CN106318901A - Method for culturing in vitro three-dimensional respiratory tract stem/ancestral cells - Google Patents
Method for culturing in vitro three-dimensional respiratory tract stem/ancestral cells Download PDFInfo
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- 210000002345 respiratory system Anatomy 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000000338 in vitro Methods 0.000 title abstract description 3
- 238000012258 culturing Methods 0.000 title abstract 2
- 239000001963 growth medium Substances 0.000 claims abstract description 11
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 10
- 108010082117 matrigel Proteins 0.000 claims abstract description 8
- 239000012981 Hank's balanced salt solution Substances 0.000 claims abstract description 4
- 239000000203 mixture Substances 0.000 claims abstract description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 10
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 10
- 241000699670 Mus sp. Species 0.000 claims description 6
- 229930182555 Penicillin Natural products 0.000 claims description 6
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 6
- FHYUGAJXYORMHI-UHFFFAOYSA-N SB 431542 Chemical compound C1=CC(C(=O)N)=CC=C1C1=NC(C=2C=C3OCOC3=CC=2)=C(C=2N=CC=CC=2)N1 FHYUGAJXYORMHI-UHFFFAOYSA-N 0.000 claims description 6
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 229940049954 penicillin Drugs 0.000 claims description 6
- 229960001471 sodium selenite Drugs 0.000 claims description 6
- 235000015921 sodium selenite Nutrition 0.000 claims description 6
- 239000011781 sodium selenite Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 102000004877 Insulin Human genes 0.000 claims description 5
- 108090001061 Insulin Proteins 0.000 claims description 5
- 102000004338 Transferrin Human genes 0.000 claims description 5
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- 229940125396 insulin Drugs 0.000 claims description 5
- 229960005322 streptomycin Drugs 0.000 claims description 5
- 239000012581 transferrin Substances 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 3
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- 238000004113 cell culture Methods 0.000 abstract description 6
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- 108091003079 Bovine Serum Albumin Proteins 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- 238000012136 culture method Methods 0.000 abstract 1
- 239000012894 fetal calf serum Substances 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 55
- 210000000130 stem cell Anatomy 0.000 description 8
- 108091005735 TGF-beta receptors Proteins 0.000 description 4
- 102000016715 Transforming Growth Factor beta Receptors Human genes 0.000 description 4
- 101000994437 Homo sapiens Protein jagged-1 Proteins 0.000 description 3
- 102100032702 Protein jagged-1 Human genes 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 230000009668 clonal growth Effects 0.000 description 2
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- 238000012549 training Methods 0.000 description 2
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 210000001552 airway epithelial cell Anatomy 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
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- 208000030603 inherited susceptibility to asthma Diseases 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0688—Cells from the lungs or the respiratory tract
- C12N5/0689—Stem cells; Progenitors
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- C12N2500/00—Specific components of cell culture medium
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- C12N2501/30—Hormones
- C12N2501/33—Insulin
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- C12N2502/00—Coculture with; Conditioned medium produced by
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Abstract
The invention relates to a method for culturing in vitro three-dimensional respiratory tract stem/ancestral cells. The method comprises the following steps: (1) uniformly mixing respiratory tract stem/ancestral cells and MLg cells in 50mu l of an HBSS buffer solution containing 10% of fetal calf serum, adding 50mu l of Matrigel, continuously mixing the materials uniformly, and then adding the mixture into a small cell culture chamber; (2) performing standing for 30 minutes in an incubator at 37 DEG C to enable the Matrigel to be solidified; and (3) adding 410mu l of a DMEM/F12 culture medium into a corresponding pore plate below the small cell culture chamber, placing the pore plate into a cell incubator for culture, and replacing the culture medium once every other day, wherein the respiratory tract stem/ancestral cells can grow into spherical structures after 14 days of culture. By use of the culture method, before phenotype identification of the respiratory tract stem/ancestral cells, functions, regulation and control of the respiratory tract stem/ancestral cells can be researched.
Description
Technical field
The present invention relates to a kind of cell culture processes, the cultivation side of a kind of external three-dimensional respiratory tract ancestral cells
Method.
Background technology
Respiratory tract ancestral cells is the important cells of airway epithelial reparation and regeneration.With airway epithelial cell mucosa
It is closely related that damage, hypertrophy or canceration can cause many major diseases such as bronchial asthma, chronic obstructive pulmonary disease or pulmonary carcinoma.
