CN104403992A - Method for primary culture of rat mammary epithelial cells - Google Patents
Method for primary culture of rat mammary epithelial cells Download PDFInfo
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- CN104403992A CN104403992A CN201410738484.XA CN201410738484A CN104403992A CN 104403992 A CN104403992 A CN 104403992A CN 201410738484 A CN201410738484 A CN 201410738484A CN 104403992 A CN104403992 A CN 104403992A
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Abstract
The invention provides a method for primary culture of rat mammary epithelial cells. The method comprises the following steps: digesting rat breast tissue pieces; washing, re-suspending, blowing away and filtering cells subjected to centrifugal sedimentation; carrying out multiple times of instantaneous centrifugation on the cells, preparing the precipitated cells into suspension liquid, adjusting the densities of the mammary epithelial cells of the suspension liquid and carrying out inoculated culture. The method has the benefits that the steps are simple, and a large amount of cells with higher vitality can be obtained only through three hours of enzymic digestion; in addition, the fibroblast contamination is reduced to a great degree, and keratin and casein detection results show that the mammary epithelial cells obtained by the method have high purity and good secretion functions.
Description
Technical field
The invention belongs to technical field of cell culture, particularly relate to a kind of method that rat mammary gland epithelial cells of primary culture is supported.
Background technology
Existing rat mammary gland epithelial cell cultural method is attachmentblook culture and collagenase digestion, and wherein the attachmentblook culture time is long, complex steps, and cell easily pollutes, and the epithelial cell heterogeneity obtained, the repeatability of impact experiment; Although collagenase digestion is cultivated can obtain a large amount of cells in the short period of time, a large amount of inoblasts is digested to get off, and mammary epithelial cell purity is low, after adherent, mammary epithelial cell finally replace by the inoblast that more easily breeds.The shortcoming instant invention overcomes attachmentblook culture complex steps, easily polluting, take collagenase and Unidasa simultaneous digestion and combine repeatedly brief centrifugation separating mammary epithelial cells, eliminate the inoblast in cell precipitation as much as possible, in nutrient solution, add the somatomedins such as Regular Insulin, hydrocortisone, Transferrins,iron complexes simultaneously, facilitate the growth of mammary epithelial cell, maintain the insulin secreting function of mammary epithelial cell.
Summary of the invention
The object of the present invention is to provide a kind of method that rat mammary gland epithelial cells of primary culture is supported, the mammary epithelial cell purity being intended to solve primary separation and Culture is low, easily by problem that inoblast pollutes.
The present invention is achieved in that and comprises the following steps a kind of method that rat mammary gland epithelial cells of primary culture is supported:
(1) gather rat breast tissue, shred after rat breast tissue pre-treatment as 1mm
3the rat breast tissue fritter of size, is suspended in Digestive system by described fritter of organizing, at 37 DEG C of digestion 3h;
(2), after digestion terminates, centrifuging and taking cell precipitation, washs 3 times to remove fat with the nutrient solution containing 10% foetal calf serum, is resuspended in by cell in nutrient solution, dispels cell, through 100 order screen filtration to remove indigested tissue and fragment;
(3) cell is through repeatedly brief centrifugation, supernatant discarded, adds DMEM/F12 nutrient solution and be prepared into cell suspension in the epithelial cell of precipitation;
(4) the mammary epithelial cell density of suspension is adjusted to 5 × 10
5mL
-1, be inoculated in six orifice plates with every hole 2mL or every hole 100 μ L is inoculated in 96 orifice plates, put 37 DEG C, 5%CO
2cultivate under condition, change liquid the 1st time after 48h, remove not adherent red corpuscle and fragment, every 3d changes liquid 1 time later.
Preferably, in step (1), described rat breast tissue is gestation rat fourth, fifth pair of mammary tissue of 15 ~ 18 days;
Described pre-treatment is that the mammary tissue gathered is peeled off fatty tissue and reticular tissue, is placed in sterilizing culture dish, D-Hank ' the s liquid rinsing of precooling three times.
Preferably, in step (1), consisting of of described Digestive system: 1ml DMEM/F12 comprises 2mg type i collagen enzyme, 100U Unidasa and 100U mycillin.
