CN103361299A - Method and kit for separating stem cells - Google Patents
Method and kit for separating stem cells Download PDFInfo
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- CN103361299A CN103361299A CN2012100969660A CN201210096966A CN103361299A CN 103361299 A CN103361299 A CN 103361299A CN 2012100969660 A CN2012100969660 A CN 2012100969660A CN 201210096966 A CN201210096966 A CN 201210096966A CN 103361299 A CN103361299 A CN 103361299A
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- 238000000034 method Methods 0.000 title claims abstract description 36
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- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 54
- 102000013275 Somatomedins Human genes 0.000 claims description 54
- 210000002950 fibroblast Anatomy 0.000 claims description 49
- 210000004700 fetal blood Anatomy 0.000 claims description 32
- 239000012981 Hank's balanced salt solution Substances 0.000 claims description 29
- 235000010981 methylcellulose Nutrition 0.000 claims description 28
- 238000004062 sedimentation Methods 0.000 claims description 28
- 239000002504 physiological saline solution Substances 0.000 claims description 23
- 239000012530 fluid Substances 0.000 claims description 22
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- 239000000872 buffer Substances 0.000 claims description 17
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 16
- 108010039811 Starch synthase Proteins 0.000 claims description 12
- 239000007864 aqueous solution Substances 0.000 claims description 12
- 235000015097 nutrients Nutrition 0.000 claims description 12
- 238000001556 precipitation Methods 0.000 claims description 12
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 8
- 239000012930 cell culture fluid Substances 0.000 claims description 8
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- NTHXOOBQLCIOLC-UHFFFAOYSA-N iohexol Chemical compound OCC(O)CN(C(=O)C)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NTHXOOBQLCIOLC-UHFFFAOYSA-N 0.000 abstract description 31
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- 238000005119 centrifugation Methods 0.000 description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 9
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Abstract
The invention relates to a method and a kit for separating stem cells and provides a composition for separating stem cells. The composition comprises iohexol and balanced salt solution, wherein the iohexol is dissolved in the balanced salt solution. With the adoption of the composition, the stem cells can be effectively separated.
Description
Technical field
The present invention relates to field of biomedicine technology, especially the method and the test kit that relate to separate stem cells, more specifically, the present invention relates to the purposes of Schering AG) in preparation stem cell refined solution, the composition for separating of stem cell, the method for separate stem cells, test kit and the purposes of this test kit in separate stem cells of separate stem cells.
Background technology
Contain a large amount of hematopoietic cells and all kinds of stem cell and a large amount of mature cells in people's marrow and the Cord blood.Each lineage stem cells and ripe white corpuscle are karyocyte.Comprise multiple stem cell and list/syncyte in the karyocyte, also have lymphocyte and mesenchymal cell etc.
The separation method of external erythrocyte and karyocyte comprises machine centrifuging and settling process.The machine centrifuging is extensively used abroad with the separating effect of its totally closed operation and stability and safety.But because equipment price is expensive, at present domestic still difficult popularizing.Also has in addition several different methods: paramagnetic particle method, immunofluorescence analysis, flow cytometer method etc.The common problem of these methods is: the stem cell that 1) obtains is impure, and 2) use inconvenience, involve great expense 3) technical equipment requirement height, in the operation technician is required height.
Thereby the technology of separate stem cells still remains to be improved at present.
Summary of the invention
The present invention is intended to solve at least one of technical problem that exists in the prior art.For this reason, one object of the present invention is to propose a kind of means that can the effective separation stem cell.
The present invention is based on contriver's following discovery and finishes: the soluble in water and damping fluid of Schering AG), and concentration can reach 80 % by weight, and the solution of Schering AG) can pass through autoclaving, and nontoxic low viscosity does not affect Premeabilisation of cells and presses.The contriver is surprised to find, and when Schering AG) is applied to refined solution as the density gradient centrifugation separating medium, can be widely used in the various types of cells of purifying, subcellular organelle and macromolecule complex, and efficient obviously is better than Percoll.
For this reason, in a first aspect of the present invention, the present invention proposes the purposes of Schering AG) in preparation stem cell refined solution.By Schering AG) being applied to prepare the stem cell refined solution, can effectively improve the efficient of separate stem cells.
In a second aspect of the present invention, the present invention proposes a kind of composition for separating of stem cell.According to embodiments of the invention, said composition comprises: Schering AG); And balanced salt solution, wherein, described Schering AG) is dissolved in the described balanced salt solution.Utilize said composition as the stem cell refined solution, can effectively improve the efficient of separate stem cells from the biological sample that contains stem cell.
In a third aspect of the present invention, the present invention proposes a kind of method of separate stem cells.According to embodiments of the invention, the method comprises following: the biological specimen that will contain stem cell mixes with the red corpuscle parting liquid, and standing sedimentation is in order to obtain containing the upper strata enchylema of stem cell; With the described upper strata enchylema centrifugal concentrating that contains stem cell, obtain cell precipitation; With the first stem cell refined solution described cell precipitation is carried out resuspension, obtain suspension; Join in the second stem cell refined solution described suspension centrifugal, and collection stem cell, wherein, described red corpuscle parting liquid is the methylated cellulose aqueous solution of 0.2-0.7 % by weight, and described the first stem cell refined solution is that the composition that balanced salt solution and described the second stem cell refined solution are foregoing separate stem cells (namely comprises Schering AG); And balanced salt solution, wherein, described Schering AG) is dissolved in the described balanced salt solution).The contriver finds can effectively improve the efficient of separate stem cells by adopting the method for above-mentioned separate stem cells.
