CN104771412A - Novel platelet freeze-drying treating fluid - Google Patents
Novel platelet freeze-drying treating fluid Download PDFInfo
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Abstract
The invention discloses a novel platelet freeze-drying treating fluid. The fluid is composed of two parts: (1) a platelet freeze-drying pretreatment liquid, which is prepared by adding mycose and PGE1 into a basic buffer liquid; (2) a platelet freeze-drying buffer liquid, which is prepared by adding blood plasma into a freeze-drying pretreatment liquid. Preferably 30%v/v-40%v/v blood plasma is adopted to replace human serum albumin, a good effect is achieved, and the blood plasma is taken as the platelet protein protective agent to replace the pricy albumin and is economic. The osmotic pressure of the pretreatment liquid is 311.6 mOsm, is similar to the blood plasma osmotic pressure, and thus will not generate adverse effect on platelet. The weight concentration of the freeze-drying buffer liquid is 29%w/v, and the obtained freeze-dried platelet has a qualified appearance. The freeze-dried platelet can be stably preserved at a room temperature for at least 6 months, and the platelet can be used to prepare hemostatic or platelet products used in operation.
Description
Technical field
The present invention relates to platelet lyophilization and preserve field, be specifically related to a kind of platelet frozen dried liquid.
Background technology
Hematoblastic lyophilization research starts from the 1950's, but result of study not success as people's expection at that time.Until calendar year 2001, Wolkers WF etc. have carried out to platelet the research that lyophilizing preserves again, use trehalose to freeze pre-treatment to platelet, successful lyophilizing platelet first.The trial of forefathers to platelet lyophlization imply that the probability of platelet lyophlization, lyophilizing is preserved platelet and has been become study hotspot, domestic Yi Youduojia unit has carried out lyophilizing research to platelet, but the lyophilizing formula reported at present comes with some shortcomings, if protective agent protein-based in formula is with human serum albumin, expensive, and not easily obtain; Also useful dimethyl sulfoxide (dimethyl sulfoxide, DMSO) is pretreated, and it has certain toxicity, and treatment step is loaded down with trivial details etc.At present still that generally acknowledge, the reliable and stable and platelet lyophlization method of economy of neither one.
Be further noted that platelet lyophilizing buffer osmotic pressure and concentration problems, the research of current lyophilized platelet all seldom mentions this point.Lyophilizing buffer osmotic pressure is too high or too low all unfavorable to platelet.Lyophilizing buffer concentration is the key factor determining lyophilizing success or failure and freeze-drying prods quality.Freeze-dry process requires that lyophilizing buffer weight concentration (w/v) is advisable between 4% ~ 25%, and optium concentration is 10% ~ 15%.If buffer concentration is too high, time dry, medicinal liquid surface is first dry, and form the top layer of compact structure, this top layer hole is tiny, poor air permeability, and the aqueous vapor is therefore difficult to through drying layer, thus rises along bottle wall, causes goods from wall, atrophy; Or making aqueous vapor at drying layer overstand, the dry top layer goods moisture absorption again, causes goods to subside, volume-diminished; In addition, when concentration is too high, medicinal liquid easily lumps in dry run, and resulting product outward appearance is uneven; When buffer concentration is too low, resulting product structure comparison is loose, mechanical strength is low, is subject to vibrating easy atrophy or powdering, affects the outward appearance of goods, solute even can be caused to fly away with steam in packaging and transportation, causes lyophilizing failure.
Summary of the invention
The object of the present invention is to provide a kind of platelet frozen dried liquid;
Another object of the present invention is to provide a kind of platelet frozen dried method.
The technical solution used in the present invention is:
A kind of platelet frozen dried liquid, is made up of following two parts:
1) platelet lyophilizing pretreatment fluid: add 40 ~ 60mmol/L trehalose and reversibility platelet aggregation inhibitors by basis buffer, regulates pH to be 6.6 ~ 6.8, prepares after filtration;
2) platelet lyophilizing buffer: add 40 ~ 60mmol/L trehalose, reversibility platelet aggregation inhibitors and protein-based protective agent by basis buffer, adjusts pH to be 6.6 ~ 6.8, prepares after filtration.
As the further improvement of above-mentioned platelet frozen dried liquid, the constituent of basis buffer is: 55mmol/LNaCl, 2.1mmol/L KCl, 0.7mmol/L CaCl
2, 1.1mmol/L MgSO
4, 31mmol/L NaHCO
3, 3.4mmol/L citric acid, 6.7mmol/L sodium citrate, 15mmol/L sodium acetate, 10.7mmol/L glucose.
As the further improvement of above-mentioned platelet frozen dried liquid, the concentration of trehalose is 50mmol/L.
As the further improvement of above-mentioned platelet frozen dried liquid, reversibility platelet aggregation inhibitors is 0.8 ~ 1.2ug/mL PGE1 or 5mmol/L L-Arg.
