CN110592011A - Method for preparing umbilical cord blood plasma - Google Patents
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- 210000004700 fetal blood Anatomy 0.000 title claims description 60
- 210000002381 plasma Anatomy 0.000 title claims description 48
- 238000000034 method Methods 0.000 title claims description 17
- 210000004369 blood Anatomy 0.000 claims description 187
- 239000008280 blood Substances 0.000 claims description 187
- 239000003146 anticoagulant agent Substances 0.000 claims description 62
- 229940127219 anticoagulant drug Drugs 0.000 claims description 62
- 239000000243 solution Substances 0.000 claims description 44
- 239000006228 supernatant Substances 0.000 claims description 34
- 239000004227 calcium gluconate Substances 0.000 claims description 26
- 229960004494 calcium gluconate Drugs 0.000 claims description 26
- 235000013927 calcium gluconate Nutrition 0.000 claims description 26
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 claims description 26
- 210000004027 cell Anatomy 0.000 claims description 23
- 210000005087 mononuclear cell Anatomy 0.000 claims description 19
- 208000007536 Thrombosis Diseases 0.000 claims description 18
- 239000001509 sodium citrate Substances 0.000 claims description 15
- 238000002156 mixing Methods 0.000 claims description 13
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical group O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 12
- 230000000295 complement effect Effects 0.000 claims description 7
- 230000002779 inactivation Effects 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 210000000601 blood cell Anatomy 0.000 claims description 5
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- 230000007480 spreading Effects 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 17
- 229910001424 calcium ion Inorganic materials 0.000 description 17
- 239000002244 precipitate Substances 0.000 description 13
- 230000003321 amplification Effects 0.000 description 11
- 238000003199 nucleic acid amplification method Methods 0.000 description 11
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 10
- 230000000694 effects Effects 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 6
- 230000004936 stimulating effect Effects 0.000 description 5
- 208000032467 Aplastic anaemia Diseases 0.000 description 4
- 210000001185 bone marrow Anatomy 0.000 description 4
- 230000015271 coagulation Effects 0.000 description 4
- 238000005345 coagulation Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000022534 cell killing Effects 0.000 description 3
- 230000001684 chronic effect Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 230000011132 hemopoiesis Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 210000000822 natural killer cell Anatomy 0.000 description 3
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- 238000011160 research Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
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- 238000012423 maintenance Methods 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 201000010000 Agranulocytosis Diseases 0.000 description 1
- 229910014812 CaC6 Inorganic materials 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108010014172 Factor V Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 206010018687 Granulocytopenia Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- FNAQSUUGMSOBHW-UHFFFAOYSA-H calcium citrate Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FNAQSUUGMSOBHW-UHFFFAOYSA-H 0.000 description 1
- 239000001354 calcium citrate Substances 0.000 description 1
- 229940070006 calcium gluconate injection Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- 230000001737 promoting effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000013337 tricalcium citrate Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
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Abstract
The invention discloses a method for preparing cord blood plasma. According to the invention, the seventh blood sample is obtained by adding the calcium gluconate solution into the sixth blood sample, and calcium ions in the calcium gluconate solution are complexed with the anticoagulant to generate a precipitate. The seventh blood sample is centrifuged to obtain an eighth blood sample, all precipitates generated by calcium ions and the anticoagulant are settled at the lower part of the eighth blood sample, and the supernatant of the eighth blood sample does not contain the anticoagulant, namely the ninth blood sample does not contain the anticoagulant, so that the influence of the anticoagulant on the amplification culture of the umbilical cord blood is effectively avoided.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for preparing umbilical cord blood plasma.