In a fourth aspect of the present invention, the present invention proposes a kind of test kit for separating of stem cell.According to embodiments of the invention, this test kit comprises: the red corpuscle parting liquid; The first stem cell refined solution; And the second stem cell refined solution, wherein, described red corpuscle parting liquid is the methylated cellulose aqueous solution of 0.2-0.7 % by weight, the methylated cellulose aqueous solution of preferred 0.5 % by weight, described the first stem cell refined solution are that the composition that balanced salt solution and described the second stem cell refined solution are foregoing separate stem cells (namely comprises Schering AG); And balanced salt solution, wherein, described Schering AG) is dissolved in the described balanced salt solution).Utilize this test kit, can effectively implement the method for aforementioned separate stem cells, thus, can effectively improve the efficient of separate stem cells.
Additional aspect of the present invention and advantage in the following description part provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Description of drawings
Above-mentioned and/or additional aspect of the present invention and advantage are from obviously and easily understanding becoming the description of embodiment in conjunction with following accompanying drawing, wherein:
Fig. 1 is the schematic flow sheet that has shown according to the method for embodiments of the invention separate stem cells.
Embodiment
The below describes embodiments of the invention in detail, and the example of described embodiment is shown in the drawings, and wherein identical or similar label represents identical or similar element or the element with identical or similar functions from start to finish.Be exemplary below by the embodiment that is described with reference to the drawings, only be used for explaining the present invention, and can not be interpreted as limitation of the present invention.
The present invention is based on contriver's following discovery and finishes: existing gradient centrifugation reagent for example Percoll, Filcoll solution is being applied to the purifying karyocyte especially during stem cell, and efficient is lower.Because cell is long in the external time, be subject to the impact of temperature and reagent in the sepn process, the recovery rate of nucleated cell that prior art obtains can only maintain 80-90%, and erythrocytic survival rate can reach about 4%, and since prior art the manipulation in vitro time above 2 hours, can greatly affect the differentiation versatility of resulting stem cell, reduce the differentiation potential of stem cell.Schering AG) (Iohexol, chemical name: Ominipaque Solution, trade(brand)name: Nycodenz, CAS no.66108-95-0) is that a kind of molecular weight is 821, and density is 2.1g/ml, be non-ionic type, to the avirulent compound of cell.The contriver finds Schering AG) to be effectively applied to separating or purification of the various biological substances such as cell, organoid, biomacromolecule and virus, and Schering AG) is stable under High Temperature High Pressure, thereby can carry out autoclave sterilization to the solution that is made into by Schering AG), thereby Schering AG) is suitable for the gradient centrifugation reagent as separate stem cells.
In a first aspect of the present invention, the present invention proposes the purposes of Schering AG) in preparation stem cell refined solution.Stem cell refined solution by utilizing the Schering AG) preparation to obtain can effectively for separating of the purifying stem cell, especially be applicable to from spinal fluid and Cord blood separation and purification stem cell, preferably from anti-freezing spinal fluid and Cord blood.
In a second aspect of the present invention, the present invention proposes a kind of composition for separating of stem cell.According to embodiments of the invention, should be the solution of Schering AG) in balanced salt solution for separating of the composition of stem cell, namely the composition of this separate stem cells comprises Schering AG) and balanced salt solution, and wherein said Schering AG) is dissolved in the described balanced salt solution.Can be with said composition as gradient centrifugation reagent, effectively by gradient centrifugation separate stem cells from the sample that contains stem cell.
According to embodiments of the invention, the type of balanced salt solution also is not particularly limited, as long as it can keep the Premeabilisation of cells pressure balanced.According to one embodiment of present invention, the balanced salt solution that can adopt is HBSS (Hank ' s Balanced Salt Solution, Hank ' s balanced salt solution) and GBSS's (GEY ' S balanced salt solution) is at least a.Preferred balanced salt solution is HBSS.According to embodiments of the invention, the pH of said composition is 7-8.Thus, can be further separate stem cells effectively.
According to embodiments of the invention, the concentration of Schering AG) also is not particularly limited.According to one embodiment of present invention, every 100ml balanced salt solution contains the 8-15g Schering AG), preferred 13g Schering AG).Thus, can further improve efficient by the gradient centrifugation separate stem cells.According to embodiments of the invention, can be balanced easily the composition that contains Schering AG) in the salt, for example can by with a certain amount of Schering AG) for example 13 gram Schering AG)s and 100mlHBSS solution (can obtain by commercial means, for example can be available from invitrogen company), and stirring certain hour, for example stir more than 1 hour, keep in Dark Place in 4 ℃ afterwards, for subsequent use.
According to embodiments of the invention, in said composition, cell growth factor be can also further comprise in addition, the human alkaline fibroblast somatomedin of 10-30ng/ml and human stem cell growth-α of 10-30ng/ml preferably further comprised.Thus, the contriver finds, by add somatomedin, especially stem cell factor in composition, can effectively improve the stem cell in vitro survival rate, and can keep the differentiation potential of stem cell.