As the further improvement of above-mentioned platelet frozen dried liquid, reversibility platelet aggregation inhibitors is 1ug/mLPGE1.
As the further improvement of above-mentioned platelet frozen dried liquid, protein-based protective agent is the blood plasma of BSA or the 30%v/v ~ 40%v/v of 2%v/v.
As the further improvement of above-mentioned platelet frozen dried liquid, protein-based protective agent is the blood plasma of 30%v/v.
A kind of platelet frozen dried liquid, is made up of following two parts:
(1) platelet lyophilizing pretreatment fluid: comprise 55mmol/L NaCl, 2.1mmol/L KCl, 0.7mmol/L CaCl
2, 1.1mmol/L MgSO
4, 31mmol/L NaHCO
3, 3.4mmol/L citric acid, 6.7mmol/L sodium citrate, 15mmol/L sodium acetate, 10.7mmol/L glucose, 50mmol/L trehalose, 1ug/mL PGE1; Regulate pH to be 6.6 ~ 6.8, prepare after filtration;
(2) platelet lyophilizing buffer: comprise 55mmol/L NaCl, 2.1mmol/L KCl, 0.7mmol/L CaCl
2, 1.1mmol/L MgSO
4, 31mmol/L NaHCO
3, 3.4mmol/L citric acid, 6.7mmol/L sodium citrate, 15mmol/L sodium acetate, 10.7mmol/L glucose, 50mmol/L trehalose, 1ug/mL PGE1,30% blood plasma; Regulate pH to be 6.6 ~ 6.8, prepare after filtration.
A kind of platelet frozen dried method, comprises the following steps:
1) get platelet sample, centrifugal supernatant of abandoning stays precipitation, and by the resuspended platelet of platelet lyophilizing pretreatment fluid with supernatant discarded same volume, 37 DEG C of shaking bath 3h ~ 6h, make platelet keep suspended state;
2) after water-bath, platelet suspension is centrifugal again, abandon supernatant and stay precipitation, by the resuspended platelet of platelet lyophilizing buffer with supernatant discarded same volume;
3) pretreated platelet suspension is transferred in lyophilizing bottle, spend the night in-80 DEG C of ultralow temperature pre-freezes, the freeze dryer proceeding to pre-cooling next day carries out vacuum drying, condenser temperature is-45 ± 5 DEG C, vacuum is < 133mbar, take out after vacuum drying 24h, sealed under being placed on room temperature and preserve;
Wherein said platelet lyophilizing pretreatment fluid and platelet lyophilizing buffer described above.
The invention has the beneficial effects as follows:
The invention discloses a kind of platelet frozen dried liquid and frozen dried method, namely 50mmol/L trehalose and 1 μ g/ml PGE1 is added to water-bath trehalose before platelet lyophilizing with basis buffer, add lyophilizing after 50mmol/L trehalose, 1 μ g/ml PGE1 and 30%v/v ~ resuspended platelet of 40%v/v blood plasma with basis buffer again, prepare the lyophilized platelet at room temperature can stablized and preserve.
The present invention adopts PGE1 to be reversibility platelet aggregation inhibitors, and PGE1 can effective anticoagulant, reduces thromboxane A2 level, also can suppress platelet activation, promote that the platelet activated around thrombosis reverses; Find that PGE1 is good to recovery rate of blood platelet effect after lyophilizing by research.
Albumin is a kind of conventional protein-based freeze drying protectant, due to human serum albumin costly, and be difficult to, the BSA of the preferred 2%v/v of the present invention replaces human serum albumin to prepare lyophilized platelet, can meet the user demand of various outside.If the use in operation transfusion, consider that BSA is bovine serum albumin, use in human body and may there is rejection or anaphylactoid risk, the preferred 30%v/v ~ 40%v/v blood plasma of the present invention replaces human serum albumin, and achieves good effect.Blood plasma is very common and more cheap raw material, and containing protein such as albumin, replaces expensive albumin as blood platelet albumen matter class protective agent with it, economical and practical.
Platelet frozen dried liquid of the present invention comprises lyophilizing pretreatment fluid and lyophilizing buffer, and its osmotic pressure is 311.6mOsm, close with plasma osmotic pressure (290 ~ 310mOsm), can not produce harmful effect to platelet.In addition lyophilizing buffer weight concentration (w/v) is about 29%; The lyophilized platelet outward appearance obtained still can.
0d ~ 180d is preserved under lyophilized platelet of the present invention being placed in respectively-20 DEG C, 4 DEG C and room temperature, when finding that the holding time is identical, there are no significant for somatomedin PDGF-BB, TGF-β and the VEGF difference discharged after recovery rate of blood platelet, MPV, the relative aggregation rate of platelet and platelet activation after lyophilized platelet rehydration, and namely preservation effect is suitable at three temperatures for lyophilized platelet.
