 0.05% clostridiopetidase A II（Roche company）, 0.05% trypsase（Sigma
Company）,DMEM（Hyclone company）, 10% fetal calf serum（Hyclone company）.
Two, experimental procedure
It takes Rats Spleen sample under aseptic condition, the PBS containing 1% blueness-streptomysin is pre-chilled and rinses, with eye scissors by rat skin
Tissue is cut into about 1 mm3Fritter is added and the isometric digestion enzymatic mixture of tissue block（0.05% clostridiopetidase A II（Roche is public
Department）, 0.05% trypsase（Sigma company）.
The configuration of tissue digestion liquid：The powdered II Collagenase Type of 50mg is weighed respectively, and 50mg trypsase is sufficiently molten
Solution is in 100ml PBS（PH 7.2）, 0.22 zut filter degerming, cryopreservation tube 1ml packing, -20 DEG C save backup；37 DEG C,
10 min are blown and beaten 100-120 times with suction pipe, 37 DEG C, stand 5 min, draw supernatant；It repeats above step 3-5 times, until tissue
Block digestion is complete.
Isometric [DMEM of complete medium containing low sugar is added in the supernatant of collection（Hyclone company）+ 10% fetal calf serum
（Hyclone company）] terminate digestion；Cell suspension is filtered with 200 mesh filter screens, filters out the tissue block not digested；1,200 rpm
5 min are centrifuged, complete medium, which is resuspended, to be precipitated, cell count, and 1 × 105The concentration of a/mL is seeded to 25 cm2In culture bottle, 30
Min differential velocity adherent removes fibroblast, and complete medium is added and continues to cultivate.
Complete medium is replaced for the first time after 24 h, to remove not adherent cell and cell fragment, under inverted microscope
TCs representative configuration feature and growth conditions are observed, and are photographed to record as shown in Figure 1.
Wherein A is separately cultured 3 days skin TCs to be primary（0.05% clostridiopetidase A II, 0.05% trypsase simultaneous digestion method）；
B is separately cultured 3 days skin TCs to be primary（0.05% collagenase P and 0.1% trypsase）, ↑ indicate cell space, is in shuttle row more, compared with
It is small, ▲ indicate that stretch out on cell space 1 arrives multiple elongated protrusions.
It is observed that compared with 0.05% collagenase P of conventional method and 0.1% trypsase, 0.05% glue of our uses
The newborn rat skin TCs that protoenzyme II, 0.05% trypsase simultaneous digestion method are separately cultured obtains cell purity height, vigor
Good, quantity is big, and cellular morphology is stablized after passage.
Janus green B（Janus Green B）Dyeing liquor is to originally culture TCs visitain, as shown in Fig. 2, primary point
5 days from culture skins TCs, Fig. 2A are the TCs of cluster distribution, and Fig. 2 B is that a cell space, the TCs of four protrusions, and protrusion are in two
The growth of branch sample.↑ indicate cell space, it is in shuttle row more, it is smaller, ▲ indicate that stretch out on cell space 1 arrives multiple elongated protrusions.It was found that
The newborn rat skin TCs that 0.05% clostridiopetidase A II, 0.05% trypsase simultaneous digestion method are separately cultured, cell space and protrusion it is swollen
Most of Distribution of mitochondria is abundant, meets TCs morphological feature.
Every 48 h changes liquid, when cell collects close to 85%, pancreatin cell dissociation buffer（Containing 0.25% pancreatin, 0.02%EDTA）, 37
DEG C, 2 min digest attached cell, with 1:3 inoculation passages expand culture, and 2-3 in orifice plate, carries out cell and climb for cell inoculation
Piece,
When cell collects close to 70%, 2.5% glutaraldehyde fixed 30min, PBS are washed 3 times, alcohol serial dehydration, through low temperature drying
Instrument is dry, and scanning electron microscope captures image, as a result as shown in figure 3, it is whole septal cell that figure A, which is two TCs, B and C arranged in parallel,
Protrusion and flanking cell be connected with each other, protrusion has miniliform expand.Wherein ↑ and indicate cell space, it is in shuttle row, smaller, ▲ table more
Show the protrusion stretched out from cell space.
For the above experimental result, it was confirmed that separated using 0.05% clostridiopetidase A II, 0.05% trypsase simultaneous digestion method
The newborn rat skin TCs of culture has typical morphological feature, as shown in figure 4, A, E are nucleus（DAPI is dyed, and is in
Blue）；B, F are respectively c-kit and CD34 dyeing（Cy3 fluorescent marker, takes on a red color）；C, G are vimentin dyeing（FITC fluorescence
Label, in green）；D, H are respectively first three columns picture stacking chart, scale：50μm；Cell space is smaller, stretches out one or more from cell space
A TPs different in size has in protrusion（Podomer) and（Podom it) is alternately arranged, shows beads shape characteristic structural.Exempt from
Epidemic disease fluorescent staining shows that TCs expresses skelemin vimentin, stem cell factor c-kit and transmembrane glycoprotein CD34, and then demonstrate,proves
The real character of the skin TCs of this method separation.
Currently, be separately cultured for skin TCs is primary, tissue digestion liquid is 0.05% collagenase P and 0.1% trypsase,
Cell culture fluid is DMEM in high glucose culture medium, the primary whole septal cell negligible amounts of culture, with enormous amount and multiplication rate compared with
Together, after secondary culture, fibroblast proliferation is significant for fast fibroblast, and the quantity of whole septal cell will be increasingly
It is few.This kind of method obtains TCs and is not able to satisfy cell concentration needed for airflow classification, is unfavorable for TCs and isolates and purifies.
