CN106609259A - Mouse thyroid epithelial cell isolation and culture method - Google Patents
Mouse thyroid epithelial cell isolation and culture method Download PDFInfo
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- CN106609259A CN106609259A CN201510702243.4A CN201510702243A CN106609259A CN 106609259 A CN106609259 A CN 106609259A CN 201510702243 A CN201510702243 A CN 201510702243A CN 106609259 A CN106609259 A CN 106609259A
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Abstract
The invention provides a mouse thyroid epithelial cell isolation and culture method. The method includes the steps of: (a) conducting intraperitoneal injection of pentobarbital sodium to kill a mouse, performing alcohol disinfection, than taking out a mouse thyroid tissue connected to a trachea, conducting soaking washing in a precooled sterile double-antibody-containing PBS buffer solution, and stripping the thyroid tissue; (b) carrying out mechanical dissociation of the thyroid tissue, then preheating a mixed enzyme solution and performing shock digestion for 30-40min, using Ficoll to separate thyroid mononuclear cells, and conducting centrifugation to obtain cell precipitate; (c) employing a completely mixed medium to resuspend the cell precipitate, performing inoculation in a treated cell bottle, and carrying out differential adhesion to obtain thyroid epithelial cells; and (d) culturing the purified thyroid epithelial cells in the completely mixed medium. The mouse thyroid epithelial cell isolation and culture method provided by the invention can acquire mouse thyroid epithelial cells with the characteristics of high yield, high purity and long survival time.
Description
Technical field
The invention belongs to cell biology, and in particular to a kind of Mouse thyroid epithelial cell is separated and cultural method.
Background technology
Thyroid is the very important body of gland of vertebratess, belongs to endocrine organ, below mammal cervical region thyroid cartilage, trachea both sides.Wherein, Thyroid follicular epithelial cell (also referred to as follicular cellss or main cell) in thyroid cell is responsible for producing and secretes thyroxin, including tetraiodothyronine (T4) and 3 (T3), with the important physiological function for adjusting animal body growth promoter, basal metabolism and breeding activity.Abnormal thyroid function can cause thyroid adenoma, thyroid carcinoma, primary hyperthyroidism and the disease such as go down, and the In vitro culture of thyroid cell is a kind of important method of thyroid disease research.Have drawn from the tissue of relevant thyroid original cuiture at present the larger mammal of build, such as pig, Canis familiaris L., sheep, rabbit etc. more, relatively costly, and the thyroid cell of culture the survival in vitro time and function difference it is also larger.Due to the parathyroid tissue of mice it is less, draw materials, but MOUSE REPRODUCTION rate is high, experimental cost can be reduced, and biological property is clear and is easy to the advantages such as operation, it is to study one of optimal model organism of human diseasess, the isolated culture method for obtaining Thyroid follicular epithelial cell provided by the present invention can obtain high yield, purity height and the longer Mouse thyroid epithelial cell of life span.
The content of the invention
The purpose of the present invention is to set up a kind of method for obtaining high yield, purity height and the longer Mouse thyroid epithelial cell separation and Culture of life span.
Above-mentioned involved in order to solve the problems, such as, the present invention takes following method to obtain Mouse thyroid epithelial cell：
The step of Mouse thyroid epithelial cell separation method, includes：A () lumbar injection pentobarbital sodium puts to death mice, take out the Mouse thyroid that is connected with trachea and organize after alcohol disinfecting, and it is aseptic containing washing by soaking in dual anti-PBS to be placed in pre-cooling, and peels off parathyroid tissue；B, after () mechanical dissociation parathyroid tissue, preheating mixed enzyme solution concussion digestion 30-40min, Ficoll separate thyroid mononuclearcell, and centrifugation obtains cell precipitation；C () mixing complete medium re-suspended cell precipitation, is inoculated in the cell bottle after processing, differential attachment method obtains Thyroid follicular epithelial cell；Thyroid follicular epithelial cell after (d) mixing complete medium culture purified.
Optional mice is body weight 20-25g, the mice of 6-8 week old.
Optionally digestion digestion enzyme liquid used is that digestion time is 30-40min in advance in the enzyme mixation of 37 DEG C of 0.05%-0.1% (m/v) pancreatin for preheating, 0.1%-0.2% (m/v) type i collagen enzyme, 0.05%-0.15% (m/v) II Collagenase Types and 1-2U/mL neutral protease；
The method of optional concussion digestion is 37 DEG C of water-bath concussions, and concussion frequency is 250-300 time/min；
The processing method of optional cell bottle is containing 5% 37 DEG C of overnight incubations of mice serum；
Optional differential velocity adherent method in 37 DEG C of incubators, after adherent 20-30min, not adherent cell suspension is transferred in new coating plate again, the step is repeated once for cell suspension.
The basal medium of optional mixing complete medium is RPMI1640, DMEM/F12 and α-MEM (1: 1: 1～1: 1.5: 1).
The addition factor in optional mixing complete medium is 100 μ g/mL Niu Chuiti and inferior colliculus brain extract, 10ng/mL hydrocortisone, 10 μ g/mL insulins, 20-50ng/mL trilutes, the mice serum of 20-50ng/mL epidermal growth factor EGF and 5%-10%.
