CN110604745B - Method for producing platelet dry powder - Google Patents
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- CN110604745B CN110604745B CN201810615278.8A CN201810615278A CN110604745B CN 110604745 B CN110604745 B CN 110604745B CN 201810615278 A CN201810615278 A CN 201810615278A CN 110604745 B CN110604745 B CN 110604745B
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- 239000000843 powder Substances 0.000 title claims abstract description 55
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 13
- 210000001772 blood platelet Anatomy 0.000 claims abstract description 132
- 210000002381 plasma Anatomy 0.000 claims abstract description 38
- 229910001424 calcium ion Inorganic materials 0.000 claims abstract description 34
- 239000007864 aqueous solution Substances 0.000 claims abstract description 32
- 239000008280 blood Substances 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 29
- 210000004369 blood Anatomy 0.000 claims abstract description 28
- -1 calcium ion compound Chemical class 0.000 claims abstract description 24
- 210000002966 serum Anatomy 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 23
- 239000011259 mixed solution Substances 0.000 claims abstract description 16
- 239000002002 slurry Substances 0.000 claims abstract description 14
- 239000000203 mixture Substances 0.000 claims abstract description 12
- 238000002156 mixing Methods 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 9
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 9
- 239000012530 fluid Substances 0.000 claims abstract description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 11
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 claims description 4
- 238000005086 pumping Methods 0.000 claims description 2
- 229940003871 calcium ion Drugs 0.000 claims 4
- 229940043430 calcium compound Drugs 0.000 claims 3
- 150000001674 calcium compounds Chemical class 0.000 claims 3
- 238000004378 air conditioning Methods 0.000 claims 1
- 239000002504 physiological saline solution Substances 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 10
- 239000007788 liquid Substances 0.000 description 9
- 239000003146 anticoagulant agent Substances 0.000 description 8
- 229940127219 anticoagulant drug Drugs 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 238000001223 reverse osmosis Methods 0.000 description 8
- 238000005119 centrifugation Methods 0.000 description 7
- 108090000190 Thrombin Proteins 0.000 description 5
- 229960004072 thrombin Drugs 0.000 description 5
- 241000283690 Bos taurus Species 0.000 description 4
- 210000000601 blood cell Anatomy 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- 239000000644 isotonic solution Substances 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000000735 allogeneic effect Effects 0.000 description 3
- 230000015271 coagulation Effects 0.000 description 3
- 238000005345 coagulation Methods 0.000 description 3
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- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 244000058281 Ulmus pumila Species 0.000 description 2
- 235000001547 Ulmus pumila Nutrition 0.000 description 2
- 230000004520 agglutination Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
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- 239000003102 growth factor Substances 0.000 description 2
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- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229960003766 thrombin (human) Drugs 0.000 description 2
- 208000025978 Athletic injury Diseases 0.000 description 1
- 208000009084 Cold Injury Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000002595 cold damage Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000032798 delamination Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012213 gelatinous substance Substances 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 210000004623 platelet-rich plasma Anatomy 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 230000007838 tissue remodeling Effects 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/19—Platelets; Megacaryocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
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- Life Sciences & Earth Sciences (AREA)
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- Pharmacology & Pharmacy (AREA)
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- Animal Behavior & Ethology (AREA)
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- Immunology (AREA)
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Abstract
A method of manufacturing a platelet dry powder, comprising: obtaining a platelet-containing plasma fluid, wherein the plasma fluid has substantially been depleted of red blood cells; preparing 0.00001-0.03M calcium ion compound aqueous solution; a small amount of this serum was taken at 1: 2-1: 200, adding the mixture into the aqueous solution of the calcium ion compound, and uniformly mixing to form a mixed solution; 1, the method comprises the following steps: 1to 1:50, adding the mixed solution into the plasma solution, and uniformly mixing to form mixed blood slurry; centrifugally separating the mixed blood serum to obtain platelet wet powder; and drying the platelet wet powder to obtain platelet dry powder.
Description
Technical Field
The present invention relates to a method for producing a platelet dry powder, and more particularly, to a method for producing a platelet dry powder by using calcium ions (an aqueous solution of a calcium ion compound).