At present the phenotype of respiratory tract ancestral cells is not disclosed the most completely, to the research of its function and regulation and control by one
Fixed restriction.Hence set up the cultural method of a kind of external three-dimensional respiratory tract ancestral cells by dry for respiratory tract of going a long way greatly/ancestral
Cell function is studied.
The cultural method of present stage respiratory tract ancestral cells being supported, cell derived becomes fiber finer in the primary lung of mice
Born of the same parents, these cells need to utilize airflow classification to obtain, and owing to content is the highest, once test and usually need many mices, not only take
Time laborious, also increase a lot of reagent cost.
Summary of the invention
The technical problem to be solved is to provide the cultivation side of a kind of external three-dimensional respiratory tract ancestral cells
Method.
For solving above-mentioned technical problem, the technical scheme is that
The cultural method of a kind of external three-dimensional respiratory tract ancestral cells, specifically comprises the following steps that
(1) respiratory tract ancestral cells and MLg cell (mouse lung fibroblast) are evenly mixed in 50 μ l containing 10%
In the HBSS buffer of hyclone, wherein, the consumption of respiratory tract ancestral cells is 1000-10000 cell/cell, and MLg is thin
Born of the same parents' consumption is 200000 cells/cell, adds 50 μ l Matrigel matrigels, is added into cell training after continuing mix homogeneously
Support little indoor；
(2) in 37 DEG C of incubators, stand 30 minutes, make Matrigel matrigel solidify；
(3) in the corresponding orifice plate that cell is cultivated below cell, add 410 μ l DMEM/F12 culture medium, orifice plate is placed in
Cultivating in cell culture incubator, every other day change a subculture, after cultivating 14 days, respiratory tract ancestral cells grows up to chondritic.
Preferably, the cultural method of above-mentioned external three-dimensional respiratory tract ancestral cells, described respiratory tract ancestral cells is outstanding
Floating people, mice or the respiratory tract ancestral cells of other mammal.
Preferably, the cultural method of above-mentioned external three-dimensional respiratory tract ancestral cells, it is Germany that described cell cultivates cell
The circular repeatedly used cell in Greiner Bio-One 1 hole cultivates cell.
Preferably, the cultural method of above-mentioned external three-dimensional respiratory tract ancestral cells, described DMEM/F12 culture medium includes
10% hyclone, penicillin, streptomycin, insulin, transferrin, sodium selenite, SB431542 are (for TGF-β Receptor
Inhibitor) and distilled water, wherein, every 100ml DMEM/F12 culture medium hyclone Han 10ml, 10000U penicillin, 10mg chain
Mycin, 1mg insulin, 0.55mg transferrin, 0.5 μ g sodium selenite, 10 μMs of SB431542 (suppress for TGF-β Receptor
Agent), remaining is distilled water.
The invention has the beneficial effects as follows:
The cultural method of described external three-dimensional respiratory tract ancestral cells, with Apoptosis for supporting cell, greatly
Reduce greatly experimental cost, and the time be quick, respiratory tract ancestral cells grow up to chondritic needed for time by existing 4
Shorten in week 2 weeks, its function and regulation and control can be studied before respiratory tract ancestral cells phenotypic evaluation.
Accompanying drawing explanation
Fig. 1 is the chondritic figure of respiratory tract ancestral cells；
Fig. 2 is the clonal growth situation map of respiratory tract stem cell.
Detailed description of the invention
In order to make those skilled in the art be better understood from technical scheme, below in conjunction with detailed description of the invention
Technical scheme of the present invention is described in further detail.
Embodiment 1
The cultural method of a kind of external three-dimensional respiratory tract ancestral cells, specifically comprises the following steps that
(1) Respiratory Tract of Mice ancestral cells is evenly mixed in containing of 50 μ l with MLg cell (mouse lung fibroblast)
In the HBSS buffer of 10% hyclone, wherein, the consumption of respiratory tract ancestral cells is 1000-10000 cell/cell,
MLg cell consumption is 200000 cells/cell, adds 50 μ l Matrigel matrigels, is added into thin after continuing mix homogeneously
Born of the same parents cultivate little indoor, and it is the circular repeatedly used cell training of Germany Greiner Bio-One 1 hole that described cell cultivates cell
Support cell；
(2) in 37 DEG C of incubators, stand 30 minutes, make Matrigel matrigel solidify；
(3) in the corresponding orifice plate that cell is cultivated below cell, add 410 μ l DMEM/F12 culture medium, orifice plate is placed in
Cultivating in cell culture incubator, every other day change a subculture, after cultivating 14 days, respiratory tract ancestral cells can grow up to chondritic
(Fig. 1 is shown in by globuli cell picture), wherein, described DMEM/F12 culture medium includes 10% hyclone, penicillin, streptomycin, pancreas
Island element, transferrin, sodium selenite, SB431542 (for TGF-β Receptor inhibitor) and distilled water, wherein, every 100ml
DMEM/F12 culture medium hyclone Han 10ml, 10000U penicillin, 10mg streptomycin, 1mg insulin, 0.55mg transports ferrum egg
In vain, 0.5 μ g sodium selenite, 10 μMs of SB431542 (for TGF-β Receptor inhibitor), remaining is distilled water.