In the Mouse thyroid epithelial cell separation method that the present invention is provided, Digestive system selects 0.05%-0.1% (m/v) pancreatin, 0.1%-0.2% (m/v) type i collagen enzyme, 0.05%-0.15% (m/v) II Collagenase Types and the 1-2U/mL neutral protein enzyme mixations of preheating to peptic cell, on the one hand reduce the consumption of pancreatin, reduce damage of the pancreatin to Thyroid follicular epithelial cell, the survival rate of cell is improve, the time required on the other hand saving digestion.
The present invention provides a kind of method of mixing complete medium culture thyroid cell, increased the life cycle of Thyroid follicular epithelial cell, and reduces the species for adding the factor, reduces cost.
Further, the present invention from add containing 100 μ g/mL Niu Chuiti and inferior colliculus brain extract, 10ng/mL hydrocortisone, 10 μ g/mL insulins, 20-50ng/mL trilutes, the mice serum of 20-50ng/mL epidermal growth factors EGF and 10% mixing complete medium as early growth period cell inoculation liquid, when cell grows up to monolayer, the mixing complete medium containing 5% mice serum is replaced by, promotes Mouse thyroid cell to keep the Epithelial of long period.
The present invention solves that Thyroid follicular epithelial cell separation and Culture process cost is high, time-to-live short problem, and the Mouse thyroid epithelial cell yield for making acquisition is high, purity is high and life span is longer.
Description of the drawings
Fig. 1：The culture Mouse thyroid epithelial cell of 5 days；
Fig. 2：Mouse thyroid epithelial cell identified by immunofluorescence.
Specific embodiment
In order that the purpose of the present invention and advantage represent more pure and freshly, now specific embodiment is expanded on further.Specific embodiment set forth herein is explained only for the present invention, is not intended to limit the present invention.
The present invention selects the male mice of 6-8 week old, separates Thyroid follicular epithelial cell.Concrete operations are as follows：
1st, take the male mice of 6-8 week old, lumbar injection pentobarbital sodium puts to death mice, and fixed mice takes out the Mouse thyroid tissue being connected with trachea after 75% alcohol disinfecting, it is placed in pre-cooling aseptic containing washing by soaking in dual anti-PBS buffer, and peels off parathyroid tissue；
2nd, the attachments such as peplos on thyroid, hemocyte and connective tissue are promptly removed on ice under microscope, the thyroid after stripping is gone in new PBS and is cleaned 2 times；
3rd, the broken parathyroid tissue of dissecting scissorss mechanical shear, size about 1mm are used on ice3The fragment of left and right；
The 4th, the tissue for shredding is added the digestion mixed liquor of 4-5 times of volume of preheating, in 37 DEG C of concussion digestion 30-40min, concussion frequency is 250-300 time/min；The digestion mixed liquor includes 0.05%-0.1% (m/v) pancreatin, 0.1%-0.2% (m/v) type i collagen enzyme, 0.05%-0.15% (m/v) II Collagenase Types and 1-2U/mL neutral protease；
5th, piping and druming disperses cell, Jing after 200 mesh sieve net filtrations, adds into the separating liquid containing Ficoll, 2000r/min centrifugations, takes middle white layer, and 1500rpm/min centrifugation 5min abandon supernatant；
6th, prepare the mixing complete medium containing 10% mice serum：By RPMI1640, DMEM/F12 with α-MEM culture medium according to 1: 1: 1～1: 1.5: 1 proportional arrangement add 100 μ g/mL Niu Chuiti and inferior colliculus brain extract, 10ng/mL hydrocortisone, 10 μ g/mL insulins, 20-50ng/mL trilutes, the mice serum of 20-50ng/mL epidermal growth factors EGF and 10% in mixed base culture medium into mixed base culture medium.
Prepare the mixing complete medium containing 5% mice serum：By RPMI1640, DMEM/F12 with α-MEM culture medium according to 1: 1: 1～1: 1.5: 1 proportional arrangement add 100 μ g/mL Niu Chuiti and inferior colliculus brain extract, 10ng/mL hydrocortisone, 10 μ g/mL insulins, 20-50ng/mL trilutes, the mice serum of 20-50ng/mL epidermal growth factors EGF and 5% in mixed base culture medium into mixed base culture medium.
7th, precipitated with the mixing complete medium re-suspended cell of 10% mice serum, be inoculated in the cell bottle Jing after 5% 37 DEG C of overnight incubations of mice serum are processed, be placed in 37 DEG C, the CO of 5% (m/v)2After 20-30min is cultivated in the incubator of saturated humidity, not adherent cell suspension is transferred in the cell bottle after new process, above-mentioned differential velocity adherent step is repeated once.
8th, after cultivating 24h, the mixing complete medium of 5% mice serum is replaced by, visible cell is adherent under microscope, in aggregation growth, cell is rounded or oval.
