CN102747035A - Kit for in-vitro separation of karyocytes and removal of erythrocytes and use method thereof - Google Patents
Kit for in-vitro separation of karyocytes and removal of erythrocytes and use method thereof Download PDFInfo
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- CN102747035A CN102747035A CN2011100973163A CN201110097316A CN102747035A CN 102747035 A CN102747035 A CN 102747035A CN 2011100973163 A CN2011100973163 A CN 2011100973163A CN 201110097316 A CN201110097316 A CN 201110097316A CN 102747035 A CN102747035 A CN 102747035A
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Abstract
The invention discloses a kit for in-vitro separation of karyocytes and removal of erythrocytes and a use method thereof. The kit comprises a precipitation solution, a purification solution, a suspension and an erythrocyte lysis solution, wherein the precipitation solution, the purification solution, the suspension and the erythrocyte lysis solution are packaged separately. The precipitation solution is composed of normal saline and hydroxyethyl starch dissolved in the normal saline and has a weight volume concentration of 6%. The purification solution is a mixture of polysucrose and sodium diatrizoate. The suspension is normal saline. The erythrocyte lysis solution comprises ammonium chloride having a concentration of 7.47g/L and trihydroxymethylaminomethane having a concentration of 2.6g/L. The invention also discloses a use method of the kit.
Description
Technical field
The present invention relates to a kind ofly be used for in-vitro separation purifying marrow, Cord blood or peripheral blood nucleated cell and can remove erythrocytic method and test kit.
Background technology
Stem cell is the initiating cell with self-replacation and multidirectional differentiation potential, is the cells of origin of body, is the initiating cell that forms the various histoorgans of human body.Stem cell can be from marrow, Cord blood and peripheral blood the separation and purification stem cell; The separation method that mononuclearcell is commonly used mainly contains following several kinds: 6% hydroxyethylamyle settling process, methylcellulose gum settling process, Ficoll partition method, Percoll partition method, 3% gelatin partition method, chloride leach red corpuscle method, direct centrifuging etc.Monocytic separation method commonly used is Ficoll or Percoll direct method of isolation.Be about to directly be added on the parting liquid through density gradient centrifugation acquisition mononuclearcell after the hemodilution.Ficoll and Percoil are lymphocyte separation medium commonly used, the people are arranged once to these two kinds of parting liquid comparative studies, and it is the purest to think that Ficoll separates the mononuclearcell that obtains.
In the prior art from marrow, Cord blood and peripheral blood the method for separation and purification stem cell exist shortcomings such as stem cell motility rate and the recovery be lower, and can not cracking red corpuscle wherein.
Our experiment is thought through comparative study, adopts 6% hydroxyethylamyle to combine Ficoll to be used for the separating blood monocyte and has improved the monocytic recovery, but still be mixed with red corpuscle.Utilize chloride leach red corpuscle method, all the method for splitting erythrocyte for this reason.
Summary of the invention
The invention provides a kind of method of use that is used for in-vitro separation purifying marrow, Cord blood or peripheral blood nucleated cell and can removes erythrocytic test kit and this test kit.
Test kit of the present invention comprises precipitated liquid, refined solution, suspension-s and the erythrocyte cracked liquid of barrier packaging separately; Wherein precipitated liquid is the hydroxyethylamyle that is dissolved in saline water; Its bulking value concentration is 6%; Refined solution is the ficoll thypaque sodium, and suspension-s is saline water, and erythrocyte cracked liquid is the Tutofusin tris of ammonium chloride+2.6g/L of 7.47g/L.So-called barrier packaging is meant that precipitated liquid, refined solution and suspension-s are sealed in the not connected container separately, preferably, and independent packaging separately.
Test kit of the present invention, wherein said precipitated liquid: refined solution: suspension-s: and the volume ratio of erythrocyte cracked liquid is (40-180): (60-180): 20: 1.