In a third aspect of the present invention, the present invention proposes a kind of method of separate stem cells.With reference to figure 1, the method for this separate stem cells may further comprise the steps:
At first, separating red corpuscle.According to embodiments of the invention, can mix with the red corpuscle parting liquid by the biological specimen that will contain stem cell, and standing sedimentation, so that the lower floor of erythrocyte sedimentation liquid, and obtain containing the upper strata enchylema of stem cell.Here the type of employed term " red corpuscle parting liquid " and being not particularly limited is as long as can fully separate red corpuscle with the karyocyte that comprises stem cell by sedimentation.According to one embodiment of present invention, the red corpuscle parting liquid of employing is the methylated cellulose aqueous solution of 0.2-0.7 % by weight, the methylated cellulose aqueous solution of preferred 0.5 % by weight.According to embodiments of the invention, the settling time also is not particularly limited, and according to a particular embodiment of the invention, can implement sedimentation 45-60 minute.Thus, can be effectively so that red corpuscle fully separate with the karyocyte that comprises stem cell.Thereby obtain containing the upper strata enchylema of stem cell.According to embodiments of the invention, contain stem cell biological specimen type and be not particularly limited, can be any from the organism biological specimen that contains stem cell that separates of any position of human body for example.According to one embodiment of present invention, the biological specimen that contains stem cell can be for being selected from least a of spinal fluid and Cord blood, preferred anti-freezing spinal fluid or Cord blood.Thus, can obtain a large amount of stem cells, especially mescenchymal stem cell.According to embodiments of the invention, contain the blending ratio of the biological specimen of stem cell and red corpuscle parting liquid and be not particularly limited.According to one embodiment of present invention, select such blending ratio, so that final mixed solution contains the methylcellulose gum of 0.1 % by weight.For example, for anti-freezing spinal fluid or Cord blood, according to 4: 1 volume ratio, anti-freezing spinal fluid or Cord blood are mixed with the methylated cellulose aqueous solution (red corpuscle parting liquid) of 0.5 % by weight.Thus, can further improve the efficient of separating red corpuscle, thereby further improve the efficient of final separate stem cells.According to embodiments of the invention, can the upper strata enchylema that contains that separate be repeated sedimentation and removes erythrocytic operation, in order to further remove remaining red corpuscle.
Next, collect upper strata enchylema, and this upper strata enchylema that contains stem cell is carried out centrifugal concentrating, thereby obtain cell precipitation.And then utilize the first stem cell refined solution that resulting cell precipitation is carried out resuspension.To need to prove that employed term " first ", " second " only are in order distinguishing in the present invention, and to express never in any form or hint order between it and the difference of importance.In this article, the first stem cell refined solution that adopts is be selected from balanced salt solution and cell culture fluid at least a.According to embodiments of the invention, the type of balanced salt solution and cell culture fluid also is not particularly limited, the example of the balanced salt solution that can adopt includes but not limited to GBSS solution (sometimes also directly being called GBSS), HBSS solution (sometimes also directly being called HBSS), the balanced salt solution that preferably can adopt is HBSS, and the nutrient solution that can adopt is the MEM nutrient solution.According to embodiments of the invention, the upper strata enchylema that contains stem cell is carried out the condition of centrifugal concentrating and is not particularly limited.According to one embodiment of present invention, can adopt under 1200rpm and to realize effectively concentrated to cell in centrifugal 5 minutes.Can adopt a certain amount of balanced salt solution, for example 10ml balanced salt solution (the first stem cell refined solution) carries out resuspension to resulting cell precipitation, to obtain containing the suspension of stem cell.
Next, after obtaining containing the suspension of stem cell, resulting suspension can be joined in the second cell purification liquid and carry out gradient centrifugation, so that separate stem cells.According to embodiments of the invention, the second stem cell refined solution is foregoing composition, be the solution (in other words, the composition of separate stem cells comprise Schering AG) and balanced salt solution, wherein said Schering AG) be dissolved in described balanced salt solution) of Schering AG) in balanced salt solution.Thus, can effectively pass through gradient centrifugation, with karyocyte and other its cellular segregation, thereby be convenient to especially mescenchymal stem cell (being usually located at the middle part of centrifugate) of separate stem cells.According to embodiments of the invention, centrifugal condition also is not particularly limited.According to a particular embodiment of the invention,, can adopt centrifugal 22 minutes of 4 ℃ of Gradients.Thus, can be further separate stem cells effectively.About the second stem cell refined solution, the front can be applicable to this for the described all technical characteristic of the composition of separate stem cells and advantage, for simplicity, repeats no more.Utilize the second stem cell refined solution to carry out the condition of gradient centrifugation and be not particularly limited.
According to embodiments of the invention, after obtaining stem cell, can adopt the cell treatment solution, resulting stem cell is cleaned.According to embodiments of the invention, the cell treatment solution that can adopt is at least a of physiological saline and phosphate buffered saline buffer.According to specific examples of the present invention, can use the cell treatment solution, resulting stem cell is cleaned at least twice, thus, can guarantee the purity of resulting stem cell.According to embodiments of the invention, the type of cell treatment solution also is not particularly limited, can be for being selected from least a of phosphoric acid buffer and physiological saline.Thus, can further improve the efficient that stem cell is cleaned.Need to prove, employed phraseology " is selected from least a of phosphate buffered saline buffer and physiological saline " and refers in this article, namely can use separately phosphate buffered saline buffer, also can use separately physiological saline, can also use the combination of phosphate buffered saline buffer and physiological saline.Here employed " combination of phosphoric acid salt and physiological saline " should do broad understanding, can be to be used for stem cell is cleaned after phosphate buffered saline buffer is mixed with physiological saline, also can be to use respectively phosphate buffered saline buffer and physiological saline that stem cell is cleaned, wherein, if use respectively phosphate buffered saline buffer and physiological saline that stem cell is cleaned, sequencing and being not particularly limited then, can use first phosphate buffered saline buffer, use afterwards physiological saline that stem cell is cleaned, also can use first physiological saline, use afterwards phosphate buffered saline buffer that stem cell is cleaned.