Lyophilized platelet of the present invention at room temperature can be stablized and preserves at least 6 months, prepares platelet hemorrhage and the platelet product that uses in operation is provided convenience and may for next step.
Accompanying drawing explanation
Fig. 1 is platelet PLA2 (40000 ×) under transmission electron microscope;
Wherein: a is lyophilizing thromboblast; B is the lyophilized platelet of rehydration.
Detailed description of the invention
English initialism is as follows:
PGE1 is PGE1, and L-Arg is L-arginine, and BSA is bovine serum albumin, ADP is adenosine diphosphate (ADP), and PPP is platelet poor plasma, and PDGF-BB is PDGF-BB, VEGF is VEGF, and TGF-β is transforming growth factor β.
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited thereto.
1. main agents
Trehalose, EGTA, imidazoles (Shanghai Mei Ji Bioisystech Co., Ltd); PGE1, L-Arg, BSA, HEPES (Sigma Co., USA); ADP (Chrono-log company of the U.S.); NaCl, KCl, CaCl
2, MgSO
4, NaHCO
3, citric acid, sodium citrate, sodium acetate, glucose, sucrose (Guangzhou Chemical Reagent Factory).
2. key instrument equipment
Large capacity refrigerated centrifuge (Thermo company of the U.S.), full-automatic blood component separator (Japanese Terumo company), fully automatic blood cytoanalyze (Shenzhen MINDRAY), pH meter (German sartorius company), the dual-purpose vibration case of water-bath (Shanghai Yue Feng company), ultra cold storage freezer (Japanese Sanyo company), freeze dryer (Labconco company of the U.S.), platelet aggregation analyser (Chrono-log company of the U.S.).
3. platelet source
From the healthy Voluntary Blood Donors donated blood at the military region, Guangzhou Blood Center year February in October, 2011 ~ 2012, to donate blood first 3 days non-Aspirins or aspirin-like drugs, whole blood (400ml, 36 person-portions) is gathered with ACD 5-linked bag, all qualified by China's blood testing standard detection.In 4 ~ 6h, adopt tunica albuginea legal system for Platelet Concentrate after collecting whole blood.36 parts of every 6 bags of homotypes of platelet are collected, and adjust platelet count (platelet, PLT) to be 1000 × 10
9/ L, room temperature leaves standstill 1h, makes its depolymerization, obtains 6 parts of Pooled platelets.
4. the configuration of solution
1) configure base buffer: accurately take following component, comprises NaCl 3.214g, KCl 0.157g, CaCl
20.078g, MgSO
40.271g, NaHCO
32.604g, citric acid 0.714g, sodium citrate 1.970g, sodium acetate 1.230g, glucose 2.120g, ultra-pure water standardize solution is to 1L;
2) PGE1 stores the preparation of liquid: take PGE1:1mg, add normal saline 1ml;
3) preparation of ADP solution: take ADP:2.5mg, adds normal saline 5ml.
5. platelet suspension lyophilizing
After pretreatment following examples prepared, platelet suspension is transferred in lyophilizing bottle (siliconized glass bottles), every bottle of 4ml (thickness is no more than 1.5cm), be placed in-80 DEG C of ultra cold storage freezer pre-freezes to spend the night, first freeze dryer is opened next day after being down to-45 DEG C, again sample is taken out immigration freeze dryer from refrigerator and carry out vacuum drying, condenser temperature is-45 ± 5 DEG C, vacuum is < 133mbar, take out after vacuum drying 24h, sealed under being placed on room temperature and preserve.
6. detect after lyophilized platelet rehydration
Sample at room temperature preserves 1d again by its rehydration, and multiple hydrating fluid is PPP: sterilized water=3:1 (v/v), and each sample vibrates gently to dissolving completely after adding the multiple hydrating fluid of 4ml.
A) difference before and after recovery rate of blood platelet and MPV lyophilizing
To measure respectively before lyophilizing with blood-counter system and PLT and MPV after lyophilized platelet rehydration, and calculate difference before and after recovery rate of blood platelet and MPV lyophilizing.
B) platelet aggregation detects
With the centrifugal 15min of 1500 × g after lyophilized platelet rehydration, remove supernatant, more resuspended with PPP after do platelet aggregation detect.Regulate testing sample PLT for 200 ~ 300 × 10 with PPP
9/ L, with ADP for causing poly-agent induced platelet aggregation, upper platelet aggregation instrument detects, add bar magnet in reaction cup, get 500ul testing sample in reaction cup, then add 5ul ADP, final concentration is 10umol/L, with 500ul PPP for contrast, detects maximum platelet aggregation rate.