Preferably, in step (2), describedly contain consisting of of the nutrient solution of 10% foetal calf serum: the volume ratio of DMEM/F12 nutrient solution and foetal calf serum is 9:1.
Preferably, in step (3), described brief centrifugation be 1000 revs/min centrifugal 1 minute, repeat 10 times.
Preferably, in step (3), consisting of of described DMEM/F12 nutrient solution: 1mlDMEM/F12 comprises 100U mycillin, 5 μ g Regular Insulin, 1 μ g hydrocortisone, 5 μ g Transferrins,iron complexess, 10ng Urogastron, 2 μm of ol L-glutaminate and 0.1ml foetal calf serum.
Compared to the shortcoming and defect of prior art, the present invention has following beneficial effect: step of the present invention is simple, just can obtain the higher cell of a large amount of vigor through the enzymic digestions of 3 hours; In addition, high degree of the present invention reduce into fiber contamination, show through Keratin sulfate and casein detected result the mammary epithelial cell purity that the present invention obtains high and possess good secreting function.
Accompanying drawing explanation
Fig. 1 is rat mammary gland epithelial cell morphologic observation figure;
Fig. 2 is rat mammary gland epithelial cell Keratin sulfate fluorescence immunoassay detection figure;
Fig. 3 is the immunoblotting detected result figure of mammary epithelial cell beta-casein.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment
(1) select the gestation rat of 15 ~ 18 days, aseptic collection fourth, fifth pair of mammary tissue, peel off fatty tissue and reticular tissue as far as possible, be placed in sterilizing culture dish, D-Hank ' the s liquid rinsing of precooling three times.About 1mm is cut into eye scissors
3the mammary tissue fritter of size, is suspended in Digestive system (adding 2mg/mL type i collagen enzyme in DMEM/F12,100U/mL Unidasa, 100U/mL mycillin) 37 DEG C digestion 3h.
(2), after digestion terminates, centrifuging and taking cell precipitation, wash 3 times to remove fat with the nutrient solution containing 10% foetal calf serum, be resuspended in by cell in nutrient solution, Big caliber straw dispels cell, through 100 order screen filtration to remove indigested tissue and fragment.
(3) (1000 revs/min centrifugal 1 minute through repeatedly brief centrifugation for cell; Repeat 10 times), supernatant discarded (mostly being red corpuscle, inoblast, endotheliocyte etc.), in the epithelial cell of precipitation, add DMEM/F12 nutrient solution be prepared into cell suspension, containing 100U/mL mycillin, 5 μ g/mL Regular Insulin, 1 μ g/mL hydrocortisone, 5 μ g/mL Transferrins,iron complexess, 10ng/mL Urogastron, 2mmol/LL-glutamine and 10% foetal calf serum in nutrient solution.
(4) the mammary epithelial cell density of suspension is adjusted to 5 × 10
5mL
-1, be inoculated in six orifice plates with every hole 2mL or every hole 100 μ L is inoculated in 96 orifice plates, put 37 DEG C, 5%CO
2cultivate under condition, change liquid the 1st time after 48h, remove not adherent red corpuscle and fragment, every 3d changes liquid 1 time later.
