CN106244536A - A kind of spleen primary isolated culture method of septal cell at end - Google Patents
A kind of spleen primary isolated culture method of septal cell at end Download PDFInfo
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- CN106244536A CN106244536A CN201610833378.9A CN201610833378A CN106244536A CN 106244536 A CN106244536 A CN 106244536A CN 201610833378 A CN201610833378 A CN 201610833378A CN 106244536 A CN106244536 A CN 106244536A
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Abstract
The invention belongs to animal cell culture technology field, relate to a kind of spleen primary isolated culture method of septal cell at end.Step is as follows: the pretreatment of spleen sample；Enzymolysis sample, it is thus achieved that whole septal cell suspension；The cell inoculation of whole septal cell suspension, prepares primary cell；Primary cell pass on amplification culture, prepare passage cell；Fixing passage cell, completes the primary separation and Culture of Rats Spleen septal cell at end.The present invention is directed to the construction features of spleen tissue, use 0.1% collagenase I, 0.125% trypsin and 0.002%I type deoxyribonuclease, TCs in simultaneous digestion method separation and Culture spleen, the cell purity that the method obtains is high, vigor is good, and it is stable to pass on rear cellular morphology, vigor is high, can be widely used for effectively carrying out of follow-up relevant TCs form and functional experiment.
Description
Technical field
The invention belongs to animal cell culture technology field, relate to a kind of spleen primary isolated culture method of septal cell at end.
Background technology
Whole septal cell (Telocytes, TCs), is new discovery in recent years a kind of Interstitial cell named, widely distributed
In skin, the heart, kidney, gastrointestinal tract, bladder, the parotid gland, Placenta Hominis, lung and bronchus etc..At myocardium, endocardial, epicardial knot
Form in tissue interstitial and the interstitial in Cardiac Stem Cells pond, pulmonary vein sleeve and all confirm that it exists.Whole septal cell has typical shape
State feature: cell space is less, stretches out one or more elongated protrusion (telopodes, TPs) different in size, projection from cell space
On have narrowing portion (podomer) and bulb (podom) alternately arranged, present and read pearlitic texture.For the research of TCs, mesh
Before be concentrated mainly on the various tissue of transmission electron microscope observing and tissue different parts TCs morphological observation on, lack each device
Official TCs efficiently separates cultivation and greatly limit the further investigation to TCs.As the Interstitial cell of a kind of specific form, the most also
Not finding the specific antigen of himself, therefore researchers still can not obtain the end of purification by fluidic cell sorting technology
Septal cell is used for scientific research.Along with the people's deep understanding to its Biological characteristics, and the development of primitive cell culture technology, grind
The persons of studying carefully also gradually are exploring whole septal cell separation and the preferable method of original cuiture to obtain the cell of higher degree for shape
State and the further investigation of function assessment.As used high flux biotechnology that whole septal cell is carried out genome, the survey of protein group
Amount and analyze, study its in connective tissue with other specific type cells (endotheliocyte, fibroblast, neurocyte and
Immunocyte etc.) connect each other, find whole septal cell specific antibody etc..Therefore, for the many unknown merits of the whole septal cell of research
Can, isolation and purification culture TCs is undoubtedly an important research direction of our present stage.
At present, in heart, skin, esophagus and kidney, primary separation and Culture TCs has been reported.But the composition of Digestive system is not
The most identical.As in esophagus, being configured to Digestive system for II Collagenase Type and deoxyribonuclease, cell centrifugation speed is
2000rpm, the cell cultivation differential velocity adherent time is 60-90 min, and cell culture fluid is DMEM in high glucose culture medium, actual behaviour
Make step is also not quite similar.It is crucial that each tissue T Cs quantity of the primary separation and Culture of these methods is few, purity is relatively low, no
It is beneficial to carry out relevant cell experiment.At present, for the separation and Culture of TCs, spleen not yet has been reported that.
Summary of the invention
The present invention solves the technical problem of separation and Culture septal cell at end from spleen cell, disclose a kind of spleen eventually every
Cell primary isolated culture method.
For solve above-mentioned technical problem, the present invention by the following technical solutions:
A kind of spleen primary isolated culture method of septal cell at end, step is as follows:
(1) pretreatment of spleen sample；
(2) enzymolysis sample, it is thus achieved that whole septal cell suspension；
(3) the cell inoculation of whole septal cell suspension, prepares primary cell；
(4) primary cell pass on amplification culture, prepare passage cell；
(5) fixing passage cell, completes the primary separation and Culture of Rats Spleen septal cell at end.
In described step (1), the step of pretreatment is to take spleen sample, by spleen after rinsing with the PBS of pre-cooling
Tissue is cut into 0.5-1.5mm3Fritter.
In described step (2), the step of enzymolysis is to add the mixed enzyme with spleen tissue fritter same volume, at 37 DEG C
Processing 5min, blow and beat 80-120 time with suction pipe, 37 DEG C stand 5min, Aspirate supernatant, repeat enzymolysis piping and druming step 3-5 time.