Background
The recent curative effect of the umbilical cord blood in clinical treatment of the chronic aplastic anemia is definite (such as Zhenghuajin, Suyi, Dianya Pinna, and the like, the mechanism of the umbilical cord blood in treatment of the chronic aplastic anemia, discussion and observation of the curative effect, the report of Chengdu military Hospital, 1999, 1(3): 11-12). The possible mechanism for stimulating and recovering the bone marrow hematopoiesis function of the umbilical cord blood is that the umbilical cord blood contains a large amount of hematopoietic stem cells and progenitor cells, and the hematopoietic stem cells are implanted after the umbilical cord blood is infused. Cord blood is rich in hematopoietic stimulating factors and other factors, which have been shown to have the effect of stimulating the proliferation of myeloid-unilineage hematopoietic progenitor cells. The cord blood plasma has the function of stimulating hematopoiesis, and researches show that the cord blood plasma has the capacity of maintaining and promoting the self renewal and proliferation of hematopoietic cells in the cord blood, and the cord blood plasma is used for carrying out in-vitro colony culture on the hematopoietic stem cells and the hematopoietic progenitor cells in bone marrow, so that the cord blood plasma has the function of stimulating colony growth (such as Zhangsheng, Wang Yi Lang, Landao culture, and the like, the cord blood plasma supports the bone marrow hematopoietic progenitor cells in vitro, and the research of colony culture, the journal of Experimental hematology, 1996, 4:87-90), while the human peripheral blood plasma has no function. The factors which have been recognized in cord blood alone or in combination do not promote self-renewal and proliferation of hematopoietic stem cells in cord blood and bone marrow, but cord blood plasma has such an effect, suggesting that cord blood plasma may contain one or more hematopoietic factors which have not been found in modern medicine.
Clinical and basic research in recent years shows that umbilical cord blood plasma has specific stimulation effect and contains higher levels of G-CSF and Epo, so that the umbilical cord blood can stimulate hematopoiesis after being infused, and the curative effect for treating various anemias and granulocytopenia, particularly chronic aplastic anemia is positive (such as Cao Xiangshan, Liangjiangying, Zhang Lin, and the like, the in-vitro expansion of hematopoietic cells of the umbilical cord blood and the application in the treatment of aplastic anemia and obstructive anemia. Jiangsu medicine, 2002, 28(4):280 + 281). Therefore, the application prospect of the umbilical cord blood is wide.
In the process of collecting umbilical cord blood, an anticoagulant (such as EDTA and sodium citrate) is required to be added, after the added anticoagulant is chelated with calcium ions in the umbilical cord blood, NK cells (natural killer cells) are subjected to multiple extraction and washing, still coagulant residue exists, and the anticoagulant is an exogenous substance and is not beneficial to the amplification culture of the NK cells.
Sodium citrate has good protective effect on blood coagulation factor V, and can reduce its activity, so that it can be used for checking coagulogram and measuring erythrocyte sedimentation rate. Because of low toxicity, the composition is one of the components in the accumulation and maintenance; during the blood transportation process, anticoagulation is required to be maintained, so during the umbilical cord blood transportation process, sodium citrate anticoagulant is mainly adopted.
The sodium citrate is mainly used for the hemostasis examination and the determination of the blood sedimentation. Because of its low toxicity, it is also used in blood transfusion maintenance liquid. The anticoagulation mechanism is that sodium citrate and calcium ion in blood form soluble chelate to prevent blood coagulation, and the reaction formula is Na3C6H5O7+Ca2+→CaC6H5+3Na+. Sodium citrate (sodium citrate) with Na3C6H5O7·2H2O and Na3C6H5O7·5H2And O crystals, which can be combined with calcium ions in blood to form chelate, thereby preventing blood coagulation.
Clinically, the calcium gluconate injection is forbidden to be compatible with oxidant, citrate, soluble carbonate, phosphate and sulfate; the main reason is that citric acid can complex with calcium ions to form calcium citrate precipitate which is slightly soluble in water.
Accordingly, the prior art is deficient and needs improvement.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a method for preparing umbilical cord blood plasma, which avoids the influence of anticoagulant on the amplification culture of umbilical cord blood.
The technical scheme of the invention is as follows: provided is a method for preparing cord blood plasma, comprising the following steps.
S1: fully mixing the umbilical cord blood and the anticoagulant solution according to the volume ratio of 9-30:7 to obtain a first blood sample; the solubility range of the anticoagulant solution is 1.9 wt% -2.2 wt%. The anticoagulant is added into the cord blood to avoid the coagulation of the cord blood.
S2: the first blood sample was centrifuged at 400-.
S3: taking out the supernatant of the second blood sample, adding an equal volume of physiological saline into the second blood sample without the supernatant for replacement, and re-suspending the blood cells to obtain a third blood sample; spreading the third blood sample in ficol cell separating medium to separate out mononuclear cells; the supernatant of the second blood sample was subjected to complement inactivation to obtain a fourth blood sample. Since the anticoagulant is in solution in the third blood sample, the isolated mononuclear cells are free of anticoagulant.