With fluorescent immune method and Western blot detection angle albumen and beta-casein respectively, as shown in Figures 1 to 3, Fig. 1 is rat mammary gland epithelial cell morphologic observation figure (200 ×), and wherein, A figure is adherent in cell 24 ~ 48h, presents island shape and connects; B figure is 5d after cultivating, and cell confluent culture plate, is fused into monolayer cell.Fig. 2 is rat mammary gland epithelial cell Keratin sulfate fluorescence immunoassay detection figure (200 ×), and wherein, A figure is mammary epithelial cell is that Keratin sulfate is positive, and C figure is negative control; B, D figure is DAPI and redyes nucleus.Fig. 3 is the immunoblotting detected result figure of mammary epithelial cell beta-casein, and wherein, swimming lane 1 is Marker, and swimming lane 2,3,4 is the mammary epithelial cell extract of separation and Culture, and the secretion of result display casein is for positive.By upper figure result display separation cultivation is epithelial cell, and has mammary gland secretion function.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. the method that rat mammary gland epithelial cells of primary culture is foster, is characterized in that, comprise the following steps:
(1) gather rat breast tissue, shred after rat breast tissue pre-treatment as 1mm
3the rat breast tissue fritter of size, is suspended in Digestive system by described fritter of organizing, at 37 DEG C of digestion 3h;
(2), after digestion terminates, centrifuging and taking cell precipitation, washs 3 times to remove fat with the nutrient solution containing 10% foetal calf serum, is resuspended in by cell in nutrient solution, dispels cell, through 100 order screen filtration to remove indigested tissue and fragment;
(3) cell is through repeatedly brief centrifugation, supernatant discarded, adds DMEM/F12 nutrient solution and be prepared into cell suspension in the epithelial cell of precipitation;
(4) the mammary epithelial cell density of suspension is adjusted to 5 × 10
5mL
-1, be inoculated in six orifice plates with every hole 2mL or every hole 100 μ L is inoculated in 96 orifice plates, put 37 DEG C, 5%CO
2cultivate under condition, change liquid the 1st time after 48h, remove not adherent red corpuscle and fragment, every 3d changes liquid 1 time later.
2. the method that rat mammary gland epithelial cells of primary culture as claimed in claim 2 is foster, is characterized in that, in step (1), described rat breast tissue is gestation rat fourth, fifth pair of mammary tissue of 15 ~ 18 days;
Described pre-treatment is that the mammary tissue gathered is peeled off fatty tissue and reticular tissue, is placed in sterilizing culture dish, D-Hank ' the s liquid rinsing of precooling three times.
3. the method that rat mammary gland epithelial cells of primary culture as claimed in claim 2 is foster, it is characterized in that, in step (1), consisting of of described Digestive system: 1ml DMEM/F12 comprises 2mg type i collagen enzyme, 100U Unidasa and 100U mycillin.
4. the rat mammary gland epithelial cells of primary culture as claimed in claim 3 method of supporting, is characterized in that, in step (2), describedly contains consisting of of the nutrient solution of 10% foetal calf serum: the volume ratio of DMEM/F12 nutrient solution and foetal calf serum is 9:1.
5. the rat mammary gland epithelial cells of primary culture as claimed in claim 4 method of supporting, is characterized in that, in step (3), described brief centrifugation be 1000 revs/min centrifugal 1 minute, repeat 10 times.
6. the method that rat mammary gland epithelial cells of primary culture as claimed in claim 5 is foster, it is characterized in that, in step (3), consisting of of described DMEM/F12 nutrient solution: 1mlDMEM/F12 comprises 100U mycillin, 5 μ g Regular Insulin, 1 μ g hydrocortisone, 5 μ g Transferrins,iron complexess, 10ng Urogastron, 2 μm of ol L-glutaminate and 0.1ml foetal calf serum.
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| CN201410738484.XA CN104403992A (en) | 2014-12-05 | 2014-12-05 | Method for primary culture of rat mammary epithelial cells |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115074312A (en) * | 2022-07-12 | 2022-09-20 | 北京大学口腔医学院 | Preparation method of primary epithelial cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484424A (en) * | 2013-10-10 | 2014-01-01 | 山东农业大学 | Single cell cloning method for obtaining goat mammary epithetical cells |
| CN103525752A (en) * | 2013-09-21 | 2014-01-22 | 山东农业大学 | Separation and purification method of goat mammary epithelial cells |
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2014
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525752A (en) * | 2013-09-21 | 2014-01-22 | 山东农业大学 | Separation and purification method of goat mammary epithelial cells |
| CN103484424A (en) * | 2013-10-10 | 2014-01-01 | 山东农业大学 | Single cell cloning method for obtaining goat mammary epithetical cells |
Non-Patent Citations (2)
| Title |
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| 张以涛: "小鼠乳腺上皮细胞培养体系的建立及初步应用", 《中国优秀硕士学位论文全文数据库基础科学辑（电子期刊）》 * |
| 李震等: "牛乳腺上皮细胞的分离培养", 《上海农业学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115074312A (en) * | 2022-07-12 | 2022-09-20 | 北京大学口腔医学院 | Preparation method of primary epithelial cells |
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Application publication date: 20150311 |