C) ultrastructure under platelet transmission electron microscope
Get 1ml platelet sample, with the centrifugal 15min of 1500 × g, remove supernatant, add before at 3% glutaraldehyde 4 DEG C and fix 2 ~ 4h, PBS washs 2 times, fixes 1.5 ~ 2h after 1% osmic acid, PBS washs 2 times, the ethanol of variable concentrations dewaters step by step, and displacement, soaks into, semithin section is made after epoxy resin embedding, make ultrathin section, electron staining after HE dyeing, after drying, under transmission electron microscope, observe hematoblastic ultrastructure.
D) detection of somatomedin PDGF-BB, VEGF, TGF-β burst size after platelet activation
Platelet sample mixes in 10:1 ratio with calcium gluconate, and after leaving standstill 30min, by it with the centrifugal 5min of 5000 × g, get supernatant, carry out detection by quantitative by ELISA kit, experimental implementation is carried out in strict accordance with test kit description.
Embodiment 1
1) configure platelet lyophilizing pretreatment fluid: basis buffer adds 50mmol/L trehalose and 1ug/mL PGE1, adjust pH to be for subsequent use after 6.6 ~ 6.8,0.22um frit.
2) configure platelet lyophilizing buffer: basis buffer adds 50mmol/L trehalose, 1ug/mL PGE1 and 30%v/v blood plasma, adjust pH to be for subsequent use after 6.6 ~ 6.8,0.22um frit.
3) platelet lyophilizing pretreatment:
1 part of Pooled platelets, the centrifugal 15min of 1500 × g, abandons supernatant and stays precipitation, and by the resuspended platelet of platelet lyophilizing pretreatment fluid with supernatant discarded same volume, 37 DEG C of shaking bath 4h, make platelet keep suspended state.After water-bath, by platelet suspension again with the centrifugal 15min of 1500 × g, abandon supernatant and stay precipitation, by the resuspended platelet of platelet lyophilizing buffer with supernatant discarded same volume.
4) detect after frozen dried and lyophilized platelet rehydration being carried out to platelet suspension.
5) interpretation of result: after rehydration, recovery rate of blood platelet is (91.0 ± 4.0) %; Before and after MPV lyophilizing, difference is (2.0 ± 0.3) fl; After rehydration, the relative aggregation rate of platelet is (80.8 ± 6.3) %; Under transmission electron microscope, Ultrastructure of Platelets as shown in Figure 1: compared with lyophilizing thromboblast, and the lyophilized platelet of rehydration has slight swelling, but membrane structure is substantially complete, and born of the same parents' endoparticle structure still retains, and similar is in lyophilizing thromboblast.
Through calculating, platelet frozen dried liquid of the present invention comprises lyophilizing pretreatment fluid and lyophilizing buffer, and its osmotic pressure is 311.6mOsm, close with plasma osmotic pressure (290 ~ 310mOsm), can not produce harmful effect to platelet.In addition lyophilizing buffer concentration (w/v) is about 29%; Slightly higher than lyophilizing buffer optimum concentration range (w/v) 4% ~ 25%, but the lyophilized platelet outward appearance obtained as can be seen from Figure 1 still can.It can thus be appreciated that the present invention's preferred platelet frozen dried formula of liquid is reasonable.
Embodiment 2
1) configure platelet lyophilizing pretreatment fluid: basis buffer adds 50mmol/L trehalose and 1ug/mL PGE1, adjust pH to be for subsequent use after 6.6 ~ 6.8,0.22um frit.
2) configure platelet lyophilizing buffer: basis buffer adds 50mmol/L trehalose, 1ug/mL PGE1 and 40%v/v blood plasma, adjust pH to be for subsequent use after 6.6 ~ 6.8,0.22um frit.
3) platelet lyophilizing pretreatment:
1 part of Pooled platelets, the centrifugal 15min of 1500 × g, abandons supernatant and stays precipitation, and by the resuspended platelet of platelet lyophilizing pretreatment fluid with supernatant discarded same volume, 37 DEG C of shaking bath 4h, make platelet keep suspended state.After water-bath, by platelet suspension again with the centrifugal 15min of 1500 × g, abandon supernatant and stay precipitation, by the resuspended platelet of platelet lyophilizing buffer with supernatant discarded same volume.
4) detect after frozen dried and lyophilized platelet rehydration being carried out to platelet suspension.
5) interpretation of result: after rehydration, recovery rate of blood platelet is (87.7 ± 2.4) %; Before and after MPV lyophilizing, difference is (1.9 ± 0.4) fl; After rehydration, the relative aggregation rate of platelet is (81.1 ± 9.6) %.
Comparative example 1
1) configure platelet lyophilizing pretreatment fluid: basis buffer adds 40mmol/L trehalose and 1ug/mL PGE1 and 5mmol/LL-Arg, adjust pH to be for subsequent use after 6.6 ~ 6.8,0.22um frit.