S4: centrifuging the fourth blood sample at 3000 rpm for 20-50 minutes at 1000-; taking a supernatant of a fifth blood sample, said supernatant of said fifth blood sample being a sixth blood sample. To ensure uniformity of the sixth blood sample, the fifth blood sample was transferred to a T175 flask and mixed uniformly.
S5: adding a calcium gluconate solution into the sixth blood sample and fully mixing, wherein the volume ratio of the calcium gluconate solution to the sixth blood sample is 1: 3-10; a seventh blood sample was obtained. Calcium ions in the calcium gluconate solution are complexed with the anticoagulant to generate precipitate.
S6: centrifuging the seventh blood sample at 4000-. And (3) centrifuging the seventh blood sample to obtain an eighth blood sample, wherein all precipitates generated by calcium ions and the anticoagulant are settled at the lower part of the eighth blood sample, and the supernatant of the eighth blood sample does not contain the anticoagulant, namely the ninth blood sample does not contain the anticoagulant, so that the influence of the anticoagulant on the subsequent amplification culture is eliminated.
S7: subjecting the mononuclear cells obtained in step S3 to 1 × 106-2*106The tenth blood sample was obtained by adding 3% -20% by volume of the ninth blood sample to each 1ml of CIK whole culture medium.
S8: the tenth blood sample was cultured to obtain cord blood plasma.
Further, the solubility of the calcium gluconate solution is as follows: 0.01g/ml to 1 g/ml.
Further, the anticoagulant is sodium citrate or EDTA.
Further, step S2 is to check whether the first blood sample has blood clot, and if there is no blood clot, centrifuging the first blood sample at 400-; after removing the blood clot, if any, from the first blood sample, the first blood sample with the blood clot removed is centrifuged at 400-.
Further, the complement inactivation was performed in a water bath at 56 ℃ for 30 minutes.
By adopting the scheme, the seventh blood sample is obtained by adding the calcium gluconate solution into the sixth blood sample, and calcium ions in the calcium gluconate solution are complexed with the anticoagulant to generate precipitate. The seventh blood sample is centrifuged to obtain an eighth blood sample, all precipitates generated by calcium ions and the anticoagulant are settled at the lower part of the eighth blood sample, and the supernatant of the eighth blood sample does not contain the anticoagulant, namely the ninth blood sample does not contain the anticoagulant, so that the influence of the anticoagulant on the amplification culture of the umbilical cord blood is effectively avoided.
Drawings
FIG. 1 is a flow chart of example 1 of the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments.
Example 1
Referring to fig. 1, the technical solution of the present invention is as follows: provided is a method for preparing cord blood plasma, comprising the following steps.
S1: fully mixing the umbilical cord blood with a sodium citrate solution according to a volume ratio of 15:7 to obtain a first blood sample; the solubility range of the anticoagulant solution is 2 wt%. The anticoagulant is added into the cord blood to avoid the coagulation of the cord blood.
S2: checking the first blood sample for a blood clot, and if the first blood sample does not have a blood clot, centrifuging the first blood sample at 800 rpm for 10 minutes to obtain a second blood sample; after removing the blood clot, if any, from the first blood sample, the first blood sample from which the blood clot was removed was centrifuged at 800 rpm for 10 minutes to obtain a second blood sample.
S3: taking out the supernatant of the second blood sample, adding an equal volume of physiological saline into the second blood sample without the supernatant for replacement, and re-suspending the blood cells to obtain a third blood sample; spreading the third blood sample in ficol cell separating medium to separate out mononuclear cells; the supernatant of the second blood sample was subjected to complement inactivation at 56 ℃ for 30 minutes in a water bath to obtain a fourth blood sample. Since the anticoagulant is in solution in the third blood sample, the isolated mononuclear cells are free of anticoagulant.
S4: centrifuging the fourth blood sample at 2000 rpm for 30 minutes to obtain a fifth blood sample; taking the supernatant of a fifth blood sample into the culture bottle, and uniformly mixing, wherein the supernatant of the fifth blood sample is a sixth blood sample. The culture flask is a T175 culture flask.