According to embodiments of the invention, can further the comprising one of at least of the red corpuscle parting liquid that adopts in the present invention, the first stem cell refined solution, the second stem cell refined solution and cell treatment solution: the human alkaline fibroblast somatomedin of 10-30ng/ml and human stem cell growth-α of 10-30ng/ml.Preferred red corpuscle parting liquid, the first stem cell refined solution, the second stem cell refined solution and cell treatment solution all further comprise: the human alkaline fibroblast somatomedin of 10-30ng/ml and human stem cell growth-α of 10-30ng/ml.Thus, the contriver finds, by adding somatomedin, especially stem cell factor, can effectively improve the stem cell in vitro survival rate, and can keep the differentiation potential of stem cell.
Utilization can realize separating the stem cell in spinal fluid and the Cord blood according to the method for the separate stem cells of the embodiment of the invention, and the stem cell survival of separating reaches more than 99%, and the stem cell yield reaches 90%.According to embodiments of the invention, aforesaid operations all can operate in Biohazard Safety Equipment according to working specification, separates and can not pollute because of reagent.Simultaneously according to according to the isolated stem cell surface of the method for the embodiment of the invention without any marker, do not change the biological activity of cell.In addition, according to the method for the embodiment of the invention, owing to added somatomedin in the reagent in sepn process, thereby, stem cell has been improved survival rate and kept stem cell in sepn process versatility.
In a fourth aspect of the present invention, the present invention proposes a kind of test kit for separating of stem cell.According to embodiments of the invention, this test kit comprises: red corpuscle parting liquid, the first stem cell refined solution and the second stem cell refined solution.According to embodiments of the invention, can further include the cell treatment solution in this test kit.
According to embodiments of the invention, the red corpuscle parting liquid is the methylated cellulose aqueous solution of 0.2-0.7 % by weight, and the methylated cellulose aqueous solution of preferred 0.5 % by weight can be used for by the settlement separate red corpuscle of removing effectively.According to embodiments of the invention, the first stem cell refined solution is be selected from balanced salt solution and cell culture fluid at least a.According to embodiments of the invention, the type of balanced salt solution and cell culture fluid also is not particularly limited, according to one embodiment of present invention, the balanced salt solution that can adopt is HBSS (Hank ' s Balanced Salt Solution, Hank ' s balanced salt solution) and GBSS's (GEY ' S balanced salt solution) is at least a.Preferred balanced salt solution is HBSS, and the nutrient solution that can adopt is the MEM nutrient solution.Can be effectively the cell precipitation that contains stem cell of centrifugal concentrating be carried out resuspension, in order to carry out down-stream.According to embodiments of the invention, the second stem cell refined solution is foregoing composition, be the solution (in other words, the composition of separate stem cells comprise Schering AG) and balanced salt solution, wherein said Schering AG) be dissolved in described balanced salt solution) of Schering AG) in balanced salt solution.Thus, can effectively pass through gradient centrifugation, with karyocyte and other cellular segregation, thereby be convenient to especially mescenchymal stem cell (being usually located at the middle part of centrifugate) of separate stem cells.About the second stem cell refined solution, the front can be applicable to this for the described all technical characteristic of the composition of separate stem cells and advantage, for simplicity, repeats no more.According to embodiments of the invention, the cell treatment solution is at least a of phosphoric acid buffer and physiological saline.Thus, can utilize easily this cell treatment solution that the stem cell that separation obtains is cleaned.Thus, utilize this test kit, can effectively implement the method for aforementioned separate stem cells, thus can be effectively from containing the biological specimen separate stem cells of stem cell.
According to embodiments of the invention, can further the comprising one of at least of the red corpuscle parting liquid that adopts in the present invention, the first stem cell refined solution, the second stem cell refined solution and cell treatment solution: the human alkaline fibroblast somatomedin of 10-30ng/ml and human stem cell growth-α of 10-30ng/ml.Preferred red corpuscle parting liquid, the first stem cell refined solution, the second stem cell refined solution and cell treatment solution all further comprise: the human alkaline fibroblast somatomedin of 10-30ng/ml and human stem cell growth-α of 10-30ng/ml.Thus, the contriver finds, by adding somatomedin, especially stem cell factor, can effectively improve the stem cell in vitro survival rate, and can keep the differentiation potential of stem cell.According to embodiments of the invention, described red corpuscle parting liquid, the first stem cell refined solution and the second stem cell refined solution are separately positioned in the different containers.Thus, can conveniently use this test kit separate stem cells.Test kit of the present invention is suitability for industrialized production easily, utilizes this test kit, can obtain easily high quality and highly purified mescenchymal stem cell and be used for scientific research or clinical treatment.
Thus, in a fifth aspect of the present invention, the present invention proposes the purposes of mentioned reagent box in separate stem cells.Adopt the test kit of the embodiment of the invention, can effectively implement the method for the separate stem cells of the embodiment of the invention.Whole feature and advantage about this separation method all are fit to herein, for the sake of simplicity, repeat no more.
Below with reference to specific embodiment, the present invention will be described, need to prove, these embodiment only are illustrative, and can not be interpreted as limitation of the present invention.
If do not specialize, the conventional means that the technique means that adopts among the embodiment is well known to those skilled in the art can carry out with reference to " molecular cloning experiment guide " third edition or related products, and the reagent that adopts and product also are and can commercial obtain.Various processes and the method do not described in detail are ordinary methods as known in the art, the source of agents useful for same, trade(brand)name and be necessary to list its moiety person, all when occurring first, indicate, thereafter used identical reagent if no special instructions, all identical with the content of indicating first.