Embodiment 2
Using method described in embodiment 1 to carry out respiratory tract hematopoietic stem/progenitor cells in vitro Three-dimensional cell culture, this method helps
Still function and the regulation and control of air flue stem cell can cannot be studied in the case of purification stem cell.Specific experiment process is as follows:
Matched group and drug treating group (JAG1) is set for the Respiratory Tract of Mice ancestral cells after cultivating.Cultivate 14 days
After, examine under a microscope and record clonal growth situation.After finding to add JAG1 in respiratory tract stem cell media, breathe
The cloning efficiency of road stem cell is decreased obviously (see Fig. 2), the increasing of these the results shows JAG1 suppression respiratory tract stem cell
Grow, illustrate that Notch1 signal path directly or indirectly regulates and controls the function of respiratory tract stem cell.
Above-mentioned retouch in detail what the cultural method of this external three-dimensional respiratory tract ancestral cells was carried out with reference to detailed description of the invention
State, be illustrative rather than determinate, can according to restriction scope list several embodiments, therefore without departing from this
Changing and modifications under invention general plotting, within should belonging to protection scope of the present invention.
Claims (4)
1. the cultural method of an external three-dimensional respiratory tract ancestral cells, it is characterised in that: specifically comprise the following steps that
(1) respiratory tract ancestral cells and MLg cell are evenly mixed in the HBSS buffer containing 10% hyclone of 50 μ l
In, wherein, the consumption of respiratory tract ancestral cells is 1000-10000 cell/cell, and MLg cell consumption is 200000 cells/little
Room, adds 50 μ l Matrigel matrigels, is added into cell and cultivates little indoor after continuing mix homogeneously；
(2) in 37 DEG C of incubators, stand 30 minutes, make Matrigel matrigel solidify；
(3) in the corresponding orifice plate that cell is cultivated below cell, add 410 μ l DMEM/F12 culture medium, orifice plate is placed in cell
Cultivating in incubator, every other day change a subculture, after cultivating 14 days, respiratory tract ancestral cells grows up to chondritic.
The cultural method of external three-dimensional respiratory tract ancestral cells the most according to claim 1, it is characterised in that exhale described in:
Inhale the respiratory tract ancestral cells that road ancestral cells is suspension people, mice or other mammal.
The cultural method of external three-dimensional respiratory tract ancestral cells the most according to claim 1, it is characterised in that: described carefully
Born of the same parents cultivate cell and cultivate cell for the circular repeatedly used cell in Germany Greiner Bio-One 1 hole.
The cultural method of external three-dimensional respiratory tract ancestral cells the most according to claim 1, it is characterised in that: described
DMEM/F12 culture medium include 10% hyclone, penicillin, streptomycin, insulin, transferrin, sodium selenite,
SB431542 and distilled water, wherein, every 100ml DMEM/F12 culture medium hyclone Han 10ml, 10000U penicillin, 10mg
Streptomycin, 1mg insulin, 0.55mg transferrin, 0.5 μ g sodium selenite, 10 μMs of SB431542, remaining is distilled water.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877964A (en) * | 2015-04-24 | 2015-09-02 | 赵振民 | In vitro construction method for salivary glands organs and acinus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877964A (en) * | 2015-04-24 | 2015-09-02 | 赵振民 | In vitro construction method for salivary glands organs and acinus |
Non-Patent Citations (1)
| Title |
|---|
| ROXANA M. TEISANU等: "Functional Analysis of Two Distinct Bronchiolar Progenitors during Lung Injury and Repair", 《AM J RESPIR CELL MOL BIOL.》 * |
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