9th, identified by immunofluorescence：(1) after cell growth 5d, culture medium being discarded, cell 2 times being rinsed with the PBS for incubating, then each 10min fixes cell 15min at ambient temperature with 4% paraformaldehyde；(2) PBS flushings cell 2 times, each 10min, then under the conditions of 4 DEG C, with 0.1%Triton X-100 permeable membrane 15min；(3) PBS flushings cell 2 times, each 10min, then at ambient temperature, with 4%BSA closing cell 30min；(4) resist by 1: 100 dilution proportion monoclonal Cytokeratin-18 mono-, be then placed on overnight incubation in 4 DEG C of refrigerators；(5) PBS flushings cell 3 times, each 10min are resisted by 1: 100 dilution proportion two, place 1h under the conditions of 37 DEG C；(6) rinsed 3 times with PBS, each 10min, last DAPI dye nucleus are simultaneously taken pictures with fluorescence microscope.
Below the preferred embodiment to the invention is described in detail; but the invention is not limited to above-described embodiment; all to make any modification within the spirit and principles in the present invention, equivalent and improvement etc. are should be included in protection scope of the present invention.
Claims (9)
1. a kind of Mouse thyroid epithelial cell is separated and cultural method, it is characterised in that including step：(a) abdomen
Chamber injection pentobarbital sodium puts to death mice, and the Mouse thyroid tissue being connected with trachea is taken out after alcohol disinfecting,
It is placed in pre-cooling aseptic containing washing by soaking in dual anti-PBS, and peels off parathyroid tissue；(b) machinery
After dissociation parathyroid tissue, preheating mixed enzyme solution concussion digestion 30-40min, Ficoll separate the single core of thyroid
Cell, centrifugation obtain cell precipitation；(c) mixing complete medium re-suspended cell precipitation, after being inoculated in process
In cell bottle, differential attachment method obtains Thyroid follicular epithelial cell；After (d) mixing complete medium culture purified
Thyroid follicular epithelial cell.
2. Mouse thyroid epithelial cell according to claim 1 is separated and cultural method, and its feature exists
In, described mice be 20-25g, the mice of 6-8 week old.
3. Mouse thyroid epithelial cell according to claim 1 is separated and cultural method, and its feature exists
In step b digestion digestion enzyme liquid used is that, in advance in the mixed liquor of 37 DEG C of preheatings, digestion time is
30-40min；The mixed liquor include 0.05%-0.1% (m/v) pancreatin, 0.1%-0.2% (m/v) type i collagen enzyme,
0.05%-0.15% (m/v) II Collagenase Types and 1-2U/mL neutral protease.
4. Mouse thyroid epithelial cell according to claim 1 is separated and cultural method, and its feature exists
In the method for the step b concussion digestion is 37 DEG C of water-bath concussions, and concussion frequency is 250-300 time/min.
5. Mouse thyroid epithelial cell according to claim 1 is separated and cultural method, and its feature exists
In in step c, the processing method of cell bottle is containing 5% 37 DEG C of overnight incubations of mice serum.
6. Mouse thyroid epithelial cell according to claim 1 is separated and cultural method, and its feature exists
In the differential velocity adherent of, step c be cell suspension in 37 DEG C of incubators after adherent 20-30min, will not
Adherent cell suspension is transferred in new cell bottle again, is repeated once differential velocity adherent, collects cell suspension.
7. Mouse thyroid epithelial cell according to claim 1 is separated and cultural method, and its feature exists
In, in step c and step d mix complete medium basal medium be RPMI 1640,
DMEM/F 12 and α-MEM (1: 1: 1～1: 1.5: 1), the addition factor are that 100 μ g/mL Niu Chuiti and hypothalamuses are carried
Take thing, 10ng/mL hydrocortisone, 10 μ g/mL insulins, 20-50ng/mL trilutes,
20-50ng/mL epidermal growth factor EGF.
8. Mouse thyroid epithelial cell according to claim 1 is separated and cultural method, and its feature exists
In the serum in step c in mixing complete medium is the mice serum of 10% concentration.
9. Mouse thyroid epithelial cell according to claim 1 is separated and cultural method, and its feature exists
In the serum in step d in mixing complete medium is the mice serum of 5% concentration.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109423475A (en) * | 2017-09-05 | 2019-03-05 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of mouse skeletal muscle satellite cells |
| CN114317399A (en) * | 2021-08-18 | 2022-04-12 | 川北医学院 | Thyroid organoid culture medium, thyroid organoid culture and passage method |
-
2015
- 2015-10-23 CN CN201510702243.4A patent/CN106609259A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 刘泽兵等: "小鼠甲状腺细胞原代培养及鉴定", 《中国地方病学杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109423475A (en) * | 2017-09-05 | 2019-03-05 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of mouse skeletal muscle satellite cells |
| CN114317399A (en) * | 2021-08-18 | 2022-04-12 | 川北医学院 | Thyroid organoid culture medium, thyroid organoid culture and passage method |
| CN114317399B (en) * | 2021-08-18 | 2024-04-05 | 川北医学院 | Thyroid organoid culture medium, thyroid organoid culture and passage method |
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