2) configure platelet lyophilizing buffer: basis buffer adds the BSA of 50mmol/L trehalose, 1ug/mL PGE1,5mmol/L L-Arg and 2%v/v, adjust pH to be for subsequent use after 6.6 ~ 6.8,0.22um frit.
3) platelet lyophilizing pretreatment:
1 part of Pooled platelets, the centrifugal 15min of 1500 × g, abandons supernatant and stays precipitation, and by the resuspended platelet of platelet lyophilizing pretreatment fluid with supernatant discarded same volume, 37 DEG C of shaking bath 4h, make platelet keep suspended state.After water-bath, by platelet suspension again with the centrifugal 15min of 1500 × g, abandon supernatant and stay precipitation, by the resuspended platelet of lyophilizing buffer with supernatant discarded same volume.
4) detect after frozen dried and lyophilized platelet rehydration being carried out to platelet suspension.
5) interpretation of result: after rehydration, recovery rate of blood platelet is (75.4 ± 3.7) %, with the result difference extremely remarkable (P < 0.01) of embodiment 1; Before and after MPV lyophilizing, difference is (2.4 ± 0.4) fl; After rehydration, the relative aggregation rate of platelet is (29.0 ± 4.2) %, with the result difference extremely remarkable (P < 0.01) of embodiment 1.
Comparative example 2
1) configure platelet lyophilizing pretreatment fluid: 35mmol/L trehalose, 100mmol/L NaCl, 10mmol/L KCl, 10mmol/L EGTA, 10mmol/L imidazoles, 10ug/mL PGE1, regulate pH to be for subsequent use after 6.8,0.22um frit.
2) platelet lyophilizing buffer is configured: 9.5mmol/L HEPES, 142.5mmol/L NaCl, 4.8mmol/L KCl, 1mmol/L MgCl
2, 30mmol/L trehalose, the BSA of 1%v/v, regulates pH to be for subsequent use after 6.8,0.22um frit.
3) platelet lyophilizing pretreatment:
1 part of Pooled platelets, the centrifugal 15min of 1500 × g, abandons supernatant and stays precipitation, and by the resuspended platelet of platelet lyophilizing pretreatment fluid with supernatant discarded same volume, 37 DEG C of shaking bath 4h, make platelet keep suspended state.After water-bath, by platelet suspension again with the centrifugal 15min of 1500 × g, abandon supernatant and stay precipitation, by the resuspended platelet of lyophilizing buffer with supernatant discarded same volume.
4) detect after rehydration after frozen dried and lyophilizing being carried out to platelet suspension.
5) interpretation of result: after rehydration, recovery rate of blood platelet is (69.7 ± 1.9) %, with the result difference extremely remarkable (P < 0.01) of embodiment 1; Before and after MPV lyophilizing, difference is (3.6 ± 0.6) fl, with the result difference extremely remarkable (P < 0.01) of embodiment 1.
The stability study experiment that lyophilized platelet is preserved
Preserve 0d, 10d, 30d, 90d and 180d recurrence of disease at the same time next year aquation under being divided by the lyophilized platelet prepared in embodiment 1 three groups to be placed in-20 DEG C, 4 DEG C and room temperature (25 DEG C) to detect.
1) recovery rate of blood platelet
Result is as shown in table 1:
The different storage temperature of table 1 is on the impact of the lyophilized platelet response rate
As shown in Table 1: the fluctuation of-20 DEG C of group recovery rate of blood platelet is between (90.2 ± 3.3) % to (92.5 ± 3.3) %; The fluctuation of 4 DEG C of group recovery rate of blood platelet is between (89.5 ± 2.1) % to (92.5 ± 2.6) %; The fluctuation of room temperature (25 DEG C) group recovery rate of blood platelet is between (90.8 ± 4.5) % to (92.7 ± 4.2) %; The results of analysis of variance shows: in storage life 180d, As time goes on, there was no significant difference (P > 0.05) between recovery rate of blood platelet after the lyophilized platelet rehydration of preserving under three kinds of different storage temperatures; When preserving the identical time, three kinds of different storage temperatures affect there was no significant difference (P > 0.05) to recovery rate of blood platelet.
2) platelet MPV
Result is as shown in table 2:
The different storage temperature of table 2 is on the impact of lyophilized platelet MPV
(note: * is P < 0.05, compared with lyophilizing thromboblast MPV)
As shown in Table 2 :-20 DEG C of group platelet MPV fluctuation is between (8.1 ± 0.3) fl to (9.6 ± 0.7) fl; 4 DEG C of group platelet MPV fluctuation is between (8.2 ± 0.4) fl to (9.7 ± 0.5) fl; Room temperature (25 DEG C) group platelet MPV fluctuation is between (7.9 ± 0.5) fl to (9.6 ± 0.5) fl.The results of analysis of variance shows: in storage life 180d, As time goes on, platelet MPV there was no significant difference (P > 0.05) after the lyophilized platelet rehydration of preserving under three kinds of different storage temperatures; Preserve the identical time, MPV there was no significant difference (P > 0.05) between three groups of different storage temperature, but all have significant difference (P < 0.05) with lyophilizing thromboblast MPV.