Also comprise working instructions in the wherein said test kit of test kit of the present invention, said specification sheets has stated clearly the method for use of said test kit, and said method comprises the steps:
Marrow/Cord blood/the peripheral blood that (1) will pass through the anti-freezing processing is poured in the precipitated liquid reagent bottle, tightens bottle cap and puts upside down mixing 5 times, unscrews bottle cap, leaves standstill 22-25 minute.
(2) transfer pipet with 10ml forwards supernatant in the centrifuge tube of 50ml, and every pipe 25ml tries not to be drawn onto erythroprecipitin, and trim.
(3) with 4 ℃ of two centrifuge tubes, cf-710xg (rotating speed 2000rpm), 5 minutes are centrifugal, and behind centrifugal the finishing, supernatant discarded is opened deposition with pointing shake gently, and every pipe adds 25ml saline water re-suspended cell.
(4) refined solution is added in the new centrifuge tube of two other 50ml by every pipe 25ml; Cell suspension in the 3rd step slowly is added to transfer pipet on the liquid level of refined solution, guarantees that the liquid level between two kinds of liquid is clear, then; 4 ℃; Cf-710xg (rotating speed 2000rpm), 20 minutes centrifugal, closes the whizzer brake function.
(5) after centrifugal, draw intermediate layer cell, be added in the new centrifuge tube, dilute with 50ml injection saline water, 4 ℃, trim.Cf-400xg (rotating speed 1500rpm), the centrifuge washing cell was twice in 8 minutes.
(6) abandon supernatant, in cell precipitation, add erythrocyte cracked liquid and blow and beat 15～20s mixing gently.
(7) 4 ℃, cf-400xg (rotating speed 1500rpm), the centrifuge washing cell was three times in 8 minutes;
(8) cell after will washing is added in the suspension-s, and is subsequent use.
Test kit of the present invention is used for in-vitro separation purifying marrow/Cord blood/peripheral blood nucleated cell.The principle that it utilizes cell and inorganic ion associating aggregate and precipitate adopts reverse collecting method separation and purification nucleated cell.Test kit of the present invention is usually 2-8 ℃ of preservation.
Test kit of the present invention, the sample type that uses is marrow/Cord blood/peripheral blood, can collect required marrow/Cord blood/peripheral blood sample according to method in common; Guarantee to change disposable anticoagulation bag over to after marrow/Cord blood/peripheral blood is collected; Marrow/Cord blood/peripheral blood should carry out the separation and purification nucleated cell after collecting in 6 hours.
Test kit of the present invention, the recovery can reach " 90%, isolating nucleated cell motility rate >=96% has farthest been kept stem cell constant in external microenvironment.
The invention also discloses the method for use of said test kit, said method comprises the steps:
Marrow/Cord blood/the peripheral blood that (1) will pass through the anti-freezing processing is poured in the precipitated liquid reagent bottle, tightens bottle cap and puts upside down mixing 5 times, unscrews bottle cap, leaves standstill 22-25 minute.
(2) transfer pipet with 10ml forwards supernatant in the centrifuge tube of 50ml, and every pipe 25ml tries not to be drawn onto erythroprecipitin, and trim.
(3) with 4 ℃ of two centrifuge tubes, cf-710xg (rotating speed 2000rpm), 5 minutes are centrifugal, and behind centrifugal the finishing, supernatant discarded is opened deposition with pointing shake gently, and every pipe adds 25ml saline water re-suspended cell.
(4) refined solution is added in the new centrifuge tube of two other 50ml by every pipe 25ml; Cell suspension in the 3rd step slowly is added to transfer pipet on the liquid level of refined solution, guarantees that the liquid level between two kinds of liquid is clear, then; 4 ℃; Cf-710xg (rotating speed 2000rpm), 20 minutes centrifugal, closes the whizzer brake function.
(5) after centrifugal, draw intermediate layer cell, be added in the new centrifuge tube, dilute with 50ml injection saline water, 4 ℃, trim.Cf-400xg (rotating speed 1500rpm), the centrifuge washing cell was twice in 8 minutes.
