CN103834612A - Cell co-culturing method - Google Patents
Cell co-culturing method Download PDFInfo
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- CN103834612A CN103834612A CN201210481063.4A CN201210481063A CN103834612A CN 103834612 A CN103834612 A CN 103834612A CN 201210481063 A CN201210481063 A CN 201210481063A CN 103834612 A CN103834612 A CN 103834612A
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Abstract
The invention belongs to the biotechnical field, and aims to provide a cell co-culturing method. The observation of the interaction between two different types of cells in a cell co-culturing way is helpful for researching the future correlation between the cells in the medical field and the biological field.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method that cell is supported altogether, contribute to the research to iuntercellular mutual relationship from now on of medical science and biology aspect.
Background technology
Cell is the fundamental unit of the organic morphology and function of composition, and self is made up of mass part again.Not only to know about the research of structure it which part is made up of, and will further get the composition of each part clear.Correspondingly, not only to know that about function cell makes as a whole function, and will understand the mutual relationship of various piece in function.Organic physiological function and all life phenomenon are all taking cell as basal expression.Therefore, no matter the understanding to organic heredity, growth and physiological function, or for the breeding of pathology, pharmacology etc. as medical treatment basis and agricultural etc., cytology is all most important.
What played huge pushing effect for research cell is that M.J. Shi Laideng and T.A.H. execute ten thousand.The former described in 1838 cell be in a kind of muciform matrix through a kind of similarly be that the process of crystalline produces, and first produce core (also finding kernel).He and plant is regarded as to the community of cell, the colony that just looks like hydroid is the same.Under his inspiration, execute ten thousand and believe firmly that animal and plant are all by cellularity.He has accumulated a host of facts, points out the two consistence in structure and growth, has proposed cell theory in 1839.Meanwhile, Czech's animal physiological scholar J.E. Purkinje proposes plasmic concept; Germany's zoologist's C.T.E.von Seybold (1845) concludes that protozoon is all single celled.German pathologist R.C. Fei Erxiao (1855) proposes the well-known saying of " all cells are from cell " on the basis of research reticular tissue, and has founded cytopathology.Germany zoologist M. Shu Erce 1861 under cell definition: " cell is a protoplasma with all life feature, and nucleus is in wherein.”
Iuntercellular mutual relationship is an important directions of current medical science and biological study.The research of carrying out this respect often be unable to do without cell cultures, particularly co-culture of cells (coculture).We introduce a kind of simple co-culture of cells method.
Summary of the invention
A kind of method that the object of the present invention is to provide cell to support altogether, means by co-culture of cells are observed the interaction between two kinds of dissimilar cells, effectively improve the bioavailability of medicine, delay the release of medicine, strengthen drug effect, reduce toxic side effect, reach the object of slowly-releasing, long-acting, target.The co-culture of cells method of introducing, its feature is: (1) is simple, can carry out in common laboratory; (2) in common culturing process, can clearly observe the growing state of 2 kinds of cells, the observation such as be convenient to take pictures, does not resemble micropore counterdie cover ware and is difficult for observation of cell growing state; (3) in the time that cell is inoculated, slide size is the same, and the cell count on slide can be deducted the remaining cell count in every hole and be obtained by the total cell count in every hole, can obtain several times as long as test before experiment; (4) what cultivate altogether is attached cell, and cell is difficult for crossed contamination; (5) by the also common cultivation situation of 3 kinds of attached cells of observable of the method for fixed cap slide again on screen cloth; (6) experiment can be in researchs such as the enterprising line scanning Electronic Speculum of slide, immunohistochemical methodss after finishing.
Embodiment
(1) preparation of cultivating altogether, cover glass, 24 orifice plates, stainless steel mesh;
(2) cell single culture is got cell sample isolation identification and is cultivated from tissue;
(3) cell co-culture experiment, is inoculated in 24 orifice plates after getting the cell counting that single culture is good, adds serum free culture system liquid, cultivate 24 hours, when cell is fusion growth state soon, change the DMEM of calf serum, every hole adds gentamicin after 24 hours, to carry out common cultivation.
embodiment 1
(1) preparation of cultivating altogether:: cover glass is cut into the square that can just put into 24 orifice plates with glass cutter, after cleaning by cell culture materials requirement, sterilization is for subsequent use, stainless steel mesh is cut into square with scissors, again the steel wire of screen cloth is curved to the leg perpendicular to wire side, size is advisable can put into 24 orifice bores, make upper and lower two confluent monolayer cells differ 1mm, after cleaning, sterilization is for subsequent use.
(2) cell single culture: the cultivation of kidney of rats Stromal fibroblasts and rat renal tubular epithelial cells is undertaken by bibliographical information with qualification, after cultivating successfully, carry out following experiment, to observe the feasibility of said apparatus culturing cell, before experiment, rat renal tubular epithelial cells and kidney of rats Stromal fibroblasts are all done Trypan Blue (0.4 %) dyeing, detect the active condition of cell.
