CN1101931C - Heart pouring process of eliminating red blood cell from tissue and preparing dispersed cell suspension - Google Patents
Heart pouring process of eliminating red blood cell from tissue and preparing dispersed cell suspension Download PDFInfo
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- CN1101931C CN1101931C CN00122410A CN00122410A CN1101931C CN 1101931 C CN1101931 C CN 1101931C CN 00122410 A CN00122410 A CN 00122410A CN 00122410 A CN00122410 A CN 00122410A CN 1101931 C CN1101931 C CN 1101931C
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Abstract
The present invention relates to a method for preparing dispersive cell suspension by removing red blood cells in tissue with a heart perfusion method, which belongs to the field of biological medicine. The difference between the present invention and the known method lies in that red blood cells in tissue are separated by heart perfusion. The present invention has the advantages of simple and rapid method, good effect of removing the red blood cells in the tissue, high survival rate of dispersive tissue cells, etc. The method can be used for preparing the dispersive cell suspension of the tissue of various kinds.
Description
The present invention relates to a kind of heart perfusion that adopts and remove the method that red blood cell prepares the isolated cell suspension, belong to biomedical sector.
Helping to understand startup, reinforcement or the inhibition of cell function with the variation of fluorometric determination intracellular cation concentration, is the effective ways of some disease of research and drug mechanism.The quality of cell preparation is the key issue that ion concentration changes in the research born of the same parents, and the survival rate of stipulating prepared cell in the cell suspension usually is greater than 90%, and the problem of another no less important is can not be by red blood cell in the prepared cell suspension; Otherwise will influence its measured value.The erythrocytic separation method of reporting in the document of organizing is after tissue is taken out, to use the flushing of Hank ' s liquid for several times, under dissecting microscope blood vessel is rejected, and erythrocytic content is reduced in the flushing back.This method is difficult to reach removes erythrocytic requirement, and because of long slightly as if flush time, then cell mortality increases, if the flush time deficiency, too much red blood cell will have a strong impact on the reliability and the cultivation results of experimental result in the tissue, and operate extremely inconvenience at microscopically.
The objective of the invention is to overcome the deficiency of prior art, and provide a kind of simple and efficient, the employing heart perfusion of removing in the tissue that the red blood cell effect is good, cell survival rate is high is removed the method that red blood cell prepares the isolated cell suspension.
The present invention is achieved in that this method, and red blood cell separates by organizing, tissue digestion is handled, centrifugal and piping and druming operation is made.Wherein organize red blood cell to separate and intend adopting the cardiac perfusion method, be about to Hank ' s liquid or physiological saline slowly push the animal of surveying by the left ventricle apex heart, when treating that the liquid that flows out from the heart right ventricle is colourless, stop perfusion, this moment, each internal organs of animal were become colorless by redness, take out the tissue that needs to survey the position subsequently, the double dish of containing Hank ' s liquid in placing washes, careful knockout cell film, the small beaker of containing Hank ' s liquid in placing again shreds into rotten shape, use trypsinization, the DMEM that contains calf serum stops digestion, obtains cell suspension again after Pasteur manages piping and druming and centrifugal filtration.Said process is except that heart perfusion, and all the other each operations are all carried out according to a conventional method.
Embodiments of the invention are as follows:
Example one: (red blood cell of removing in the cerebral tissue with heart perfusion prepares the method for separating suspension) removes living 0-2 days Kunming mouse, cut off rib after the wipes of alcohol wash disinfection, open flatly at the right auricle of heart place of exposing heart, from the left ventricle apex syringe is inserted, slowly push Hank ' s liquid, this moment, visible right auricle of heart flowed out blood, flow out colourless perfusate at last, tissue becomes colorless, and every mouse needs perfusion 0.5-1ml, lasts 10～15 seconds.After finishing, gets perfusion the double dish that cerebral tissue places the interior Hank ' of Sheng s liquid, reject tissue film's separation and take out required tissue, with iris scissors tissue is shredded into rotten shape, digest under 37 ° of conditions with 0.125% trypsase, when tissue becomes transparent summary when sticking, stop digestion with the DMEM nutrient culture media that contains 10% calf serum.Filter with 200 purposes sieves then, filtrate is through 1000 rev/mins speed centrifuging, and precipitation Hank ' the s liquid of abandoning behind the supernatant is washed once again, adds the DMEM nutrient solution that contains serum at last, and to blow and beat into the cell suspension of dispersion standby through clinging to blood vessel.
Embodiment two (red blood cell of removing in the organs and tissues with heart perfusion prepares the method for separating suspension): whole process is substantially with embodiment one, and difference is: perfusate is a physiological saline, and the tissue at taking internal organ position carries out red blood cell to be separated.
Cell mortality and the data of red blood cell removing degree and being compared as follows of conventional method gained data of the cell suspension that the employing heart perfusion preparation that the inventor obtains through example disperses:
The present invention compared with prior art, it is simple and efficient to have method, the red blood cell effect of removing in the tissue is good, The histocyte survival rate advantages of higher of disperseing is applicable to the histiocytic cell suspension that preparation disperses, for The interior ion measurement of the cultivation of cell and cell provides satisfactory experiment material.
Claims (1)
- A kind of method of removing the cell suspension that the red blood cell preparation disperses in the tissue, by organizing red blood cell to separate, tissue digestion handles, centrifugal and piping and druming operation is made, it is characterized in that organizing the red blood cell separation circuit to adopt heart perfusion, be about to perfusate slowly pushes the animal of surveying through the left ventricle apex heart, when treating that the liquid that flows out from the heart right ventricle is colourless, stop perfusion, this moment, each internal organs of animal were become colorless by redness, take out to need survey the tissue at position subsequently and it is cut into rotten shape, be placed in the double dish of perfusion stand-by then.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN00122410A CN1101931C (en) | 2000-07-28 | 2000-07-28 | Heart pouring process of eliminating red blood cell from tissue and preparing dispersed cell suspension |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN00122410A CN1101931C (en) | 2000-07-28 | 2000-07-28 | Heart pouring process of eliminating red blood cell from tissue and preparing dispersed cell suspension |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1336540A CN1336540A (en) | 2002-02-20 |
| CN1101931C true CN1101931C (en) | 2003-02-19 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN00122410A Expired - Fee Related CN1101931C (en) | 2000-07-28 | 2000-07-28 | Heart pouring process of eliminating red blood cell from tissue and preparing dispersed cell suspension |
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| CN (1) | CN1101931C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104880546B (en) * | 2015-04-28 | 2017-12-12 | 中国科学院近代物理研究所 | The sample preparation methods of Mice brain tissues Proteomic analysis |
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2000
- 2000-07-28 CN CN00122410A patent/CN1101931C/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 福建医药杂志 19 (1) 2097-01-01 孙墉 等胎肝细胞悬液的制备及17例重症肝炎临床疗效观察 * |
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| Publication number | Publication date |
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| CN1336540A (en) | 2002-02-20 |
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