In described step (3), the inoculation step of whole septal cell suspension is, adds equal-volume and contain the complete of low sugar in supernatant
Full culture medium, terminates digestion, obtains cell suspension, with 200 mesh filter screen filtration cell suspensions, filter the piece of tissue not digested；
1200 rpm are centrifuged 5 min, and by the resuspended precipitation of complete medium, cell counting, with 1 × 105The concentration of individual/mL is seeded to cultivate
In Ping.
In described step (3), the step of primitive cell culture is that 40 min differential velocity adherents remove fibroblast adherent on culture bottle
Dimension cell, overturns culture bottle, adds complete medium and continues to cultivate；Culture medium is changed first after 24 h, the most adherent to remove
Cell and cell debris, later every 48 h change liquid, when cell collects close to 85%, digest attached cell with pancreatin cell dissociation buffer.
In described step (3), the step of passage cell is for passing on amplification culture with 1:3 inoculation, and 2-3 is inoculated in hole for cell
In plate, row cell climbing sheet, when cell collects close to 70%, 4% paraformaldehyde is fixed standby under the conditions of lucifuge.
The beneficial effects of the present invention is: the present invention is directed to the construction features of spleen tissue, use 0.1% collagenase I,
0.125% trypsin and 0.002%I type deoxyribonuclease, the TCs in simultaneous digestion method separation and Culture spleen, the method
The cell purity obtained is high, and vigor is good, and passes on rear cellular morphology stably, and vigor is high, can be widely used for follow-up relevant TCs form
With effectively carrying out of functional experiment.
Accompanying drawing explanation
Fig. 1 is the whole septal cell of different cultivation periods under light microscopic, and wherein A is original cuiture 3 days；B is original cuiture 5 days；C
For Secondary Culture first generation cell, 5 days；D is Secondary Culture second filial generation cell, 3 days.
Fig. 2 is the spleen septal cell at end of separation and Culture under scanning electron microscope.
Fig. 3 is the spleen septal cell at end under transmission electron microscope.
Spleen septal cell at the end Double immunofluorescence figure of Fig. 4 Isolation and culture.
Detailed description of the invention
Taking Rats Spleen sample under aseptic condition, the PBS that pre-cooling contains 1% green grass or young crops-streptomycin rinses, with eye scissors by spleen
Tissue is cut into about 1 mm3Fritter, adds and piece of tissue isopyknic digestive enzyme mixture: it is public that 0.1% collagenase I(is purchased from Roche
Department), 0.125% trypsin be purchased from Sigma company) and 0.002%I type deoxyribonuclease (being purchased from Sigma company)；Disappear
Change the configuration of enzyme: weigh the type i collagen enzyme of the powder of 100mg, 12.5mg trypsin and 0.2mg I type deoxyribose respectively
Nuclease, is dissolved in 100ml PBS(pH 7.2), stirring and evenly mixing, 0.22 zut filter is degerming, cryopreservation tube 1mL subpackage, 20 DEG C
Save backup；Piece of tissue and isopyknic mixture slaking enzyme, in 37 DEG C, are blown and beaten 100 times with suction pipe in 5 min, 37 DEG C, standing 5
Min, draws supernatant；Repeat above step 3-5 time, until piece of tissue digestion is completely.The supernatant collected adds equal-volume containing low
Sugar complete medium [DMEM(Hyclone company)+10% hyclone (Hyclone company)] terminate digestion；Cell suspension is used
200 mesh filter screens filter, and filter the piece of tissue not digested；1,200 rpm is centrifuged 5 min, the resuspended precipitation of complete medium, cell
Counting, 1 × 105The concentration of individual/mL is seeded to 25 cm2In culture bottle, 40 min differential velocity adherents remove fibroblast, have added
Full culture medium continues to cultivate.Change complete medium after 24 h first, to remove the most adherent cell and cell debris, exist every day
Observe TCs representative configuration feature and growth conditions under inverted microscope, and Taking Pictures recording is as shown in Figure 1.
The most every 48 h change 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, pass on amplification culture with 1:3 inoculation, 2-3 is inoculated in orifice plate for cell, OK
Cell climbing sheet, when cell collects close to 70%, 4% paraformaldehyde is fixed standby under the conditions of lucifuge, result as in figure 2 it is shown, its
Middle A is the whole septal cell that a cell space has six projections, and B is that the projection of whole septal cell is connected with each other with flanking cell, interweaves
Reticulate, and projection is two branched growths；C is the spleen septal cell at end of separation and Culture, and projection is different in size, from some tens of pm
To hundreds of microns；D is the whole septal cell having two projections, and one of them projection is up to 297.5 microns.Wherein ↑ represent born of the same parents
Body, how in shuttle row, less, ▲ represent the enlargement being distributed in projection in beads shape.Spleen under transmission electron microscope is eventually every carefully
Born of the same parents as it is shown on figure 3, wherein A be the whole septal cell being connected with lymphocyte by projection；B be projection surround leukocyte eventually every
Cell；C be the projection of whole septal cell be two branched distributions；D be between leukocyte and hemocyte cluster distribution end every carefully
Born of the same parents.