(3) cell co-culture experiment: by counting after the rat renal tubular epithelial cells digestion of former culture, by every hole 1 × 10
5individual cell is inoculated in 24 orifice plates that are placed with in advance cover glass for subsequent use, cultivate 24 hours with the DMEM nutrient solution containing 10% calf serum, when cell is fusion growth state soon, changing nutrient solution is the DMEM containing 1% calf serum, and every hole adds 200 μ g/ml gentamicins after 24 hours, to carry out common cultivation.Get the kidney of rats Stromal fibroblasts in the 3rd generation, by every hole 1 × 10
5cell count is implanted in 24 orifice plates and is cultivated 12 hours with the DMEM nutrient solution containing 10% calf serum, after cell attachment growth, nutrient solution is replaced by containing the DMEM of 1% calf serum and continues to cultivate 24 hours, put into stainless steel mesh for subsequent use, putting into above-mentioned length has the cover glass of rat renal tubular epithelial cells again, cultivate altogether after 48 hours, remove cover glass and stainless steel mesh, mix the propagation situation of method mensuration kidney of rats Stromal fibroblasts with tetramethyl-azo polyguanidine (MTT).
Claims (4)
1. the method that cell is supported altogether, is characterized in that realizing by following steps:
(1) preparation of cultivating altogether, cover glass, 24 orifice plates, stainless steel mesh;
(2) cell single culture is got cell sample isolation identification and is cultivated from tissue;
(3) cell co-culture experiment, is inoculated in 24 orifice plates after getting the cell counting that single culture is good, adds serum free culture system liquid, cultivate 24 hours, when cell is fusion growth state soon, change the DMEM of calf serum, every hole adds gentamicin after 24 hours, to carry out common cultivation.
2. cell as claimed in claim 1 breeding method altogether, it is characterized in that: the preparation that step (1) is cultivated altogether: cover glass glass cutter is cut into the square that can just put into 24 orifice plates, after cleaning by cell culture materials requirement, sterilization is for subsequent use, stainless steel mesh is cut into square with scissors, again the steel wire of screen cloth is curved to the leg perpendicular to wire side, size is advisable can put into 24 orifice bores, makes upper and lower two confluent monolayer cells differ 1mm, and after cleaning, sterilization is for subsequent use.
3. cell as claimed in claim 1 breeding method altogether, it is characterized in that: step (2) cell single culture: the cultivation of kidney of rats Stromal fibroblasts and rat renal tubular epithelial cells is undertaken by bibliographical information with qualification, after cultivating successfully, carry out following experiment, to observe the feasibility of said apparatus culturing cell, before experiment, rat renal tubular epithelial cells and kidney of rats Stromal fibroblasts are all done Trypan Blue (0.4%) dyeing, detect the active condition of cell.
4. cell as claimed in claim 1 breeding method altogether, is characterized in that: the experiment of step (3) cell co-culture: by counting after the rat renal tubular epithelial cells digestion of former culture, by every hole 1 × 10
5individual cell is inoculated in 24 orifice plates that are placed with in advance cover glass for subsequent use, cultivate 24 hours with the DMEM nutrient solution containing 10% calf serum, when cell is fusion growth state soon, changing nutrient solution is the DMEM containing 1% calf serum, every hole adds 200 μ g/ml gentamicins after 24 hours, to carry out common cultivation, get the kidney of rats Stromal fibroblasts in the 3rd generation, by every hole 1 × 10
5cell count is implanted in 24 orifice plates and is cultivated 12 hours with the DMEM nutrient solution containing 10% calf serum, after cell attachment growth, nutrient solution is replaced by containing the DMEM of 1% calf serum and continues to cultivate 24 hours, put into stainless steel mesh for subsequent use, putting into above-mentioned length has the cover glass of rat renal tubular epithelial cells again, cultivate altogether after 48 hours, remove cover glass and stainless steel mesh, mix the propagation situation of method mensuration kidney of rats Stromal fibroblasts with tetramethyl-azo polyguanidine (MTT).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210481063.4A CN103834612A (en) | 2012-11-23 | 2012-11-23 | Cell co-culturing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201210481063.4A CN103834612A (en) | 2012-11-23 | 2012-11-23 | Cell co-culturing method |
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| CN103834612A true CN103834612A (en) | 2014-06-04 |
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| CN201210481063.4A Pending CN103834612A (en) | 2012-11-23 | 2012-11-23 | Cell co-culturing method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104403991A (en) * | 2014-11-27 | 2015-03-11 | 苏州嘉禧萝生物科技有限公司 | Method for co-culturing cells |
| CN106754663A (en) * | 2016-11-30 | 2017-05-31 | 中国人民解放军第三军医大学第三附属医院 | The method for promoting the extracorporeal culturing method and monitoring Adult Mammals cardiomyocyte proliferation of Adult Mammals myocyte survival |
-
2012
- 2012-11-23 CN CN201210481063.4A patent/CN103834612A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104403991A (en) * | 2014-11-27 | 2015-03-11 | 苏州嘉禧萝生物科技有限公司 | Method for co-culturing cells |
| CN106754663A (en) * | 2016-11-30 | 2017-05-31 | 中国人民解放军第三军医大学第三附属医院 | The method for promoting the extracorporeal culturing method and monitoring Adult Mammals cardiomyocyte proliferation of Adult Mammals myocyte survival |
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Application publication date: 20140604 |