Experimental Of Large Volume Freeze-dried Platelets

ObjectiveIn this study,three components of the platelet protective agent,platelet activated inhibitor and protein protectant were changed,the optimized of platelet protect solution was selected by orthogonal design.The optimized platelet protective solution was used to detect the 100 mL platelets freeze dried process,and the preparation technology of this study was determined.The cell proliferation experiment of freeze-dried platelets to certify the effectiveness of promoting wound healing of the product prepared by this method.To evaluate the preliminary safety of the product,the toxicity of freeze-dried platelets was evaluated by acute toxicity test of skin and multiple-dose toxicity study.We conclude the four part of this study in order to obtain a safe and effective platelets lyophilized.Method1.Optimization of freeze-dried protective liquid: the optimized of protective solution consisting of three components of platelet membrane protector,platelet activated inhibitor and protein protector was made by orthogonal design of 3 factors and 3 levels.Platelet aggregation rate was selected as the optimal index,and combined with the three indexes of platelet activated markers PAC-1,CD62 P positive expression rate and platelet recovery rate,the optimal freeze-dried platelets protection solution was screened by comparing the difference.The optimal protective solution was used to freeze dried platelets.To verify the feasibility of the optimal freeze-drying protection solution,the experimental group was compared with fresh platelets and original protectant solutions,the maximum aggregation rate of platelets,the positive expression of platelet activated markers PAC-1,CD62 P,the rate of platelet recovery and uLtrastructural differences under electron microscope were detected.2.Study on the process of platelet freeze-drying: The original material of platelet freeze-drying special bag was selected according to the characteristics of platelets,and the special bag of platelet freeze-drying special bag was designed by freeze-drying experiment and the bacteria of the bags were detected.On the basis of small volume freeze-dried platelets,the parameters such as temperature,time and vacuum degree of freeze-drying of large capacity platelets were explored.The water content of freeze-dried platelets with larger capacity was detected by Carle Fisher method and decompression drying method.The difference between 100 mL freeze-dried platelets and fresh platelets was compared by the maximum platelet aggregation rate、PAC-1、CD62P.3.The cell proliferation promoting effect of freeze-dried platelets: The freeze-dried platelets were rehydrated with equal volume of PPP,then rehydration of freeze-dried platelets were activated by the calcium gluconate thrombin activator at 10:1,the supernatant was centrifuged.Human umbilical vein endothelial cells(HUVEC)was cuLtured 5d,and then 5%,10% and 20% supernatants were added to cuLture HUVEC 24 h,48h,72 h,and the cell density was detected by CCK8.The fresh platelets and 5%FBS medium of 1640 were used as controls.The OD values of cultured cells were detected by enzyme labelling instrument,and the results were analyzed statistically.4.Safety evaluation:(1)Acute skin irritation test.SD rats were selected as experimental subjects,and FDP rehydration suspension was used as experimental group,and physiological saline(NS)was used as a blank control group.After the skin was depilated on both sides of the back of the rat,the experimental group was repeatedly smeared with rehydration solution on the left side,and the right side was repeatedly smeared with saline.The four time points of 1,24,48 and 72 h were observed and recorded respectively.The edema and allergic erythema of the wound were observed and compared.(2)Multiple-dose toxicity study.SD rats were selected as experimental subjects,and FDP rehydration suspension was used as experimental group,and rats without any treatment were used as blank control group.The FDP rehydration solution 0.5mL and 2.0mL were injected intraperitoneally on the zeroth and fourteenth days respectively.In the 7 、14 、21 and 28 days,blood routine and five biochemical indexes of ALT,AST,BUN,Cr and UA were examined,and the statistical analysis of the experimental data was carried out.Result1.The optimization results of freeze-dried protection solution:(1)Orthogonal design experiment results: the highest platelet aggregation rate(74.33±24.01)% among groups,platelet activated markers PAC-1 and CD62 P lower than the average level of 9 groups,the platelet recovery rate is higher than the average level of the 9 groups.The maximum aggregation rate of platelets,which mainly embodies the function of platelets,is taken as the priority index,combined with the combined analysis of the other three items of detection.On the basis of the original protective liquid,the best combination of 2% DMSO,second messenger reguLator,2mmol/L vitamin B6 and 75% PPP protection solution is selected.