For above experimental result it was confirmed use 0.1% collagenase I, 0.125% trypsin and 0.002%I type deoxidation
Ribonuclease simultaneous digestion method, we are the TCs in separation and Culture spleen first, it may have typical morphological feature such as Fig. 4
Shown in, wherein A, E, I are that (A is Cy3 fluorescent labeling, takes on a red color, E and I, for FITC fluorescent labeling, in green in Vimentin dyeing
Color)；B, F, J. be respectively CD34, nanog and sca-1 dyeing (B is FITC fluorescent labeling, in green, F and J, glimmering for Cy3
Signal, takes on a red color)；C, G, K are nucleus (DAPI dyes, in blueness)；D, H, L are respectively first three columns picture stacking chart, mark
Chi: 20 μm；Cell space is less, stretches out one or more TPs different in size from cell space, and projection has (podomer) and (podom)
Alternately arranged, present beads shape characteristic structural.Immunofluorescence dyeing, similar with TCs in heart, splenic T Cs expresses skeleton
Albumen vimentin, multipotency transcription factor nanog and transmembrane glycoprotein CD34, and then confirm splenic T Cs that the method separates
Character.
In a word, 0.1% collagenase I, 0.125% trypsin and 0.002%I type deoxyribonuclease simultaneous digestion are used
Method, the first TCs in separation and Culture spleen, the cell purity obtained is high, and vigor is good, can be widely used for subsequent experimental.
Claims (6)
1. the spleen primary isolated culture method of septal cell at end, it is characterised in that: step is as follows:
(1) pretreatment of spleen sample；
(2) enzymolysis sample, it is thus achieved that whole septal cell suspension；
(3) the cell inoculation of whole septal cell suspension, prepares primary cell；
(4) primary cell pass on amplification culture, prepare passage cell；
(5) fixing passage cell, completes the primary separation and Culture of Rats Spleen septal cell at end.
2. the spleen primary isolated culture method of septal cell at end as claimed in claim 1, it is characterised in that: in described step (1)
The step of pretreatment is, takes spleen sample, after rinsing with the PBS of pre-cooling, spleen tissue is cut into 0.5-1.5mm3Little
Block.
3. the spleen primary isolated culture method of septal cell at end as claimed in claim 1, it is characterised in that: in described step (2)
The step of enzymolysis is to add the mixed enzyme with spleen tissue fritter same volume, processes 5min, blow and beat 80-with suction pipe at 37 DEG C
120 times, 37 DEG C stand 5min, Aspirate supernatant, repeat enzymolysis piping and druming step 3-5 time.
4. the spleen primary isolated culture method of septal cell at end as claimed in claim 1, it is characterised in that: in described step (3)
The inoculation step of whole septal cell suspension is, adds the equal-volume complete medium containing low sugar, terminate digestion, obtain carefully in supernatant
Born of the same parents' suspension, with 200 mesh filter screen filtration cell suspensions, filters the piece of tissue not digested；1200 rpm are centrifuged 5 min, with completely
The resuspended precipitation of culture medium, cell counting, with 1 × 105The concentration of individual/mL is seeded in culture bottle.
5. the spleen primary isolated culture method of septal cell at end as claimed in claim 1, it is characterised in that: in described step (3)
The step of primitive cell culture is that 40 min differential velocity adherents remove fibroblast adherent on culture bottle, overturns culture bottle, adds
Enter complete medium to continue to cultivate；Culture medium is changed first, to remove the most adherent cell and cell debris, the most often after 24 h
48 h change liquid, when cell collects close to 85%, digest attached cell with pancreatin cell dissociation buffer.
6. the spleen primary isolated culture method of septal cell at end as claimed in claim 1, it is characterised in that: in described step (3)
The step of passage cell is for passing on amplification culture with 1:3 inoculation, and 2-3 is inoculated in orifice plate for cell, row cell climbing sheet, works as cell
When collecting close to 70%, 4% paraformaldehyde is fixed standby under the conditions of lucifuge.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865977A (en) * | 2018-07-24 | 2018-11-23 | 新乡医学院 | A kind of newborn rat skin end septal cell massive amplification method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894056A (en) * | 2015-06-19 | 2015-09-09 | 中国长江三峡集团公司中华鲟研究所 | Method for constructing spleen tissue cell line of acipenser dabryanus |
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2016
- 2016-09-20 CN CN201610833378.9A patent/CN106244536A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894056A (en) * | 2015-06-19 | 2015-09-09 | 中国长江三峡集团公司中华鲟研究所 | Method for constructing spleen tissue cell line of acipenser dabryanus |
Non-Patent Citations (2)
| Title |
|---|
| YUQIAO CHANG等: "Telocytes in the Spleen", 《PLOS ONE》 * |
| 赵金茹等主编: "《基础医学实验基本技能》", 30 September 2009, 中国医药科技出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865977A (en) * | 2018-07-24 | 2018-11-23 | 新乡医学院 | A kind of newborn rat skin end septal cell massive amplification method and application |
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Application publication date: 20161221 |