Embodiment 1 (general material and general method)
Test kit:
Reagent A: red corpuscle parting liquid
0.5-0.6% methylcellulose gum (MC) (Switzerland FLUN K company).
The methylcellulose gum average viscosity is 4,000cP, at first, with 4 ℃ of physiological saline preparation 0.4-0.6 % by weight methocel solutions of precooling, puts 4 ℃ and spends the night, and vibration shakes up repeatedly again, dissolves fully to methylcellulose gum.With resulting methocel solution autoclave sterilization 15 minutes under 0.1MPa, use again forced oscillation after being cooled to 4 ℃.Be the water white transparency shape after methylcellulose gum dissolves fully, pH 7-8 puts 4 ℃ and saves backup.
Reagent B: stem cell refined solution I
HBSS solution (available from Invitrogen company) or GBSS solution or MEM nutrient solution
Reagent C: stem cell refined solution II
8-15 % by weight Schering AG) solution: with the Schering AG) (Nycodenz) of predetermined amount (8-15g), (Invitrogen company) mixed with 100mlHBSS solution, and stirs more than 1 hour, keeps in Dark Place in 4 ℃ at last.pH?7-8。
Schering AG) (Nycodenz) is the pulvis of a kind of white, high-hydrophilic, and chemical ingredients is Schering AG) (Iohexol, CAS no.66108-95-0), can be made into the nonionic density gradient separation liquid of Almightiness type.
The molecular weight of Schering AG) (Nycodenz) is 821, and density is 2.1g/ml, be non-ionic type, to the avirulent compound of cell, can be used for separation or the purification of the various biological substances such as cell, organoid, biomacromolecule, virus.Parting liquid with Schering AG) (Nycodenz) is made into can carry out the sterilization of High Temperature High Pressure.
Reagent D: cell treatment solution
Phosphoric acid buffer or physiological saline
After mentioned reagent prepared, through 121 ℃, behind 1 hour high-temperature sterilization, test endotoxin content≤0.5EU/ml.Leave in the test kit respectively.Add somatomedin before using, add final concentration in every kind of reagent and be the human alkaline fibroblast somatomedin (hFGF2) of 10-30ng/ml and 10-30ng/ml human stem cell growth-α (former stem cell factor) that final concentration is.
General method
I, separating red corpuscle:
Method 1: anti-freezing bone marrow fluid or Cord blood and red corpuscle parting liquid mixing with gathering, place in the 100ml Open system device, under the room temperature in Bechtop, suspension is left standstill, sedimentation 45-60 minute, emit the red corpuscle of lower floor fully, collect upper strata enchylema centrifugal concentrating.The cell precipitation that obtains with stem cell refined solution I resuspension centrifugal concentrating.
Method 2: with anti-freezing bone marrow fluid or Cord blood and the red corpuscle parting liquid mixing that gathers, can with sample in predetermined ratio, directly add in the red corpuscle parting liquid container, under the room temperature in Bechtop, leave standstill, sedimentation 45-60 minute, collect upper strata enchylema centrifugal concentrating.The cell precipitation that obtains with stem cell refined solution I resuspension centrifugal concentrating.
II, gradient centrifugation separate stem cells:
The resulting suspension of cell precipitation with utilizing cell purification liquid I resuspension centrifugal concentrating to obtain slowly joins on the stem cell refined solution II, drips gently, to avoid confusing boundary layer.Then with centrifugal 22 minutes of centrifuge tube, collect middle level oyster white stem cell layer, repeatedly clean cell 2 times with the cell treatment solution, obtain stem cell.
Embodiment 2
Reagent A: red corpuscle parting liquid
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the HBSS liquid (available from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, HBSS solution (invitrogen company) 100ml, stirred＞1 hour, and added 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the phosphoric acid buffer.
The 80ml anti-freezing bone marrow fluid or the Cord blood that gather are pressed 4: 1 (v: v) mixing with 20ml 0.5 % by weight red corpuscle parting liquid, making red corpuscle parting liquid final concentration is 0.1 % by weight (methylcellulose gum, together lower), and place in the 100ml Open system device.Under the room temperature, in Bechtop, suspension is left standstill, sedimentation 45-60 minute, emit the red corpuscle of lower floor fully, make red corpuscle, white corpuscle boundary liquid level be in the translucent silica gel tubing upper end that the diameter that connects under the transfusion device is 5mm, sedimentation is 5 minutes again, slowly emit lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collecting is slowly joined on the reagent C.900g, centrifugal 22 minutes of 4 ℃ of Gradients are collected middle level oyster white stem cell layer, repeatedly clean cell 2 times with the cell treatment solution.Use at last 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, detect cell survival rate with tetrabromophenol sulfonphthalein, reach 99%.Get 5 microlitre stem cell samples again, carry out cell counting, conclusion is that every 80ml marrow or Cord blood separation obtain at least 4 * 10
7Individual stem cell
Embodiment 3
Reagent A: red corpuscle parting liquid
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the HBSS liquid (available from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, HBSS solution (invitrogen company) 100ml, stirred＞1 hour, and added 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the physiological saline.
In the 80ml anti-freezing bone marrow fluid or Cord blood and 20ml reagent A bottle that gather, in Bechtop, leave standstill under the room temperature, sedimentation 45-60 minute, precipitate the red corpuscle of lower floor, collection upper strata enchylema, 1200rpm5 minute centrifugal concentrating fully.Cell mass with 10ml reagent B dilution precipitation slowly joins the cell suspending liquid of collecting on the reagent C.900g, centrifugal 22 minutes of 4 ℃ of Gradients are collected middle level oyster white stem cell layer, repeatedly clean cell 2 times with reagent D.For subsequent use with 5ml reagent D diluting cells.Getting 5 microlitres detects cell survival rate with tetrabromophenol sulfonphthalein and reaches 99% and get 5 microlitres again and carry out cell counting.Conclusion is that every 80ml marrow or Cord blood separation obtain 4 * 10
7Individual stem cell.