3) the relative aggregation rate of platelet
Result is as shown in table 3:
The different storage temperature of table 3 is on the impact of the relative aggregation rate of lyophilized platelet
As shown in Table 3 :-20 DEG C of relative aggregation rate fluctuations of group platelet are between (76.9 ± 5.0) % to (83.6 ± 7.7) %; 4 DEG C of relative aggregation rate fluctuations of group platelet are between (77.6 ± 5.3) % to (80.3 ± 8.2) %; The relative aggregation rate fluctuation of room temperature (25 DEG C) group platelet is between (76.7 ± 9.0) % to (81.3 ± 1.9) %.The results of analysis of variance shows: in storage life 180d, As time goes on, there was no significant difference (P > 0.05) between the relative aggregation rate of platelet after the lyophilized platelet rehydration of preserving under three kinds of different storage temperatures; Preserve the identical time, the relative aggregation rate there was no significant difference of platelet (P > 0.05) between three groups of different storage temperature.
4) PDGF-BB content in supernatant after platelet activation
Result is as shown in table 4:
The different storage temperature of table 4 is on the impact of lyophilized platelet PDGF-BB content
(note: * is P < 0.05, compared with lyophilizing thromboblast)
As shown in Table 4:
After-20 DEG C of group platelet activations, in supernatant, PDGF-BB content fluctuates between (5972.56 ± 649.21) pg/ml to (6624.96 ± 522.08) pg/ml;
The fluctuation of 4 DEG C of group PDGF-BB content is between (5906.36 ± 662.69) pg/ml to (6233.42 ± 672.56) pg/ml;
The fluctuation of room temperature (25 DEG C) group PDGF-BB content is between (6051.23 ± 288.01) pg/ml to (6612.33 ± 514.21) pg/ml.
The results of analysis of variance shows: in storage life 180d, three groups of PDGF-BB content and lyophilizing thromboblast PDGF-BB content there was no significant difference (P > 0.05) during preservation 0d, but when preserving 10d, 30d, 90d, 180d, three groups of PDGF-BB content and lyophilizing thromboblast PDGF-BB content have significant difference (P < 0.05); Preserve the identical time, PDGF-BB content there was no significant difference (P > 0.05) in supernatant after platelet activation between three groups of different storage temperature.
5) TGF-β content in supernatant after platelet activation
Result is as shown in table 5:
The different storage temperature of table 5 is on the impact of lyophilized platelet TGF-β content
As shown in Table 5:
After-20 DEG C of group platelet activations, in supernatant, TGF-β content fluctuates between (102.13 ± 23.35) pg/ml to (112.21 ± 27.35) pg/ml;
The fluctuation of 4 DEG C of group TGF-β content is between (108.14 ± 16.50) pg/ml to (118.03 ± 36.29) pg/ml;
The fluctuation of room temperature (25 DEG C) group TGF-β content is between (109.76 ± 19.77) pg/ml to (115.67 ± 27.87) pg/ml.
The results of analysis of variance shows: in storage life 180d, As time goes on, there was no significant difference (P > 0.05) between the TGF-β activating release after the lyophilized platelet rehydration of preserving under three kinds of different storage temperatures; Preserve the identical time, TGF-β content there was no significant difference (P > 0.05) in supernatant after platelet activation between three groups of different storage temperature, the TGF-β discharged with lyophilizing thromboblast is there was no significant difference (P > 0.05) also.
6) VEGF content in supernatant after platelet activation
Result is as shown in table 6:
The different storage temperature of table 6 is on the impact of lyophilized platelet VEGF content
As shown in Table 6:
After-20 DEG C of group platelet activations, in supernatant, VEGF content fluctuates between (98.31 ± 20.12) pg/ml to (117.08 ± 19.62) pg/ml;
The fluctuation of 4 DEG C of group VEGF content is between (99.86 ± 22.83) pg/ml to (117.63 ± 34.00) pg/ml;
The fluctuation of room temperature (25 DEG C) group VEGF content is between (104.26 ± 20.01) pg/ml to (109.64 ± 17.14) pg/ml.
The results of analysis of variance shows: in storage life 180d, As time goes on, there was no significant difference (P > 0.05) between the VEGF activating release after the lyophilized platelet rehydration of preserving under three kinds of different storage temperatures; Preserve the identical time, VEGF content there was no significant difference (P > 0.05) in supernatant after platelet activation between three groups of different storage temperature, the VEGF discharged with lyophilizing thromboblast is there was no significant difference (P > 0.05) also.
