CN107156108A - A kind of peripheral hematopoietic stem cells preserve liquid and preparation method - Google Patents
A kind of peripheral hematopoietic stem cells preserve liquid and preparation method Download PDFInfo
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- CN107156108A CN107156108A CN201710392718.3A CN201710392718A CN107156108A CN 107156108 A CN107156108 A CN 107156108A CN 201710392718 A CN201710392718 A CN 201710392718A CN 107156108 A CN107156108 A CN 107156108A
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- stem cells
- hematopoietic stem
- peripheral hematopoietic
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
Liquid is preserved the present invention relates to stem cells technology field, more particularly to a kind of peripheral hematopoietic stem cells, in parts by weight including following components：38 parts of human serum albumin, 24 parts of trehalose, 13 parts of HES, 5 15 parts of nutrient, 3 parts of dextran 1,13 parts of dimethyl sulfoxide (DMSO) and suitable quantity of water.Peripheral hematopoietic stem cells are preserved using the preservation liquid in the present invention, stem cell can be made to keep preferable stability, and the survival rate of stem cell is greatly improved, more than 98%.
Description
Technical field
Liquid and preparation method thereof is preserved the present invention relates to stem cells technology field, more particularly to a kind of peripheral hematopoietic stem cells.
Background technology
Stem-cell therapy is after the stem cell with continuous self-reproduction ability and multidirectionalization potential is cultivated in vitro, then
Be implanted into that patient is impaired or defective tissue in, so as to realize the supplement to injury tissue and reparation.Autologous peripheral blood hematopoiesis
Stem cell transplantation refers to that carrying out chemotherapy plus cells mobilization with G-CSF to the patient with some malignant hematologic diseases makes trouble
Person itself peripheral hematopoietic stem cells reach certain value, then by the collection of separator cell, the candidate stem cell of collection out is cold
Freeze and preserve, Radiotherapy chemotherapy processing then is carried out to patient, the candidate stem cell of freezen protective is defeated by rapidly oneself after recovery
Implantation method, this method has collection easy, and patient suffering is few, hematopoiesis and immunity function restructuring block, few intercurrent disease etc. after transplanting
Advantage, has been gradually available for clinic at present.It is existing, it is necessary to which adding preservation liquid carries out freezen protective after current Peripheral blood progenitor collection
There are peripheral hematopoietic stem cells to preserve liquid, nutritional ingredient is single, it is improper to match, and causes the cell survival rate of peripheral hematopoietic stem cells low, carefully
Born of the same parents' stability is poor.
The content of the invention
The technical problems to be solved by the invention are：In view of the shortcomings of the prior art there is provided one kind proportioning is appropriate, nutrition into
Divide equilibrium, cell survival rate can be improved, and the peripheral hematopoietic stem cells of cell stability can be kept to preserve liquid.
In order to solve the above technical problems, the technical scheme is that：
A kind of peripheral hematopoietic stem cells preserve liquid, in parts by weight including following components：3-8 parts of human serum albumin, sea
2-4 parts of algae sugar, 1-3 parts of HES, 5-15 parts of nutrient, -3 parts of dextran 1,1-3 parts of dimethyl sulfoxide (DMSO) and suitable quantity of water.
As an improvement technical scheme, the peripheral hematopoietic stem cells preserve liquid, in parts by weight including following
Component：5-8 parts of albumin, 3-4 parts of trehalose, 2-3 parts of HES, 8-15 parts of nutrient, 2-3 parts of dextran and diformazan
2-3 parts of base sulfoxide and suitable quantity of water.
As an improvement technical scheme, the mass concentration of the dimethyl sulfoxide (DMSO) is 5%-10%.
As an improvement technical scheme, the peripheral hematopoietic stem cells preserve liquid, 1-3 are also included in parts by weight
Part 0.5-2wt% hyaluronic acid and 1-3 parts of streptomysins.
As an improvement technical scheme, the nutrient by vitamin C, B family vitamin, essential amino acid, it is non-must
Amino acid, sodium chloride, potassium chloride and sodium gluconate are needed according to 0.1-0.5:0.1-0.3:0.3-0.7:0.2-0.5:0.3-
0.5:0.3-0.7:0.2-0.7 mass ratio composition.
As an improvement technical scheme, the essential amino acid be isoleucine, taurine, arginine in it is any one
Plant or two kinds.