(6) abandon supernatant, in cell precipitation, add erythrocyte cracked liquid and blow and beat 15～20s mixing gently.
(7) 4 ℃, cf-400xg (rotating speed 1500rpm), the centrifuge washing cell was three times in 8 minutes;
(8) cell after will washing is added in the suspension-s, and is subsequent use.
Cell motility rate of the present invention adopts following method to detect:
1. suction pipe is drawn the nucleated cell suspension after 90 microlitres separate.
2. add 0.2% trypan blue staining fluid 10 microlitres, mixing.
3. left standstill 3 minutes.
4. the absorption mixing solutions drips to cell counting count board, and after evenly spreading out, microscopically is observed.
5. 200 times of following artificial countings of microscope, the cell that indigo plant is dyed is dead cell (A), the cell of dye-free is viable cell (B).Nucleated cell motility rate=B/ (A+B) * 100%
Embodiment
Embodiment 1
The preparation method of cell processing kit:
The preparation of precipitated liquid: hydroxyethylamyle 2.40g is dissolved among the saline water 40ml, and concentration is 6%, 37 ℃ of water-bath, picks up counting 8 hours at 37 ℃ from the water-bath temperature-stable, and 37 ℃ of water-baths were taken out from water-bath after 8 hours, room temperature half a hour, preserves.
The preparation of refined solution: HISTOPAQUE1077 liquid (ficoll thypaque sodium) 60ml is stabilized in 4 ℃ from refrigerator temperature and picked up counting 8 hours.4 ℃ after 8 hours, from 4 degree refrigerators, take out, room temperature half a hour, preserve.
The preparation of suspension-s: measure 20ml saline water with graduated cylinder, preserve.
The preparation of erythrocyte cracked liquid: 3.735g ammonium chloride, 1.3g Tutofusin tris are dissolved in water and are diluted to 500ml.0.22 the degerming of μ m membrane filtration, 4 ℃ of preservations.
Claims (5)
1. in-vitro separation purifying nucleated cell and can remove erythrocytic test kit; Comprise precipitated liquid, refined solution, suspension-s and the erythrocyte cracked liquid of barrier packaging separately; Wherein precipitated liquid is the hydroxyethylamyle that is dissolved in saline water, and its bulking value concentration is 6%, and refined solution is the ficoll thypaque sodium; Suspension-s is saline water, and erythrocyte cracked liquid is the Tutofusin tris of ammonium chloride+2.6g/L of 7.47g/L.
2. test kit as claimed in claim 1, the independent packaging separately of wherein said precipitated liquid, refined solution, suspension-s and erythrocyte cracked liquid and suspension-s.
3. test kit as claimed in claim 2, wherein said precipitated liquid: refined solution: suspension-s: and the volume ratio of erythrocyte cracked liquid is (40-180): (60-180): 20: 1.
4. like the described test kit of arbitrary claim of claim 1-3, also comprise working instructions in the wherein said test kit, said specification sheets has stated clearly the method for use of said test kit, and said method comprises the steps:
Marrow/Cord blood/the peripheral blood that (1) will pass through the anti-freezing processing is poured in the precipitated liquid reagent bottle, tightens bottle cap and puts upside down mixing 5 times, unscrews bottle cap, leaves standstill 22-25 minute;
(2) transfer pipet with 10ml forwards supernatant in the centrifuge tube of 50ml, and every pipe 25ml tries not to be drawn onto erythroprecipitin, and trim;
(3) with 4 ℃ of two centrifuge tubes, cf-710xg (2000rpm), 5 minutes are centrifugal, and behind centrifugal the finishing, supernatant discarded is opened deposition with pointing shake gently, and every pipe adds 25ml saline water re-suspended cell;
(4) refined solution is added in the new centrifuge tube of two other 50ml by every pipe 25ml; Cell suspension in the 3rd step slowly is added to transfer pipet on the liquid level of refined solution, guarantees that the liquid level between two kinds of liquid is clear, then; 4 ℃; Cf-710xg (rotating speed 2000rpm), 20 minutes are centrifugal, close the whizzer brake function;
(5) after centrifugal, draw intermediate layer cell, be added in the new centrifuge tube, dilute with 50ml injection saline water, 4 ℃, trim.Cf-400xg (rotating speed 1500rpm), the centrifuge washing cell was twice in 8 minutes;
(6) abandon supernatant, in cell precipitation, add erythrocyte cracked liquid and blow and beat 15～20s mixing gently;
(7) 4 ℃, cf-400xg (rotating speed 1500rpm), the centrifuge washing cell was three times in 8 minutes;
(8) cell after will washing is added in the suspension-s, and is subsequent use.