S5: adding a calcium gluconate solution into the sixth blood sample and fully mixing, wherein the volume ratio of the calcium gluconate solution to the sixth blood sample is 1: 5; a seventh blood sample was obtained. Calcium ions in the calcium gluconate solution are complexed with the anticoagulant to generate precipitate. The solubility of the calcium gluconate solution is as follows: 0.5 g/ml.
S6: the seventh blood sample was centrifuged at 5000 rpm for 10 minutes to obtain an eighth blood sample, and the supernatant of the eighth blood sample, which was the ninth blood sample, was taken. And (3) centrifuging the seventh blood sample to obtain an eighth blood sample, wherein all precipitates generated by calcium ions and the anticoagulant are settled at the lower part of the eighth blood sample, and the supernatant of the eighth blood sample does not contain the anticoagulant, namely the ninth blood sample does not contain the anticoagulant, so that the influence of the anticoagulant on the subsequent amplification culture is eliminated.
S7: subjecting the mononuclear cells obtained in step S3 to 1.5 × 106Individual mononuclear cells/1 ml of CIK whole culture were added to the culture medium, and a ninth blood sample was added at a volume ratio of 8% to obtain a tenth blood sample.
Configuring control group: subjecting the mononuclear cells obtained in step S3 to 1.5 × 106An eleventh blood sample was obtained by adding 8% by volume of the sixth blood sample to each 1ml of CIK whole culture medium.
S8: the tenth blood sample was cultured to obtain cord blood plasma, which was the experimental group plasma. The eleventh blood sample was cultured to obtain plasma of the control group.
The total amount of cells and the killing rate of cells of the experimental group plasma and the control group plasma are tested. The test results are as follows. As can be seen from the following table, the total amount of the cells harvested from the plasma and the killing rate of the cells in the experimental group are better than those in the control group. Therefore, the added calcium gluconate solution can remove the components of the anticoagulant, and effectively avoids the influence of the anticoagulant on the amplification culture of the umbilical cord blood.
| Experimental group plasma | Control group plasma | |
| Initial cell number | 1.5*106/ml*5ml | 1.5*106/ml*5ml |
| Harvesting of cell Total | 3.23*106/ml*70ml | 3.12*106/ml*70ml |
| Cell killing rate (effective target ratio 100: 1) | 63.3％ | 55.2％ |
Example 2
The technical scheme of the invention is as follows: provided is a method for preparing cord blood plasma, comprising the following steps.
S1: fully mixing the umbilical cord blood with a sodium citrate solution according to a volume ratio of 10:7 to obtain a first blood sample; the anticoagulant solution is thoroughly mixed with a solubility in the range of 1.9 wt% to obtain a plurality of first blood samples. The anticoagulant is added into the cord blood to avoid the coagulation of the cord blood.
S2: checking the first blood sample for a blood clot, and if the first blood sample does not have a blood clot, centrifuging the first blood sample at 800 rpm for 10 minutes to obtain a second blood sample; after removing the clot, if any, from the first blood sample, from which the clot was removed, was centrifuged at 400 rpm for 30 minutes to obtain a second blood sample.
S3: taking out the supernatant of the second blood sample, adding an equal volume of physiological saline into the second blood sample without the supernatant for replacement, and re-suspending the blood cells to obtain a third blood sample; spreading the third blood sample in ficol cell separating medium to separate out mononuclear cells; the supernatant of the second blood sample was subjected to complement inactivation at 56 ℃ for 30 minutes in a water bath to obtain a fourth blood sample. Since the anticoagulant is in solution in the third blood sample, the isolated mononuclear cells are free of anticoagulant.
S4: centrifuging the fourth blood sample at 1000 rpm for 50 minutes to obtain a fifth blood sample; taking the supernatant of a fifth blood sample into the culture bottle, and uniformly mixing, wherein the supernatant of the fifth blood sample is a sixth blood sample. The culture flask is a T175 culture flask.
S5: adding a calcium gluconate solution into the sixth blood sample and fully mixing, wherein the volume ratio of the calcium gluconate solution to the sixth blood sample is 1: 3; a seventh blood sample was obtained. Calcium ions in the calcium gluconate solution are complexed with the anticoagulant to generate precipitate. The solubility of the calcium gluconate solution is as follows: 0.11 g/ml.