Background
Platelets contain a variety of different growth factors that promote cell proliferation, differentiation, and tissue remodeling and repair. Platelets have wide application in sports injuries, dentistry, orthopedics, and orthopedics. At present, the long-term preservation of the platelets still has problems, the time for which the platelet products are preserved in the environment of 22 ℃ of a blood bank is usually only 3-7 days, and the cold preservation (4 ℃) reduces the pollution probability of the platelet products to a certain extent, but leads to the cold injury of the platelets and shortens the service life of the platelets.
Therefore, some people make platelets into platelet dry powder, so that the shelf life of the platelets can be prolonged. Techniques for preparing platelet dry powder, see, for example, taiwan patent nos. I300806, I270375, 201004659, 200526680, U.S. patent nos. 7,659,052, 6,060,233, 5,736,313, 5,589,462, but either the methods or platelet cells are unprotected, so that most of the platelet cells in the process have been ruptured, causing the growth factors in the cells to be easily spoiled, such as U.S. patent No. 5,589,462; or poor protection, significantly lower platelet function, for example: us 6,060,233, 5,736,313 announces patents; or the protective agent is not easy to obtain, the dosage is not easy to control, the cost is extremely high, the process control is not easy or the cost is extremely high, and the like, such as I300806, I270375 published patent application, taiwan 201004659 and 200526680 published patent application and U.S. 7,659,052 published patent application, and the process disclosed by TW-I270375 published patent application is taken as an example, thrombin (thrombin) is used for protecting platelet cells, but the thrombin is usually different in sources (human, cow, pig..and the like), the activity variation is extremely large, and Sigma-Aldrich products are taken as examples, and human thrombin (human thrombin) is taken as an example: t1063 is >2800NIH units/mg (hereinafter abbreviated as units/mg), T7572 is 50 to 300NIH units/mg, bovine thrombin (bovine thrombin): t9549 is 1500-2500 NIH units/mg and T6200 is 40-300 NIH units/mg, so that the process control is not easy; furthermore, the stability is not high and therefore it is necessary to store it at low temperature, for example, T9326 at a storage temperature of-70 ℃. Also, as in TW-I480066, ADP is used to protect platelet cells, but ADP is expensive and causes platelet aggregation easily, so that the separation step must be completed in a short time, otherwise, platelet aggregation is difficult to separate.
Disclosure of Invention
An object of the present invention is to provide a method for producing platelet dry powder.
Another object of the present invention is to provide a method for producing a platelet dry powder using calcium ions (an aqueous solution of a calcium ion compound).
It is still another object of the present invention to provide a method for producing a platelet dry powder by using the step of indirectly adding calcium ions (an aqueous solution of a calcium ion compound).
A method of manufacturing a platelet dry powder, comprising: obtaining a platelet-containing plasma fluid, wherein the plasma fluid has substantially been depleted of red blood cells; preparing 0.00001-0.03M calcium ion compound aqueous solution; a small amount of this serum was taken at 1: 2-1: 200, adding the mixture into the aqueous solution of the calcium ion compound, and uniformly mixing to form a mixed solution; 1, the method comprises the following steps: 1to 1:50, adding the mixed solution into the plasma solution, and uniformly mixing to form mixed blood slurry; centrifugally separating the mixed blood serum to obtain platelet wet powder; and drying the platelet wet powder to obtain platelet dry powder.
The aqueous solution of the above-mentioned calcium ion compound is often "directly" added to a platelet-containing solution to be used as an activator, and the aqueous solution of the calcium ion compound is directly added to a platelet solution, and when platelets react with calcium ions, a gel-like substance is produced, and platelets cannot be separated, and platelet wet powder cannot be formed after centrifugation, see comparative example 1 below.