5. like the method for use of the test kit of claim 1-4, comprise the steps:
Marrow/Cord blood/the peripheral blood that (1) will pass through the anti-freezing processing is poured in the precipitated liquid reagent bottle, tightens bottle cap and puts upside down mixing 5 times, unscrews bottle cap, leaves standstill 22-25 minute;
(2) transfer pipet with 10ml forwards supernatant in the centrifuge tube of 50ml, and every pipe 25ml tries not to be drawn onto erythroprecipitin, and trim:
(3) with 4 ℃ of two centrifuge tubes, cf-710xg (rotating speed 2000rpm), 5 minutes are centrifugal, and behind centrifugal the finishing, supernatant discarded is opened deposition with pointing shake gently, and every pipe adds 25ml saline water re-suspended cell;
(4) refined solution is added in the new centrifuge tube of two other 50ml by every pipe 25ml; Cell suspension in the 3rd step slowly is added to transfer pipet on the liquid level of refined solution, guarantees that the liquid level between two kinds of liquid is clear, then; 4 ℃; Cf-710xg (rotating speed 2000rpm), 20 minutes are centrifugal, close the whizzer brake function;
(5) after centrifugal, draw intermediate layer cell, be added in the new centrifuge tube, dilute with 50ml injection saline water, 4 ℃, trim.Cf-400xg (rotating speed 1500rpm), the centrifuge washing cell was twice in 8 minutes;
(6) abandon supernatant, in cell precipitation, add erythrocyte cracked liquid and blow and beat 15～20s mixing gently;
(7) 4 ℃, cf-400xg (rotating speed 1500rpm), the centrifuge washing cell was three times in 8 minutes;
(8) cell after will washing is added in the suspension-s, and is subsequent use.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100973163A CN102747035A (en) | 2011-04-19 | 2011-04-19 | Kit for in-vitro separation of karyocytes and removal of erythrocytes and use method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100973163A CN102747035A (en) | 2011-04-19 | 2011-04-19 | Kit for in-vitro separation of karyocytes and removal of erythrocytes and use method thereof |
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| CN102747035A true CN102747035A (en) | 2012-10-24 |
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| CN2011100973163A Pending CN102747035A (en) | 2011-04-19 | 2011-04-19 | Kit for in-vitro separation of karyocytes and removal of erythrocytes and use method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557449A (en) * | 2017-09-19 | 2018-01-09 | 北京陆道培干细胞生物技术有限公司 | Sample for PCR amplifications and preparation method thereof, store method and application method |
| CN108265104A (en) * | 2018-01-02 | 2018-07-10 | 北京诺禾致源科技股份有限公司 | Chromosome configuration captures library and its construction method |
-
2011
- 2011-04-19 CN CN2011100973163A patent/CN102747035A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557449A (en) * | 2017-09-19 | 2018-01-09 | 北京陆道培干细胞生物技术有限公司 | Sample for PCR amplifications and preparation method thereof, store method and application method |
| CN108265104A (en) * | 2018-01-02 | 2018-07-10 | 北京诺禾致源科技股份有限公司 | Chromosome configuration captures library and its construction method |
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Application publication date: 20121024 |