Embodiment 4
Reagent A: red corpuscle parting liquid
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the GBSS liquid (available from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, GBSS solution (invitrogen company) 100ml, stirred＞1 hour, and added 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the phosphoric acid buffer.
The 80ml anti-freezing bone marrow fluid that gathers or Cord blood pressed with 20ml 0.5 % by weight red corpuscle parting liquid (v: v) mixing, making red corpuscle parting liquid final concentration was 0.1 % by weight, and placed in the 100ml Open system device in 4: 1.Under the room temperature, in Bechtop, suspension is left standstill, sedimentation 45-60 minute, emit the red corpuscle of lower floor fully, make red corpuscle, white corpuscle boundary liquid level be in the translucent silica gel tubing upper end that the diameter that connects under the transfusion device is 5mm, sedimentation is 5 minutes again, slowly emit lower floor's red corpuscle, collect 5 minutes centrifugal concentratings of upper strata enchylema 1200rpm.With 10ml reagent B diluting cells, the cell suspending liquid of collecting is slowly joined on the reagent C.900g, centrifugal 22 minutes of 4 ℃ of Gradients are collected middle level oyster white stem cell layer, repeatedly clean cell 2 times with the cell treatment solution.Use at last 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 98%. with tetrabromophenol sulfonphthalein detection cell survival rate and get 5 microlitre stem cell samples again, carry out cell counting, conclusion is that every 80ml marrow or Cord blood separation obtain 4 * 10
7Individual stem cell.
Embodiment 5
Reagent A: red corpuscle parting liquid
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the GBSS liquid (available from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, GBSS solution (invitrogen company) 100ml, stirred＞1 hour, and added 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the physiological saline.
The 80ml anti-freezing bone marrow fluid that gathers or Cord blood pressed with 20ml 0.5 % by weight red corpuscle parting liquid (v: v) mixing, making red corpuscle parting liquid final concentration was 0.1 % by weight, and placed in the 100ml Open system device in 4: 1.Under the room temperature, in Bechtop, suspension is left standstill, sedimentation 45-60 minute, emit the red corpuscle of lower floor fully, make red corpuscle, white corpuscle boundary liquid level be in the translucent silica gel tubing upper end that the diameter that connects under the transfusion device is 5mm, sedimentation is 5 minutes again, slowly emit lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collecting is slowly joined on the reagent C.900g, centrifugal 22 minutes of 4 ℃ of Gradients are collected middle level oyster white stem cell layer, repeatedly clean cell 2 times with the cell treatment solution.Use at last 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 98%. with tetrabromophenol sulfonphthalein detection cell survival rate and get 5 microlitre stem cell samples again, carry out cell counting, conclusion is that every 80ml marrow or Cord blood separation obtain 4 * 10
7Individual stem cell.
Embodiment 6
Reagent A: red corpuscle parting liquid
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the MEM nutrient solution (available from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, HBSS solution (invitrogen company) 100ml, stirred＞1 hour, and added 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the phosphoric acid buffer.
The 80ml anti-freezing bone marrow fluid that gathers or Cord blood pressed with 20ml0.5 % by weight red corpuscle parting liquid (v: v) mixing, making red corpuscle parting liquid final concentration was 0.1 % by weight, and placed in the 100ml Open system device in 4: 1.Under the room temperature, in Bechtop, suspension is left standstill, sedimentation 45-60 minute, emit the red corpuscle of lower floor fully, make red corpuscle, white corpuscle boundary liquid level be in the translucent silica gel tubing upper end that the diameter that connects under the transfusion device is 5mm, sedimentation is 5 minutes again, slowly emit lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collecting is slowly joined on the reagent C.900g, centrifugal 22 minutes of 4 ℃ of Gradients are collected middle level oyster white stem cell layer, repeatedly clean cell 2 times with the cell treatment solution.Use at last 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. with tetrabromophenol sulfonphthalein detection cell survival rate and get 5 microlitre stem cell samples again, carry out cell counting, conclusion is that every 80ml marrow or Cord blood separation obtain 4 * 10
7Individual stem cell.
Embodiment 7
Reagent A: red corpuscle parting liquid
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the MEM nutrient solution (available from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, HBSS solution (invitrogen company) 100ml, stirred＞1 hour, and added 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the physiological saline.
The 80ml anti-freezing bone marrow fluid that gathers or Cord blood pressed with 20ml0.5 % by weight red corpuscle parting liquid (v: v) mixing, making red corpuscle parting liquid final concentration was 0.1 % by weight, and placed in the 100ml Open system device in 4: 1.Under the room temperature, in Bechtop, suspension is left standstill, sedimentation 45-60 minute, emit the red corpuscle of lower floor fully, make red corpuscle, white corpuscle boundary liquid level be in the translucent silica gel tubing upper end that the diameter that connects under the transfusion device is 5mm, sedimentation is 5 minutes again, slowly emit lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collecting is slowly joined on the reagent C.900g, centrifugal 22 minutes of 4 ℃ of Gradients are collected middle level oyster white stem cell layer, repeatedly clean cell 2 times with the cell treatment solution.Use at last 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. with tetrabromophenol sulfonphthalein detection cell survival rate and get 5 microlitre stem cell samples again, carry out cell counting, conclusion is that every 80ml marrow or Cord blood separation obtain 4 * 10
7Individual stem cell.