The invention uses the step of indirectly adding calcium ions, which is that: a small amount of blood serum is firstly taken and added into an aqueous solution of a calcium ion compound, the mixture is stirred to form a substantially uniform mixed solution, then a proper amount of the mixed solution is added into the blood serum in an indirect way, after the calcium ions are added in an indirect way, the mixture is stirred to form a substantially uniform mixed blood serum, the mixed blood serum manufactured through the step of adding the calcium ions in an indirect way can effectively avoid the generation of gelatinous substances, platelet wet powder can be generated after the mixed blood serum is centrifuged, and platelet dry powder is obtained after drying, and the invention is described in examples 1-24.
The blood serum mentioned above refers to blood serum containing platelets, and particularly to blood serum from which red blood cells have been substantially removed. The method for removing red blood cells may be any known method for removing red blood cells from blood preparations, for example, a method for separating a blood serum from whole blood by one or more high-speed or low-speed centrifugation, a method for separating PRP (platelet rich plasma, platelets Rich Plasma) from whole blood by using a blood cell separator (blood cell separator), or a method for directly or indirectly obtaining PRP from various blood centers or the like.
The platelet-containing blood serum may be a blood plasma obtained by removing red blood cells from whole blood of a mammal, for example, a mammal accepted by a medical unit such as cow, pig, sheep, or human.
The platelet-containing plasma solution obtained from the mammal may be a heterogeneous platelet-containing plasma, a allogeneic platelet-containing plasma, or an autologous platelet-containing plasma, and is preferably an allogeneic platelet-containing plasma or an autologous platelet-containing plasma, more preferably an autologous platelet-containing plasma, in view of the psychological factors of the user. However, the platelet-containing plasma is preferably a allogeneic platelet-containing plasma or a xenogeneic platelet-containing plasma, and more preferably a xenogeneic platelet-containing plasma, based on ease of acquisition and mass production factors.
The platelet-containing plasma solution separated from whole blood contains anticoagulant to prevent coagulation of blood during operation. The anticoagulant can be any conventional anticoagulant such as EDTA anticoagulant, sodium citrate anticoagulant, etc., and ACD Solution-A (anti-blood coagulation citrate dextrose Solution-A) and ACD Solution-C (anti-blood coagulation citrate dextrose Solution-C) of Xindong technology Co., ltd, and sodium citrate anticoagulant prepared according to the formulations.
The concentration of the aqueous solution of the calcium ion compound may be 0.00001 to 0.03M, preferably 0.0005 to 0.01M, more preferably 0.0001 to 0.005M.
The above-mentioned calcium ion compound may be any conventional soluble calcium ion compound, and calcium chloride (CaCl 2) and calcium nitrate (Ca (NO 3) 2) are preferable, and calcium chloride (CaCl 2) is most preferable.
The solvent of the aqueous solution of the calcium ion compound is water, but in order to form an isotonic solution or a near isotonic solution, it is preferable to use physiological saline (to form an isotonic solution) or a mixed solution of reverse osmosis water and physiological saline (to form a near isotonic solution) instead of reverse osmosis water to prepare an aqueous solution of the calcium ion compound, and it is preferable to use physiological saline instead of reverse osmosis water to prepare an aqueous solution of the calcium ion compound.
The mixed solution is formed by mixing a small amount of blood slurry and an aqueous solution of a calcium ion compound, wherein the (volume) ratio of the blood slurry to the aqueous solution of the calcium ion compound is 1: 2-1: 200, 1:10 to 1:100 is more preferred.
The mixed blood slurry is formed by mixing an aqueous solution of a calcium ion compound with a plasma liquid, wherein the (volume) ratio of the aqueous solution of the calcium ion compound to the mixed blood slurry is 1: 2-1: 50, 1: 5to 1:10 is more preferable.
The platelet wet powder is a precipitate obtained by centrifuging mixed blood slurry, and after the centrifugation step is finished, the platelet wet powder can be precipitated on the pipe wall below the centrifuge pipe, and the solution on the upper layer of the centrifuge pipe is removed, so that the platelet wet powder can be obtained. The centrifugation method may be any known centrifugation method, for example, centrifugation with a centrifuge. The centrifugation speed and centrifugation time can be adjusted according to the platelet concentration.
The above-mentioned platelet dry powder is prepared by drying the platelet wet powder by any known drying method, for example, freeze-drying method using a machine such as a freeze-dryer, vacuum pumping method using a machine such as a rotary vacuum evaporator (Rotary Evaporator), and the like.