Sum up: from the above results, 0d ~ 180d is preserved under lyophilized platelet of the present invention being placed in respectively-20 DEG C, 4 DEG C and room temperature, when finding that the holding time is identical, there are no significant for somatomedin PDGF-BB, TGF-β and the VEGF difference discharged after recovery rate of blood platelet, MPV, the relative aggregation rate of platelet and platelet activation after three groups of lyophilized platelet rehydrations, and namely preservation effect is suitable at three temperatures for lyophilized platelet.
Recovery rate of blood platelet, as the monitoring to platelet counts, can reflect the during preservation quantitative situation of change of lyophilized platelet; MPV reflects mean platelet volume change, and the two combines, and objectively can reflect hematoblastic quantity and metamorphosis situation in preservation process.All more than 89.5% is maintained in recovery rate of blood platelet 180d in the present invention.Lyophilized platelet MPV comparatively all increases before platelet lyophilizing, and consideration may be that load environment causes, and also may be the impact that multiple hydration method causes it.
Platelet hemostasis is sent out and assemble (in a large number) by platelet adhesion, the gathering of platelet onset, intra platelet free calcium, platelet time, and then formed platelet plug, and a series of process composition such as hemostasis and thrombosis occurs.Platelet aggregation is the adhesion between platelet and platelet, is the basis forming platelet thrombus, is also that platelet activates and participates in second phase hemostasis further, promotes the guarantee of blood coagulation.The present invention adopts ADP to carry out induced platelet aggregation, and with lyophilizing thromboblast for contrast, the lyophilized platelet preserved under finding three kinds of storage temperatures is at the relative aggregation rate zero difference of experimental session platelet.Between storage life, the stability of platelet aggregation is the prerequisite ensureing platelet hemostatic function stable existence.
Somatomedin is the basis that platelet promotes repair in trauma, after platelet is activated, can discharges multiple somatomedin, comprise PDGF, TGF-β, VEGF, EGF, IGF etc.The present invention's ELISA detection method has carried out detection by quantitative to wherein three kinds of somatomedin PDGF, TGF-β and VEGF, finds that three kinds of different storage temperatures affect zero difference to these three kinds of somatomedin.The PDGF-BB of lyophilized platelet release comparatively slightly reduces before lyophilizing after preservation 10d, but zero difference before and after TGF-β and the lyophilizing of VEGF content.
Lyophilized platelet of the present invention at room temperature can stablize preservation at least 6 months.Lyophilized platelet at room temperature steady in a long-termly can save as next step and prepares platelet hemorrhage and the platelet product that uses in operation is provided convenience and may.
Claims (9)
1. a platelet frozen dried liquid, is made up of following two parts:
Platelet lyophilizing pretreatment fluid: add 40 ~ 60 mmol/L trehaloses and reversibility platelet aggregation inhibitors by basis buffer, regulates pH to be 6.6 ~ 6.8, prepares after filtration;
Platelet lyophilizing buffer: add 40 ~ 60 mmol/L trehaloses, reversibility platelet aggregation inhibitors and protein-based protective agent by basis buffer, adjusts pH to be 6.6 ~ 6.8, prepares after filtration.
2. a kind of platelet frozen dried liquid according to claim 1, is characterized in that: the constituent of basis buffer is: 55mmol/L NaCl, 2.1mmol/L KCl, 0.7mmol/L CaCl
2, 1.1mmol/L MgSO
4, 31mmol/L NaHCO
3, 3.4mmol/L citric acid, 6.7mmol/L sodium citrate, 15mmol/L sodium acetate, 10.7mmol/L glucose.
3. a kind of platelet frozen dried liquid according to claim 1, is characterized in that: the concentration of trehalose is 50 mmol/L.
4. a kind of platelet frozen dried liquid according to claim 1, is characterized in that: reversibility platelet aggregation inhibitors is 0.8 ~ 1.2 ug/mL PGE1 or 5mmol/L L-Arg.
5. a kind of platelet frozen dried liquid according to claim 1 or 4, is characterized in that: reversibility platelet aggregation inhibitors is 1 ug/mL PGE1.
6. a kind of platelet frozen dried liquid according to claim 1, is characterized in that: protein-based protective agent is the blood plasma of BSA or the 30%v/v ~ 40%v/v of 2%v/v.
7. a kind of platelet frozen dried liquid according to claim 1 or 6, is characterized in that: protein-based protective agent is the blood plasma of 30%v/v.