As an improvement technical scheme, the nonessential amino acid include alanine and/or tyrosine.
Another technical problem to be solved by this invention is：It is dry there is provided a kind of peripheral blood in view of the shortcomings of the prior art
The preparation method of cell-preservation liquid.
In order to solve the above technical problems, the technical scheme is that：
(1) it is proportionally successively that nutrient, trehalose and HES is soluble in water, it is well mixed, what is obtained is molten
Liquid is incubated under the conditions of 35-42 DEG C, standby；
(2) solution in step (1) is cooled to 25-30 DEG C, adds hyaluronic acid, mixed, obtained mixed liquor is in 25-
It is incubated under the conditions of 30 DEG C；
(3) dextran and human serum albumin are added into the mixed liquor of step (2), streptomysin is added after mixing, is mixed
Close uniform, filtering obtains filtered fluid；
(4) dimethyl sulfoxide (DMSO) that mass concentration is 5%-10% is added into the filtered fluid of step (3), is mixed, obtains outer
All hemocytoblasts preserve liquid.
Employ after above-mentioned technical proposal, the beneficial effects of the invention are as follows：
(1) due to many containing vitamin C, B family vitamin, inorganic salts, essential amino acid, nonessential amino acid in nutrient
Plant nutrition composition, the vital metabolic that cell can be maintained basic, the osmotic pressure of statocyte；Dimethyl sulfoxide (DMSO), hyaluronic acid, hydroxyl second
Base starch, trehalose, the use in conjunction of dextran and human serum albumin can be realized to stem cell permeability protective effect, reduction
Cell freezing point, substantially reduces the infringement to stem cell, while breed bacteria when the addition of streptomysin can prevent cell recovery, significantly
Survival rate during stem cell recovery is improved, stem cell is kept cytoactive and stability, the stem cell of preservation to greatest extent
Its survival rate is higher than 97%；
(2) consumption of dimethyl sulfoxide (DMSO) is few, and concentration is low, while itself permeability protective agent function is played, also reduces
Harm of the toxicity of dimethyl sulfoxide (DMSO) to cell, substantially increases clinical safety；
(3) liquid each component is preserved clearly, convenient configuration, it is adaptable to the Cord blood of peripheral hematopoietic stem cells.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment 1
A kind of peripheral hematopoietic stem cells preserve liquid, in parts by weight including following components：3 parts of the white egg of human blood, trehalose 2
Part, 1 part of HES, 8 parts of nutrient (vitamin C, B family vitamin, isoleucine, alanine, sodium chloride, potassium chloride and
Sodium gluconate is according to 0.2:0.1:0.3:0.2:0.3:0.3:0.2 mass ratio composition), dextran 1 part, dimethyl sulfoxide (DMSO)
1 part (mass concentration is 5%) and suitable quantity of water.
Embodiment 2
A kind of peripheral hematopoietic stem cells preserve liquid, in parts by weight including following components：5 parts of the white egg of human blood, trehalose 3
Part, 2 parts of HES, nutrient 10 parts of (vitamin C, B family vitamin, taurine, tyrosine, sodium chloride, potassium chloride and Portugals
Grape sodium saccharate is according to 0.2:0.1:0.4:0.3:0.3:0.3:0.3 mass ratio composition), 2 parts of dextran, dimethyl sulfoxide (DMSO) 2
Part (mass concentration is 5%), 2 parts of 0.5wt% hyaluronic acid, 1 part of streptomysin and suitable quantity of water.
The preparation method of above-mentioned preservation liquid：
(1) it is proportionally successively that nutrient, trehalose and HES is soluble in water, it is well mixed, what is obtained is molten
Liquid is incubated under the conditions of 37 DEG C, standby；
(2) solution in step (1) is cooled to 26 DEG C, adds hyaluronic acid, mixed, obtained mixed liquor is in 26 DEG C of bars
It is incubated under part；
(3) dextran and human serum albumin are added into the mixed liquor of step (2), streptomysin is added after mixing, is mixed
Close uniform, filtering obtains filtered fluid；
(4) dimethyl sulfoxide (DMSO) that mass concentration is 5%-10% is added into the filtered fluid of step (3), is mixed, obtains outer
All hemocytoblasts preserve liquid.