The above-mentioned platelet wet powder may be further purified, if necessary, to obtain a platelet dry powder of high purity, for example: adding water or water solution to form platelet solution or platelet dilution, and centrifuging again to remove residual peptide molecules in platelet wet powder. In this case, reverse osmosis water, physiological saline, or a mixture of reverse osmosis water and physiological saline is preferably added, and physiological saline is more preferably added.
The platelet wet powder can be directly frozen in situ (original centrifugal container) and/or subjected to a freeze-drying procedure; however, if necessary (e.g., the switching vessel is subjected to a freezing and/or freeze-drying procedure), water or an aqueous solution (e.g., physiological saline) may be added to form a platelet solution or platelet diluent, and manipulation (e.g., the switching vessel) is advantageously performed to employ the platelet diluent for the freezing and/or freeze-drying procedure. The platelet wet powder is preferably reverse osmosis water, physiological saline or a mixture of reverse osmosis water and physiological saline, more preferably reverse osmosis water. Wherein the platelet solution or platelet dilution can be optionally added with optional active ingredients having positive effects, such as antiinflammatory ingredient, analgesic ingredient, nutritional ingredient, and/or absorption enhancing ingredient; or components having substantially no adverse effects or side effects, such as pharmacologically acceptable perfumes, colorants, additives, etc.
To further illustrate the technology of the present invention, preferred embodiments are described below:
description of the embodiments
Comparative example 1:
pig whole blood was uniformly mixed with an anticoagulant ACD Solution-A (available from Xindong technology Co., ltd.) to avoid agglutination of the pig whole blood, and the whole blood was centrifuged at a low rotational speed with a high-speed refrigerated multipurpose centrifuge (Hettich, universal 320R) to precipitate most of the red blood cells in the lower layer of the centrifuge tube, and the platelet concentration of the blood slurry was measured as (platelet concentration: 374X 103/. Mu.L) by an automatic blood cell analyzer (Sysmex, hematology Analyzers XN-9000).
In a 50mL centrifuge tube, 40mL of blood slurry is added, and then 10mL of 0.03M calcium chloride (CaCl 2) aqueous solution is directly added into the centrifuge tube, and when platelets react with calcium ions, gel-like substances are formed, and the platelets cannot be centrifuged to obtain platelet wet powder.
Example 1
Pig whole blood and an anticoagulant ACD Solution-A (purchased from Xindong technology Co., ltd.) are uniformly mixed to avoid agglutination reaction of the pig whole blood, the whole blood is centrifuged at a low rotation speed by a high-speed freezing multipurpose centrifuge (Hettich, universal 320R), most of red blood cells are precipitated at the lower layer of the centrifuge tube, the upper layer of blood plasma liquid containing blood platelets is taken, and the blood plasma concentration is measured to be 374 g (103/. Mu.L) by an automatic blood cell analyzer (Sysmex, hematology Analyzers XN-9000).
10mL of 0.03M aqueous calcium chloride (CaCl 2) solution was added to 50mL of centrifuge tube A, and 500. Mu.L of plasma solution (platelet concentration: 374. Mu.L/103/. Mu.L) was added to centrifuge tube A, and the plasma solution and the aqueous calcium chloride solution were thoroughly mixed to form a ratio of 1:20, a mixed solution of 20; adding 40mL of plasma liquid (with the platelet concentration of 374 ANGSTROM 103/. Mu.L) into a 50mL centrifuge tube B, pouring 10mL of the mixed liquid in the centrifuge tube A into the centrifuge tube B, and fully mixing the mixed liquid in the centrifuge tube B with the plasma liquid to form a plasma mixed liquid with the total volume of 50mL, wherein the ratio of the mixed liquid to the plasma mixed liquid is 1:5.