S6: the seventh blood sample was centrifuged at 4000 rpm for 20 minutes to obtain an eighth blood sample, and the supernatant of the eighth blood sample, which was the ninth blood sample, was taken. And (3) centrifuging the seventh blood sample to obtain an eighth blood sample, wherein all precipitates generated by calcium ions and the anticoagulant are settled at the lower part of the eighth blood sample, and the supernatant of the eighth blood sample does not contain the anticoagulant, namely the ninth blood sample does not contain the anticoagulant, so that the influence of the anticoagulant on the subsequent amplification culture is eliminated.
S7: subjecting the mononuclear cells obtained in step S3 to 1.5 × 106Individual mononuclear cells/1 ml of CIK whole culture were added to the culture and a tenth blood sample was obtained by adding 3% by volume of the ninth blood sample.
Preparing a control group: subjecting the mononuclear cells obtained in step S3 to 1.5 × 106An eleventh blood sample was obtained by adding 3% by volume of the sixth blood sample to each 1ml of CIK whole culture medium.
S8: the tenth blood sample was cultured to obtain cord blood plasma, which was the experimental group plasma. The eleventh blood sample was cultured to obtain plasma of the control group.
The total amount of cells and the killing rate of cells of the experimental group plasma and the control group plasma are tested. The test results are as follows. As can be seen from the following table, the total amount of the cells harvested from the plasma and the killing rate of the cells in the experimental group are better than those in the control group. Therefore, the added calcium gluconate solution can remove the components of the anticoagulant, and effectively avoids the influence of the anticoagulant on the amplification culture of the umbilical cord blood.
| Experimental group plasma | Control group plasma | |
| Initial cell number | 1.5*106/ml*5ml | 1.5*106/ml*5ml |
| Harvesting of cell Total | 3.29*106/ml*70ml | 2.78*106/ml*70ml |
| Cell killing rate (effective target ratio 100: 1) | 66.5％ | 56.7％ |
Example 3
The technical scheme of the invention is as follows: provided is a method for preparing cord blood plasma, comprising the following steps.
S1: fully mixing the umbilical cord blood with a sodium citrate solution according to a volume ratio of 25:7 to obtain a first blood sample; the solubility range of the anticoagulant solution is 2.2 wt%. The anticoagulant is added into the cord blood to avoid the coagulation of the cord blood.
S2: checking the first blood sample for a blood clot, and if the first blood sample does not have a blood clot, centrifuging the first blood sample at 1200 rpm for 5 minutes to obtain a second blood sample; after removing the blood clot, if any, from the first blood sample, the first blood sample from which the blood clot was removed was centrifuged at 800 rpm for 10 minutes to obtain a second blood sample.
S3: taking out the supernatant of the second blood sample, adding an equal volume of physiological saline into the second blood sample without the supernatant for replacement, and re-suspending the blood cells to obtain a third blood sample; spreading the third blood sample in ficol cell separating medium to separate out mononuclear cells; the supernatant of the second blood sample was subjected to complement inactivation at 56 ℃ for 30 minutes in a water bath to obtain a fourth blood sample. Since the anticoagulant is in solution in the third blood sample, the isolated mononuclear cells are free of anticoagulant.
S4: centrifuging the fourth blood sample at 3000 rpm for 20 minutes to obtain a fifth blood sample; taking the supernatant of a fifth blood sample into the culture bottle, and uniformly mixing, wherein the supernatant of the fifth blood sample is a sixth blood sample. The culture flask is a T175 culture flask.
S5: adding a calcium gluconate solution into the sixth blood sample and fully mixing, wherein the volume ratio of the calcium gluconate solution to the sixth blood sample is 1: 10; a seventh blood sample was obtained. Calcium ions in the calcium gluconate solution are complexed with the anticoagulant to generate precipitate. The solubility of the calcium gluconate solution is as follows: 1 g/ml.
S6: the seventh blood sample was centrifuged at 6000 rpm for 10 minutes to obtain an eighth blood sample, and the supernatant of the eighth blood sample, which was the ninth blood sample, was taken. And (3) centrifuging the seventh blood sample to obtain an eighth blood sample, wherein all precipitates generated by calcium ions and the anticoagulant are settled at the lower part of the eighth blood sample, and the supernatant of the eighth blood sample does not contain the anticoagulant, namely the ninth blood sample does not contain the anticoagulant, so that the influence of the anticoagulant on the subsequent amplification culture is eliminated.