Embodiment 3
A kind of peripheral hematopoietic stem cells preserve liquid, in parts by weight including following components：The white protein 16.5 part of human blood, marine alga
3.5 parts of sugar, 2.5 parts of HES, 12 parts of nutrient (vitamin C, B family vitamin, taurine and arginine 1:1 ratio,
Tyrosine, sodium chloride, potassium chloride and sodium gluconate are according to 0.3:0.2:0.5:0.4:0.3:0.4:0.5 mass ratio composition),
2.5 parts of dextran, 2.5 parts of dimethyl sulfoxide (DMSO) (mass concentration is 7%), 2.5 parts of 1wt% hyaluronic acid, 2 parts of streptomysins and
Suitable quantity of water.
Embodiment 4
A kind of peripheral hematopoietic stem cells preserve liquid, in parts by weight including following components：8 parts of the white egg of human blood, trehalose 4
Part, 3 parts of HES, (vitamin C, B family vitamin, taurine and isoleucine are according to 1 for 15 parts of nutrient:1 ratio,
Tyrosine and alanine are according to 1:1 ratio, sodium chloride, potassium chloride and sodium gluconate is according to 0.4:0.3:0.5:0.4:0.5:
0.5:0.7 mass ratio composition), 3 parts of dextran, 3 parts of dimethyl sulfoxide (DMSO) (mass concentration is 9%), 3 parts of 1.8wt% it is saturating
Bright matter acid, 3 parts of streptomysins and suitable quantity of water.
Comparative example 1
A kind of peripheral hematopoietic stem cells preserve liquid, and unique difference with embodiment 3, which is to preserve in liquid, does not contain hyaluronic acid.
Comparative example 2
A kind of peripheral hematopoietic stem cells preserve liquid, and unique difference with embodiment 3, which is to preserve in liquid, does not contain taurine, essence
Propylhomoserin and tyrosine.
Comparative example 3
A kind of peripheral hematopoietic stem cells preserve liquid, and dextran is not contained only difference is that preserving in liquid with embodiment 3.
Application of the liquid in peripheral hematopoietic stem cells preservation is preserved in the embodiment 1-4 of application examples 1
The collection of peripheral hematopoietic stem cells, wherein male are carried out to the inpatient of the hematology of People's Hospital of Weifang City 200
100, women 100, the mobilization collection of autologous peripheral blood stem cells adds Recombinant Human Colony Stimulating Factors using combined chemotherapy, uses
Cell sorting machine gathers peripheral hematopoietic stem cells, and the preservation liquid of embodiment 1-4 preservation liquid and comparative example 1-3 is respectively used into periphery
The preservation of hemocytoblast, using platform expect it is blue refuse dye experiment and determine the viable count after recovery, the results are shown in Table 1.
Cell viability=viable count after defrosting/freezes preceding viable count × 100
Table 1
| Embodiment | Platform expects that orchid refuses dye rate (%) |
| Embodiment 1 | 95.7±0.5 |
| Embodiment 2 | 97.5±1.5 |
| Embodiment 3 | 98.2±0.7 |
| Embodiment 4 | 97.2±1.3 |
| Comparative example 1 | 82.3±0.7 |
| Comparative example 2 | 78.6±1.5 |
| Comparative example 3 | 80.6±1.2 |
The aggregation situation of cell is observed under the microscope, cytotostatic is good, soilless sticking phenomenon, as a result show to use this hair
The bright embodiment 1-4 preservation liquid for preserving liquid and comparative example 1-3 is contrasted, and embodiment 1-4 preservation liquid is dry thin to preserve peripheral blood
Born of the same parents, can make stem cell keep preferable stability, and substantially increase stem cell number of viable cells, and cell survival rate exists
More than 98%.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.
Claims (8)
1. a kind of peripheral hematopoietic stem cells preserve liquid, it is characterised in that in parts by weight including following components：Human serum albumin
3-8 parts, 2-4 parts of trehalose, 1-3 parts of HES, 5-15 parts of nutrient, -3 parts of dextran 1,1-3 parts of dimethyl sulfoxide (DMSO)
And suitable quantity of water.
2. a kind of peripheral hematopoietic stem cells according to claim 1 preserve liquid, it is characterised in that:The peripheral hematopoietic stem cells are protected
Liquid storage, in parts by weight including following components：5-8 parts of albumin, 3-4 parts of trehalose, 2-3 parts of HES, nutrition
Plain 8-15 parts, 2-3 parts of dextran and 2-3 parts of dimethyl sulfoxide (DMSO) and suitable quantity of water.