centrifuge tube B in a high-speed refrigerated multipurpose centrifuge (Hettich, benchtop Centrifuges, universal 320R) for 15 min at 4000rpm to generate delamination, platelet wet powder at the bottom of centrifuge tube B, removing the upper layer solution, adding 10mL of physiological saline (purchased from Gingming Utility Co., ltd., isotonic Sodium Chloride Solution, sodium Chloride 0.9 gm) into centrifuge tube B, shaking centrifuge tube B with a tube shaker to dissolve platelet wet powder in physiological saline to form platelet solution, placing 10mL of platelet solution in a-80 ℃ ultralow temperature freezer (purchased from Yongmi Ulmi technology, thermo Scientific, low Temperature Freezers, forma 910), freezing for 24 h, and drying the platelet dry powder with a freeze dryer (purchased from Jinming Utility Co., ltd., mass production type freeze dryer FD30L-6S, capable of pre-cooling at-40 ℃ and temperature control range-40 ℃ to 65 ℃ under an environment of-40 ℃ C., 0.15 torr) to obtain dry platelet powder.
Adding 10mL of physiological saline into the platelet dry powder for rehydration, measuring the platelet concentration to be 1121, and calculating the recovery rate to be 80.9% (the calculation mode is 1121/374/4, divided by 4 is that the blood slurry is 40mL, and the concentration is 10mL after rehydration); TGF-. Beta.1 was developed by enzyme immunoassay (R & D System, quantikine ELISA, mouse/Rat/Porcine/Canine TGF-. Beta.1) and the titer of TGF-. Beta.1 was analyzed by a multifunctional microplate analyzer (Thermo Scientific, varioskan Flash Multimode Reader) and the recovery was 81.4% as compared to the titer 2892 of the primary serum, which was 9416. This result was quite consistent with the platelet count calculation (80.9%).
EXAMPLE 2 ultra-high8
Examples 2 to 8 the procedure for the manufacture of platelet dry powder was similar to example 1, but with different calcium chloride concentrations, see table 1. Examples 2 to 8 were subjected to platelet concentration test in the same manner as in example 1, and the results are shown in Table 2.
From the platelet recovery rates of examples 1to 8, it can be seen that: the calcium ion concentration is 0.00001M to 0.03M, preferably 0.00005M to 0.01M, more preferably 0.0001M to 0.005M, to achieve a proper platelet recovery rate.
Table 1: caCl of examples 1to 8 2 Concentration of
Table 2: platelet concentrations (10) of examples 1to 8 3 /μL)
Examples 9 to 14:
examples 9 to 14 the procedure for the preparation of platelet dry powder was similar to example 5, except that the volume ratio between the serum in the mixed solution and the aqueous solution of calcium chloride (CaCl 2) was different, see Table 3. The platelet concentration of the blood serum in examples 9 to 14 was 582. Mu.L. Platelet solutions prepared from platelet wet powder in examples 9 to 14 were placed in an ultra-low temperature freezer (available from Ulmus pumila technology, thermo Scientific, low Temperature Freezers, forma 910) at-80℃and frozen for 24 hours, and then freeze-dried with a freeze-dryer (available from Jinming Utility Co., ltd.) at-80℃for 72 hours in an environment of 0.1torr to obtain platelet dry powder. Examples 9 to 14 were conducted in the same manner as in example 5, and platelet dry powder rehydration was conducted to test platelet concentration test after platelet dry powder rehydration, and the results are shown in Table 4.
Table 3: serum and calcium chloride (CaCl) in the mixed solution of examples 9 to 14 2 ) Volume ratio between aqueous solutions
Table 4: platelet concentrations (10) of examples 9 to 14 3 /μL)
From the platelet recovery rates of examples 9 to 14, it was found that the mixing ratio of the blood slurry to the calcium ion aqueous solution was preferably 1: 2-1: 200, 1:10 to 1:100 is preferred.