Embodiment 8
Reagent A: red corpuscle parting liquid
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the MEM nutrient solution (available from Invitrogen company).
Reagent C: stem cell refined solution II
8%Nycodenz solution: Nycodenz 8g, HBSS solution (invitrogen company) 100ml, stirred＞1 hour, and added 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the physiological saline.
The 80ml anti-freezing bone marrow fluid that gathers or Cord blood pressed with 20ml0.5 % by weight red corpuscle parting liquid (v: v) mixing, making red corpuscle parting liquid final concentration was 0.1 % by weight, and placed in the 100ml Open system device in 4: 1.Under the room temperature, in Bechtop, suspension is left standstill, sedimentation 45-60 minute, emit the red corpuscle of lower floor fully, make red corpuscle, white corpuscle boundary liquid level be in the translucent silica gel tubing upper end that the diameter that connects under the transfusion device is 5mm, sedimentation is 5 minutes again, slowly emit lower floor's red corpuscle, collect 5 minutes centrifugal concentratings of upper strata enchylema 1200rpm.With 10ml reagent B diluting cells, the cell suspending liquid of collecting is slowly joined on the reagent C.900g, centrifugal 22 minutes of 4 ℃ of Gradients are collected middle level oyster white stem cell layer, repeatedly clean cell 2 times with the cell treatment solution.Use at last 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. with tetrabromophenol sulfonphthalein detection cell survival rate and get 5 microlitre stem cell samples again, carry out cell counting, conclusion is that every 80ml marrow or Cord blood separation obtain 4 * 10
7Individual stem cell.
Embodiment 9
Reagent A: red corpuscle parting liquid
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the MEM nutrient solution (available from Invitrogen company).
Reagent C: stem cell refined solution II
11%Nycodenz solution: Nycodenz 11g, HBSS solution (invitrogen company) 100ml, stirred＞1 hour, and added 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the physiological saline.
The 80ml anti-freezing bone marrow fluid that gathers or Cord blood pressed with 20ml0.5 % by weight red corpuscle parting liquid (v: v) mixing, making red corpuscle parting liquid final concentration was 0.1 % by weight, and placed in the 100ml Open system device in 4: 1.Under the room temperature, in Bechtop, suspension is left standstill, sedimentation 45-60 minute, emit the red corpuscle of lower floor fully, make red corpuscle, white corpuscle boundary liquid level be in the translucent silica gel tubing upper end that the diameter that connects under the transfusion device is 5mm, sedimentation is 5 minutes again, slowly emit lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collecting is slowly joined on the reagent C.900g, centrifugal 22 minutes of 4 ℃ of Gradients are collected middle level oyster white stem cell layer, repeatedly clean cell 2 times with the cell treatment solution.Use at last 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. with tetrabromophenol sulfonphthalein detection cell survival rate and get 5 microlitre stem cell samples again, carry out cell counting, conclusion is that every 80ml marrow or Cord blood separation obtain 4 * 10
7Individual stem cell.
Embodiment 10
Reagent A: red corpuscle parting liquid
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the MEM nutrient solution (available from Invitrogen company).
Reagent C: stem cell refined solution II
15%Nycodenz solution: Nycodenz 15g, HBSS solution (invitrogen company) 100ml, stirred＞1 hour, and added 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the physiological saline.
The 80ml anti-freezing bone marrow fluid that gathers or Cord blood pressed with 20ml0.5 % by weight red corpuscle parting liquid (v: v) mixing, making red corpuscle parting liquid final concentration was 0.1 % by weight, and placed in the 100ml Open system device in 4: 1.Under the room temperature, in Bechtop, suspension is left standstill, sedimentation 45-60 minute, emit the red corpuscle of lower floor fully, make red corpuscle, white corpuscle boundary liquid level be in the translucent silica gel tubing upper end that the diameter that connects under the transfusion device is 5mm, sedimentation is 5 minutes again, slowly emit lower floor's red corpuscle, collect 5 minutes centrifugal concentratings of upper strata enchylema 1200rpm.With 10ml reagent B diluting cells, the cell suspending liquid of collecting is slowly joined on the reagent C.900g, centrifugal 22 minutes of 4 ℃ of Gradients are collected middle level oyster white stem cell layer, repeatedly clean cell 2 times with the cell treatment solution.Use at last 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. with tetrabromophenol sulfonphthalein detection cell survival rate and get 5 microlitre stem cell samples again, carry out cell counting, conclusion is that every 80ml marrow or Cord blood separation obtain 4 * 10
7Individual stem cell.
Embodiment 11
Reagent A: red corpuscle parting liquid
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) among the HBSS (available from Invitrogen company), add phenol red.
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, HBSS solution (invitrogen company) 100ml, stirred＞1 hour, add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor), add phenol red.
Reagent D: cell treatment solution
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in the physiological saline.
The 80ml anti-freezing bone marrow fluid that gathers or Cord blood pressed with 20ml 0.5 % by weight red corpuscle parting liquid (v: v) mixing, making red corpuscle parting liquid final concentration was 0.1 % by weight, and placed in the 100ml Open system device in 4: 1.Under the room temperature, in Bechtop, suspension is left standstill, sedimentation 45-60 minute, emit the red corpuscle of lower floor fully, make red corpuscle, white corpuscle boundary liquid level be in the translucent silica gel tubing upper end that the diameter that connects under the transfusion device is 5mm, sedimentation is 5 minutes again, slowly emit lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collecting is slowly joined on the reagent C.900g, centrifugal 22 minutes of 4 ℃ of Gradients are collected middle level oyster white stem cell layer, repeatedly clean cell 2 times with the cell treatment solution.Use at last 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. with tetrabromophenol sulfonphthalein detection cell survival rate and get 5 microlitre stem cell samples again, carry out cell counting, conclusion is that every 80ml marrow or Cord blood separation obtain 4 * 10
7Individual stem cell.