In short, using 0.05% clostridiopetidase A II, 0.05% trypsase simultaneous digestion method, low sugar DMEM culture medium culture TCs.
The newborn rat skin TCs being separately cultured by this method, it is big to obtain TCs quantity, and proliferation is rapid；Form stable after passage, it is living
Power is good, can satisfy c-kit+/CD34+Selected by flow cytometry apoptosis purifying, and then the rapid amplifying of TCs may be implemented.Massive amplification
Newborn rat skin end septal cell, requirement to cell concentration in unicellular sequencing and protein expression pedigree research can be met,
Research of the cell transplantation to injury tissue reparation can be met, provide Cytological Basis for clear whole septal cell biological function,
A kind of new seed cell is provided for clinical development application.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of newborn rat skin end septal cell massive amplification method, which is characterized in that steps are as follows：
（1）The pretreatment of newborn rat skin sample；
（2）The configuration of enzymic digestion liquid；
（3）Enzymic digestion liquid handles sample, obtains whole septal cell suspension；
（4）The cell inoculation of whole septal cell suspension, prepares primary cell；
（5）The passage of primary cell expands culture, prepares passage cell；
（6）Fixed passage cell, carries out Electronic Speculum and cellular immunofluorescence Testing and appraisal respectively；
（7）Secondary culture 2-3 generation whole septal cell, through c-kit+/CD34+Selected by flow cytometry apoptosis expands culture after purification, obtains
Obtain largely whole septal cell.
2. newborn rat skin end septal cell massive amplification method as described in claim 1, it is characterised in that：The step
（1）In pretreated step be that newborn rat skin sample is taken under aseptic condition, it is young after ice cold PBS buffer repeated flushing
It is thin to reject subcutaneous tissue, it by historrhexis is 0.5-1.0mm with eye scissors3Organize fritter.
3. newborn rat skin end septal cell massive amplification method as described in claim 1, it is characterised in that：The step
（2）The configuration of middle enzymic digestion liquid：0.05g clostridiopetidase A II and 0.05g trypsase is weighed respectively, and it is sterile to be completely dissolved in 100mL
In PBS, filter membrane aseptic filtration, tubule packing, -20 DEG C are saved backup.
4. newborn rat skin end septal cell massive amplification method as described in claim 1, it is characterised in that：The step
（3）The step of middle enzymic digestion liquid processing sample is that the mixed enzyme with skin histology fritter same volume is added, handles at 37 DEG C
10min is blown and beaten 80-120 times, 37 DEG C of standing 5min, Aspirate supernatant with suction pipe, is repeated enzymic digestion and is blown and beaten step 3-5 times.
5. newborn rat skin end septal cell massive amplification method as described in claim 1, it is characterised in that：The step
（4）The inoculation step of middle end septal cell suspension is the complete medium being added into supernatant in equal volume containing serum, and termination disappears
Change, obtains cell suspension, with 200 mesh filter screen filtration cell suspensions, filter out the tissue block not digested；1200 rpm are centrifuged 5 min,
It is resuspended and is precipitated with complete medium, cell count, with 1 × 105The concentration of a/ml is seeded in culture bottle.
6. newborn rat skin end septal cell massive amplification method as described in claim 1, it is characterised in that：The step
（4）The step of middle primitive cell culture is that 30 min differential velocity adherents remove fibroblast adherent on culture bottle, overturning culture
Bottle is added complete medium and continues to cultivate；Culture medium is replaced for the first time after 24 h, to remove not adherent cell and cell fragment,
Later every 48 h changes liquid, when cell collects for 83%-87%, with adherent containing 0.25% pancreatin and the digestion of 0.02%EDTA cell dissociation buffer
Cell carries out cell secondary culture.
7. newborn rat skin end septal cell massive amplification method as described in claim 1, it is characterised in that：The step
（5）The step of middle passage cell, is, with 1:3 inoculation passages expand culture, and 2-3 in orifice plate, carries out cell and climb for cell inoculation
Piece, when cell collects for 68%-72%, the fixation of 4% paraformaldehyde is spare.
8. newborn rat skin end septal cell massive amplification method as described in claim 1, it is characterised in that：The step
（7）Middle c-kit+/CD34+Selected by flow cytometry apoptosis technology is incubated for whole septal cell using the primary antibody with fluorescent marker, and homotype pair is arranged
According to and blank control, whole septal cell expands culture after sorting, carries out Immunohistochemical or Immunofluorescence test, verifying point
Select purity.
9. such as the application of the described in any item newborn rat skin end septal cell massive amplification methods of claim 1-8, feature
It is：The newborn rat skin end septal cell of the massive amplification answering in unicellular sequencing and protein expression pedigree research
With, and the application in injury tissue is repaired in cell transplantation.
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| CN110305857A (en) * | 2019-05-22 | 2019-10-08 | 中国人民解放军第四军医大学 | The method that mouse skin digests complex enzyme and separates skeptophylaxis cell Treg cell from mouse skin |
| CN110747159A (en) * | 2019-11-12 | 2020-02-04 | 武汉普诺赛生命科技有限公司 | Mouse or rat kidney fibroblast cell separation and subculture method |
| CN112708610A (en) * | 2021-03-26 | 2021-04-27 | 上海伯豪生物技术有限公司 | Mixed enzyme for skin tissue dissociation, preparation method thereof, dissociation kit and dissociation method |
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| CN110257353A (en) * | 2019-05-22 | 2019-09-20 | 中国人民解放军第四军医大学 | The method that application on human skin digests complex enzyme and separates skeptophylaxis cell Treg cell from people on a small quantity full pachydermia |
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