3. a kind of peripheral hematopoietic stem cells according to claim 1 preserve liquid, it is characterised in that:The matter of the dimethyl sulfoxide (DMSO)
Amount concentration is 5%-10%.
4. a kind of peripheral hematopoietic stem cells according to claim 1 preserve liquid, it is characterised in that:The peripheral hematopoietic stem cells are protected
Liquid storage, also includes 1-3 parts of 0.5-2wt% hyaluronic acid and 1-3 part streptomysin in parts by weight.
5. a kind of peripheral hematopoietic stem cells according to claim 1 preserve liquid, it is characterised in that:The nutrient is by vitamin
C, B family vitamin, essential amino acid, nonessential amino acid, sodium chloride, potassium chloride and sodium gluconate are according to 0.1-0.5:0.1-
0.3:0.3-0.7:0.2-0.5:0.3-0.5:0.3-0.7:0.2--0.7 mass ratio composition.
6. a kind of peripheral hematopoietic stem cells according to claim 5 preserve liquid, it is characterised in that:The essential amino acid is different
Any one in leucine, taurine, arginine or two kinds.
7. a kind of peripheral hematopoietic stem cells according to claim 6 preserve liquid, it is characterised in that:The nonessential amino acid bag
Include alanine and/or tyrosine.
8. a kind of peripheral hematopoietic stem cells in a kind of 1-7 such as claim as described in any claim preserve the preparation method of liquid,
It is characterized in that the preparation method comprises the following steps：
(1) it is proportionally successively that nutrient, trehalose and HES is soluble in water, it is well mixed, obtained solution exists
It is incubated under the conditions of 35-42 DEG C, it is standby；
(2) solution in step (1) is cooled to 25-30 DEG C, adds hyaluronic acid, mixed, obtained mixed liquor is at 25-30 DEG C
Under the conditions of be incubated；
(3) dextran and human serum albumin are added into the mixed liquor of step (2), streptomysin is added after mixing, mixing is equal
Even, filtering obtains filtered fluid；
(4) dimethyl sulfoxide (DMSO) that mass concentration is 5%-10% is added into the filtered fluid of step (3), mixes, obtains peripheral blood
Stem cell preserving fluid.
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107912419A (en) * | 2017-10-11 | 2018-04-17 | 重庆金时代生物技术有限公司 | The frozen stock solution and cryopreservation methods of a kind of human peripheral blood single nucleus cell |
| CN107980767A (en) * | 2018-01-10 | 2018-05-04 | 暨赛再生医学科技有限公司 | A kind of long-term preservation liquid for preserving the EPC for being overexpressed ANG-1 |
| CN108029678A (en) * | 2017-12-28 | 2018-05-15 | 重庆斯德姆生物技术有限公司 | Cell preservation method and cell transportation resources |
| CN108244096A (en) * | 2018-01-10 | 2018-07-06 | 暨赛再生医学科技有限公司 | A kind of long-term preservation liquid for preserving the endothelial progenitor cell for being overexpressed VEGF |
| CN108432742A (en) * | 2018-04-09 | 2018-08-24 | 佛山科学技术学院 | A kind of mescenchymal stem cell room temperature transport liquid |
| CN109221092A (en) * | 2018-11-14 | 2019-01-18 | 广东华夏健康生命科学有限公司 | A kind of cells frozen storing liquid and its application |
| CN109845726A (en) * | 2019-02-27 | 2019-06-07 | 李冲杰 | A kind of mescenchymal stem cell Cryopreservation protective agent |
| CN110050782A (en) * | 2019-05-07 | 2019-07-26 | 广州赛隽生物科技有限公司 | A kind of stem cell cryopreserving liquid and preparation method thereof and cryopreservation methods |
| CN110074096A (en) * | 2019-05-28 | 2019-08-02 | 苏州博特龙免疫技术有限公司 | A kind of serum-free cell frozen stock solution and its preparation method and application |