S7: subjecting the mononuclear cells obtained in step S3 to 1.5 × 106The tenth blood sample was obtained by adding 20% by volume of the ninth blood sample to each 1ml of CIK whole culture medium.
Preparing a control group: subjecting the mononuclear cells obtained in step S3 to 1.5 × 106An eleventh blood sample was obtained by adding 20% by volume of the sixth blood sample to each 1ml of CIK whole culture medium.
S8: the tenth blood sample was cultured to obtain cord blood plasma, which was the experimental group plasma. The eleventh blood sample was cultured to obtain plasma of the control group.
The total amount of cells and the killing rate of cells of the experimental group plasma and the control group plasma are tested. The test results are as follows. As can be seen from the following table, the total amount of the cells harvested from the plasma and the killing rate of the cells in the experimental group are better than those in the control group. Therefore, the added calcium gluconate solution can remove the components of the anticoagulant, and effectively avoids the influence of the anticoagulant on the amplification culture of the umbilical cord blood.
| Experimental group plasma | Control group plasma | |
| Initial cell number | 1.5*106/ml*5ml | 1.5*106/ml*5ml |
| Harvesting of cell Total | 3.31*106/ml*70ml | 2.95*106/ml*70ml |
| Cell killing rate (effective target ratio 100: 1) | 68.3％ | 59.4％ |
In summary, the present invention provides a method for preparing umbilical cord blood plasma, wherein a seventh blood sample is obtained by adding a calcium gluconate solution to a sixth blood sample, and calcium ions in the calcium gluconate solution are complexed with an anticoagulant to generate a precipitate. The seventh blood sample is centrifuged to obtain an eighth blood sample, all precipitates generated by calcium ions and the anticoagulant are settled at the lower part of the eighth blood sample, and the supernatant of the eighth blood sample does not contain the anticoagulant, namely the ninth blood sample does not contain the anticoagulant, so that the influence of the anticoagulant on the amplification culture of the umbilical cord blood is effectively avoided.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A method for preparing cord blood plasma, comprising the steps of:
s1: fully mixing the umbilical cord blood and the anticoagulant solution according to the volume ratio of 10-25:7 to obtain a first blood sample; the solubility range of the anticoagulant solution is 1.9-2.2 wt%;
s2: centrifuging the first blood sample at 400-;
s3: taking out the supernatant of the second blood sample, adding an equal volume of physiological saline into the second blood sample without the supernatant for replacement, and re-suspending the blood cells to obtain a third blood sample; spreading the third blood sample in ficol cell separating medium to separate out mononuclear cells; performing complement inactivation on the supernatant of the second blood sample to obtain a fourth blood sample;
s4: centrifuging the fourth blood sample at 3000 rpm for 20-50 minutes at 1000-; taking a supernatant of a fifth blood sample, wherein the supernatant of the fifth blood sample is a sixth blood sample;
s5: adding a calcium gluconate solution into the sixth blood sample and fully mixing, wherein the volume ratio of the calcium gluconate solution to the sixth blood sample is 1: 3-10; obtaining a seventh blood sample;
s6: centrifuging the seventh blood sample at 4000-;
s7: subjecting the mononuclear cells obtained in step S3 to 1 × 106-2*106Adding 3-20% by volume of a ninth blood sample into each 1ml of CIK complete culture solution to obtain a tenth blood sample;
s8: the tenth blood sample was cultured to obtain cord blood plasma.
2. The method for preparing cord blood plasma according to claim 1, wherein the solubility of the calcium gluconate solution is: 0.01g/ml to 1 g/ml.
3. The method for preparing cord blood plasma according to claim 1, wherein the anticoagulant is sodium citrate solution or EDTA solution.
4. The method of claim 1, wherein the step S2 is to check whether the first blood sample has blood clot, and if there is no blood clot, the first blood sample is centrifuged at 400-; after removing the blood clot, if any, from the first blood sample, the first blood sample with the blood clot removed is centrifuged at 400-.
5. The method for preparing cord blood plasma according to claim 1, wherein the complement inactivation is performed in a water bath at 56 ℃ for 30 minutes.
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| CN114058584A (en) * | 2022-01-07 | 2022-02-18 | 山东省齐鲁干细胞工程有限公司 | Preparation method of natural killer cells for clinical use |
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