Examples 15to 19:
examples 15to 19 the method of producing platelet dry powder was similar to example 5, except that the volume ratio between the mixed solution and the plasma mixed solution was different, see Table 5. The platelet concentration of the blood serum in examples 15to 19 was 633. Mu.L. The platelet solutions prepared from the platelet wet powder in examples 15to 19 were placed in an ultra-low temperature freezer (available from Ulmus pumila technology, thermo Scientific, low Temperature Freezers, forma 910) at-80℃and frozen for 24 hours, and then freeze-dried in a freeze-dryer (available from Jinming Utility Co., ltd., desktop freeze-dryer FD 3-12P-D-3S-80) at-80℃under 0.1torr for 72 hours to obtain a platelet dry powder. Examples 15to 19 were conducted in the same manner as in example 5, and platelet dry powder rehydration was conducted to test platelet concentration after platelet dry powder rehydration, and the results are shown in Table 6.
Table 5: ratio between the mixture of examples 15to 19 and the plasma mixture
Table 6: platelet concentrations (10) of examples 15to 19 3 /μL)
| Examples | 15 | 16 | 17 | 18 | 19 |
| Platelet concentration (10) 3 /μL) | 2126 | 2133 | 2206 | 2197 | 2145 |
| Recovery (%) | 83.9 | 84.2 | 87.1 | 86.7 | 84.7 |
From the platelet recovery rates of examples 15to 19, it was found that: the ratio of the mixture to the plasma mixture is preferably 1: 1to 1:50, 1: 5to 1:10 is preferred.
Examples 20 to 24:
examples 20 to 24 were similar to example 5, except that the platelet concentration of the blood slurry was different, which was a plasma solution having a platelet concentration of 1011X103/. Mu.L (example 24) and a plasma solution diluted to 1/5 (example 20), 2/5 (example 21), 3/5 (example 22), 4/5 (example 23), see Table 7. Examples 20 to 24 were conducted in the same manner as in example 5, and platelet dry powder rehydration was conducted to test platelet concentration after platelet dry powder rehydration, and the results are shown in Table 8.
Table 7: examples 20 to 24 platelet concentration ratio of blood serum
| Examples | 20 | 21 | 22 | 23 | 24 |
| Platelet concentration (10) 3 /μL) | 202 | 404 | 606 | 809 | 1011 |
Table 8: platelet concentrations (10) of examples 20 to 24 3 /μL)
| Examples | 20 | 21 | 22 | 23 | 24 |
| Platelet concentration (10) 3 /μL) | 694 | 1425 | 2060 | 2618 | 3316 |
| Recovery (%) | 85.9 | 88.2 | 85 | 80.9 | 82 |
Claims (10)
1. A method of manufacturing a platelet dry powder, comprising:
obtaining a platelet-containing plasma fluid, wherein the plasma fluid has substantially been depleted of red blood cells;
preparing 0.00001-0.03M calcium ion compound aqueous solution;
a small amount of this serum was taken at 1: 2-1: 200, adding the mixture into the aqueous solution of the calcium ion compound, and uniformly mixing to form a mixed solution;
1, the method comprises the following steps: 1to 1:50, adding the mixed solution into the plasma solution, and uniformly mixing to form mixed blood slurry;
centrifugally separating the mixed blood serum to obtain platelet wet powder; a kind of electronic device with high-pressure air-conditioning system
Drying the platelet wet powder to obtain platelet dry powder.
2. The method of claim 1, wherein the concentration of the aqueous solution of the calcium compound is 0.00005 to 0.01M.
3. The method according to claim 2, wherein the concentration of the aqueous solution of the calcium ion compound is 0.0001 to 0.005M.
4. The method of claim 1, wherein the ratio of the plasma solution to the aqueous solution of the calcium compound in the mixed solution is 1:10 to 1:100.
5. the method of claim 1, wherein the ratio of the mixed liquor in the mixed serum to the mixed serum is 1: 5to 1:10.
6. the method of claim 1, wherein the step of drying the platelet wet powder is by vacuum suction.
7. The method of claim 6, wherein the step of drying the platelet wet powder is by low temperature vacuum pumping.
8. The method of claim 1, wherein the aqueous solution of a calcium compound is an aqueous solution of calcium chloride or calcium nitrate.
9. The method of claim 8, wherein the aqueous solution of a calcium-ion compound is an aqueous solution of calcium chloride.
10. The method of claim 1, wherein the step of obtaining platelet containing blood slurry is by using a centrifuge.
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