In the description of this specification sheets, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or the example in conjunction with specific features, structure, material or the characteristics of this embodiment or example description.In this manual, the schematic statement of above-mentioned term not necessarily referred to identical embodiment or example.And the specific features of description, structure, material or characteristics can be with suitable mode combinations in any one or more embodiment or example.
Although illustrated and described embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple variation, modification, replacement and modification to these embodiment in the situation that does not break away from principle of the present invention and aim, scope of the present invention is limited by claim and equivalent thereof.
Claims (10)
1. the purposes of Schering AG) in preparation stem cell refined solution.
2. the composition for separating of stem cell is characterized in that, comprises:
Schering AG); And balanced salt solution,
Wherein, described Schering AG) is dissolved in the described balanced salt solution,
Randomly, described balanced salt solution is be selected from HBSS and GBSS at least a, preferred HBSS,
Randomly, every 100ml balanced salt solution contains the 8-15g Schering AG), and preferred every 100ml balanced salt solution contains the 13g Schering AG),
Randomly, further comprise cell growth factor, preferably further comprise the human alkaline fibroblast somatomedin of 10-30ng/ml and human stem cell growth-α of 10-30ng/ml,
Randomly, the pH of described composition is 7-8.
3. the method for a separate stem cells is characterized in that, comprises following:
The biological specimen that will contain stem cell mixes with the red corpuscle parting liquid, and standing sedimentation is in order to obtain containing the upper strata enchylema of stem cell;
With the described upper strata enchylema centrifugal concentrating that contains stem cell, obtain cell precipitation;
With the first stem cell refined solution described cell precipitation is carried out resuspension, obtain suspension;
Described suspension joined in the second stem cell refined solution and centrifugal, collecting stem cell,
Wherein,
Described red corpuscle parting liquid is the methylated cellulose aqueous solution of 0.2-0.7 % by weight, the methylated cellulose aqueous solution of preferred 0.5 % by weight,
Described the first stem cell refined solution is be selected from balanced salt solution and cell culture fluid at least a,
Randomly, described balanced salt solution is be selected from HBSS and GBSS at least a, and preferred described balanced salt solution is HBSS, and described cell culture fluid is the MEM nutrient solution,
Described the second stem cell refined solution is composition claimed in claim 2.
4. method according to claim 3 is characterized in that, the described biological specimen that contains stem cell is be selected from spinal fluid and Cord blood at least a, preferred anti-freezing spinal fluid or Cord blood.
5. method according to claim 4 is characterized in that, the described biological specimen that contains stem cell mixes according to volume ratio with described red corpuscle centrifugate at 4: 1.
6. method according to claim 4 is characterized in that, further comprises, uses the cell treatment solution that resulting stem cell is cleaned at least one times, preferably twice,
Wherein,
Described cell treatment solution is be selected from phosphoric acid buffer and physiological saline at least a.
7. method according to claim 6 is characterized in that, further the comprising one of at least of described red corpuscle parting liquid, the first stem cell refined solution, the second stem cell refined solution and cell treatment solution:
The human alkaline fibroblast somatomedin of 10-30ng/ml and human stem cell growth-α of 10-30ng/ml.
8. the test kit for separating of stem cell is characterized in that, comprising:
The red corpuscle parting liquid;
The first stem cell refined solution; And
The second stem cell refined solution,
Wherein,
Described red corpuscle parting liquid is the methylated cellulose aqueous solution of 0.2-0.7 % by weight, the methylated cellulose aqueous solution of preferred 0.5 % by weight,
Described the first stem cell refined solution is be selected from balanced salt solution and cell culture fluid at least a, randomly, described balanced salt solution is be selected from HBSS and GBSS at least a, and preferred described balanced salt solution is HBSS, described cell culture fluid is the MEM nutrient solution, and
Described the second stem cell refined solution is composition claimed in claim 2,
Randomly, described red corpuscle parting liquid, the first stem cell refined solution and the second stem cell refined solution are separately positioned in the different containers.
9. test kit according to claim 8 is characterized in that, further comprises the cell treatment solution, and described cell treatment solution is at least a of phosphoric acid buffer and physiological saline,
Randomly, described red corpuscle parting liquid, the first stem cell refined solution, the second stem cell refined solution and cell treatment solution are separately positioned in the different containers,
Randomly, further comprising one of at least of described red corpuscle parting liquid, the first stem cell refined solution, the second stem cell refined solution and cell treatment solution:
The human alkaline fibroblast somatomedin of 10-30ng/ml and human stem cell growth-α of 10-30ng/ml.
10. claim 8 or the purposes of 9 described test kits in separate stem cells.
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| CN109596687A (en) * | 2017-10-02 | 2019-04-09 | 株式会社岛津制作所 | Electrophoresis separating medium, electrophoresis kit and electrophoresis method |
| CN109596687B (en) * | 2017-10-02 | 2022-03-08 | 株式会社岛津制作所 | Separation medium for electrophoresis, kit for electrophoresis, and electrophoresis method |
| CN110283782A (en) * | 2019-06-27 | 2019-09-27 | 昆明医科大学第二附属医院 | A kind of method for building up of human marrow mesenchymal stem cell strain and induction differentiation application |
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