| CN110150267A (en) * | 2019-06-24 | 2019-08-23 | 南京沐赛生物技术有限公司 | A kind of low DMSO of clinical grade TCR-T cell freezes agent and cryopreservation methods |
| CN112806355A (en) * | 2021-01-20 | 2021-05-18 | 山东科硕生物技术有限公司 | Cell preservation solution and preparation method and application thereof |
| CN113133446A (en) * | 2021-04-30 | 2021-07-20 | 海南优尼科尔生物科技有限公司 | Peripheral blood mononuclear cell cryopreservation liquid, preparation and use method |
| WO2022067042A1 (en) * | 2020-09-24 | 2022-03-31 | Regents Of The University Of Minnesota | Cryoprotective compositions and methods for protection of a surgical site during cryosurgery |
| WO2022144186A1 (en) * | 2021-01-04 | 2022-07-07 | Xvivo Perfusion Ab | Lipid layer preservation |
| CN115251045A (en) * | 2022-08-04 | 2022-11-01 | 珠海暨创硒源纳米科技有限公司 | Cell cryopreservation liquid and preparation method thereof |
| CN115777689A (en) * | 2022-11-09 | 2023-03-14 | 武汉赛维尔生物科技有限公司 | Serum-free and protein-free non-programmed cell cryopreservation solution |
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| CN107912419A (en) * | 2017-10-11 | 2018-04-17 | 重庆金时代生物技术有限公司 | The frozen stock solution and cryopreservation methods of a kind of human peripheral blood single nucleus cell |
| CN108029678A (en) * | 2017-12-28 | 2018-05-15 | 重庆斯德姆生物技术有限公司 | Cell preservation method and cell transportation resources |
| CN107980767A (en) * | 2018-01-10 | 2018-05-04 | 暨赛再生医学科技有限公司 | A kind of long-term preservation liquid for preserving the EPC for being overexpressed ANG-1 |
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| CN108432742A (en) * | 2018-04-09 | 2018-08-24 | 佛山科学技术学院 | A kind of mescenchymal stem cell room temperature transport liquid |
| CN109221092A (en) * | 2018-11-14 | 2019-01-18 | 广东华夏健康生命科学有限公司 | A kind of cells frozen storing liquid and its application |
| CN109845726A (en) * | 2019-02-27 | 2019-06-07 | 李冲杰 | A kind of mescenchymal stem cell Cryopreservation protective agent |
| CN110050782A (en) * | 2019-05-07 | 2019-07-26 | 广州赛隽生物科技有限公司 | A kind of stem cell cryopreserving liquid and preparation method thereof and cryopreservation methods |
| CN110074096A (en) * | 2019-05-28 | 2019-08-02 | 苏州博特龙免疫技术有限公司 | A kind of serum-free cell frozen stock solution and its preparation method and application |
| CN110074096B (en) * | 2019-05-28 | 2020-05-15 | 苏州博特龙免疫技术有限公司 | Serum-free cell cryopreservation liquid and preparation method and application thereof |
| CN110150267A (en) * | 2019-06-24 | 2019-08-23 | 南京沐赛生物技术有限公司 | A kind of low DMSO of clinical grade TCR-T cell freezes agent and cryopreservation methods |
| WO2022067042A1 (en) * | 2020-09-24 | 2022-03-31 | Regents Of The University Of Minnesota | Cryoprotective compositions and methods for protection of a surgical site during cryosurgery |
| WO2022144186A1 (en) * | 2021-01-04 | 2022-07-07 | Xvivo Perfusion Ab | Lipid layer preservation |
| CN112806355A (en) * | 2021-01-20 | 2021-05-18 | 山东科硕生物技术有限公司 | Cell preservation solution and preparation method and application thereof |
| CN113133446A (en) * | 2021-04-30 | 2021-07-20 | 海南优尼科尔生物科技有限公司 | Peripheral blood mononuclear cell cryopreservation liquid, preparation and use method |
| CN115251045A (en) * | 2022-08-04 | 2022-11-01 | 珠海暨创硒源纳米科技有限公司 | Cell cryopreservation liquid and preparation method thereof |
| CN115251045B (en) * | 2022-08-04 | 2023-10-27 | 珠海暨创硒源纳米科技有限公司 | Cell freezing solution and preparation method thereof |
| CN115777689A (en) * | 2022-11-09 | 2023-03-14 | 武汉赛维尔生物科技有限公司 | Serum-free and protein-free non-programmed cell cryopreservation solution |
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Application publication date: 20170915 |