8. a platelet frozen dried liquid, is made up of following two parts:
1) platelet lyophilizing pretreatment fluid: comprise 55mmol/L NaCl, 2.1mmol/L KCl, 0.7mmol/L CaCl
2, 1.1mmol/L MgSO
4, 31mmol/L NaHCO
3, 3.4mmol/L citric acid, 6.7mmol/L sodium citrate, 15mmol/L sodium acetate, 10.7mmol/L glucose, 50 mmol/L trehaloses, 1 ug/mL PGE1; Regulate pH to be 6.6 ~ 6.8, prepare after filtration;
2) platelet lyophilizing buffer: comprise 55mmol/L NaCl, 2.1mmol/L KCl, 0.7mmol/L CaCl
2, 1.1mmol/L MgSO
4, 31mmol/L NaHCO
3, 3.4mmol/L citric acid, 6.7mmol/L sodium citrate, 15mmol/L sodium acetate, 10.7mmol/L glucose, 50 mmol/L trehaloses, 1 ug/mL PGE1,30% blood plasma; Regulate pH to be 6.6 ~ 6.8, prepare after filtration.
9. a platelet frozen dried method, is characterized in that: comprise the following steps:
1) get platelet sample, centrifugal supernatant of abandoning stays precipitation, and by the resuspended platelet of platelet lyophilizing pretreatment fluid with supernatant discarded same volume, 37 DEG C of shaking bath 3h ~ 6h, make platelet keep suspended state;
2) after water-bath, platelet suspension is centrifugal again, abandon supernatant and stay precipitation, by the resuspended platelet of platelet lyophilizing buffer with supernatant discarded same volume;
3) pretreated platelet suspension is transferred in lyophilizing bottle, spend the night in-80 DEG C of ultralow temperature pre-freezes, the freeze dryer proceeding to pre-cooling next day carries out vacuum drying, condenser temperature is-45 ± 5 DEG C, vacuum is < 133mbar, take out after vacuum drying 24h, sealed under being placed on room temperature and preserve;
Wherein said platelet lyophilizing pretreatment fluid and platelet lyophilizing buffer are as described in any one of claim 1 ~ 8.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106729955A (en) * | 2016-11-28 | 2017-05-31 | ***广州总医院 | A kind of preparation method of the shitosan soluble gauze rich in growth factor |
| CN107260639A (en) * | 2017-06-21 | 2017-10-20 | ***广州总医院 | A kind of gel rich in autologous growth factor repairs facial mask and preparation method thereof |
| CN111537326A (en) * | 2020-05-07 | 2020-08-14 | 天津德祥生物技术有限公司 | Method for preparing freeze-dried blood platelet and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101167745A (en) * | 2007-11-02 | 2008-04-30 | 中国人民解放军军事医学科学院野战输血研究所 | Small molecular sugar stable buffer used for blood platelet frozen-dried preservation |
| CN101181302A (en) * | 2007-11-29 | 2008-05-21 | 中国人民解放军军事医学科学院野战输血研究所 | Upkeep liquid as well as preservative fluid for plastocyte |
| CN101957364A (en) * | 2010-09-19 | 2011-01-26 | 李勇 | Preparation method of lyophilization type platelet antigen spectrum cells |
-
2015
- 2015-03-13 CN CN201510111401.9A patent/CN104771412B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101167745A (en) * | 2007-11-02 | 2008-04-30 | 中国人民解放军军事医学科学院野战输血研究所 | Small molecular sugar stable buffer used for blood platelet frozen-dried preservation |
| CN101181302A (en) * | 2007-11-29 | 2008-05-21 | 中国人民解放军军事医学科学院野战输血研究所 | Upkeep liquid as well as preservative fluid for plastocyte |
| CN101957364A (en) * | 2010-09-19 | 2011-01-26 | 李勇 | Preparation method of lyophilization type platelet antigen spectrum cells |
Non-Patent Citations (4)
| Title |
|---|
| PIETRAMAGGIORI G ET AL.: "Freeze-dried platelet-rich plasma shows beneficial healing properties in chronic wounds", 《WOUND REPAIR REGEN》 * |
| 刘广亚等: "影响冰冻血小板质量的因素", 《华南国防医学杂志》 * |
| 单桂秋等: "血小板冻干保存的稳定性研究", 《中国输血杂志》 * |
| 周俊: "血小板冻干保存前预处理技术的实验研究", 《中国博士学位论文全文数据库医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106729955A (en) * | 2016-11-28 | 2017-05-31 | ***广州总医院 | A kind of preparation method of the shitosan soluble gauze rich in growth factor |
| CN107260639A (en) * | 2017-06-21 | 2017-10-20 | ***广州总医院 | A kind of gel rich in autologous growth factor repairs facial mask and preparation method thereof |
| CN111537326A (en) * | 2020-05-07 | 2020-08-14 | 天津德祥生物技术有限公司 | Method for preparing freeze-dried blood platelet and application thereof |
| CN111537326B (en) * | 2020-05-07 | 2023-05-16 | 天津德祥生物技术股份有限公司 | Method for preparing freeze-dried blood platelet and application thereof |
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