(2)Experimental results: the maximum platelet aggregation rate(76.12±6.02)% of the optimized protective liquid group was statistically significant(P<0.05),compared with the fresh platelets group,the difference was not statistically significant(P >0.05).The positive expression rates of PAC-1 and CD62 P in the optimal protective group were(3.23 ± 0.49)% and(36.83 ± 8.21)% respectively,there was no significant difference between the two group and the original protective group and the fresh platelets group(P >0.05),and the rate of platelet recovery was(85.90±2.24)% after the rehydration of the freeze-dried platelets in the optimal protective liquid group.Compared with the original protection solution group,there was no significant difference(P >0.05).Transmission electron microscopy showed that the structure of the cells in the original protective solution group was released and a large number of vacuoles were formed.In the new protective group,the platelet cell membrane was complete and the inner structure of the cells was normal.Compared with the fresh platelets,the cells were dyed shallow,the cells were slightly swollen,and there were a few vacuoles formed by the release of the internal structure of the cells.2.The exploration results of lyophilization of large capacity platelets.(1)The research results of platelet freeze-drying special bag: the capacity is 400 mL,it is not easy to activate platelets,is harmless,can tolerate-80 ℃ low temperature,and does not break the bag under the vacuum of 0.001-1000 mbar,and the bag can be kept hot.The test of sealing and asepsis was negative.(2)The results of freeze-drying conditions: the cold well temperature of the freeze-dried platelets is-50℃,the partition temperature is 20℃,the vacuum is 0.1mbar,the time is 75 h,the cold well temperature of the end drying is-50℃,the partition temperature is 25℃,the vacuum degree is 0.001 mbar,and the time is 25 h.(3)Freeze-drying effect:(1)The water content of freeze-dried platelets showed that the water content was 0.The maximum aggregation rate and activation parameters of freeze-dried platelets and fresh platelets were(46.2±4.66)%,lower than that of fresh platelets(86.3 + 5.41)%,and the difference was statistically significant(P <0.05),and there was no significant difference in platelet activation.(3)platelet recovery after freeze-drying: platelet count was(974.8±52.141)x 109/L after freeze-drying,and the recovery rate was(130.08 ± 3.39)%.(4)Ultrastructure: under transmission electron microscope,the platelet cell membrane in the freeze-dried platelets group was intact and the inner structure of the cells was normal,and the staining was relatively shallow compared with the fresh platelets.The cells were slightly swollen,with a small number of vacuoles formed by the release of intracelluLar structures.3.The experimental results of promoting cell proliferation of large volume freeze-dried platelets.After cuLture of 24 h,48h and 72 h,FDP,PRP and FBS can promote the proliferation of human umbilical vein endothelial cells.When cuLtured 24 h,the difference between 20%FDP group and control group was statistically significant(P <0.05).The difference among group 10%FDP and 5%FDP and 5%FBS was statistically significant(P <0.05),and there was no significant difference with the other three contrast groups(P >0.05).When the 48 h was cuLtured,the difference between group 20%FDP and the control group 10%PRP group and 5%FBS was statistically significant(P <0.05),and there was no statistical difference between the other two contrast groups(P >0.05).The difference between group 10%FDP and group 5%FDP and group 5%PRP and 5%FBS was statistically significant(P <0.05),and there was no significant difference with the other two contrast groups(P >0.05).When cuLtured 72 h,there was a significant difference between 20%FDP group and 5%FBS group(P <0.05),and there was no significant difference with the other three contrast groups(P >0.05).The difference between group 10%FDP and group 10%PRP,group 5%PRP and 5%FBS was statistically significant(P <0.05),and there was no significant difference between group 20%PRP and control group(P >0.05).The difference between 5%FDP group and control group was statistically significant(P <0.05).4.The results of safety evaluation.(1)Skin irritation tests showed no irritation and allergic reactions such as erythema,edema and exudation in the skin of SD rats.(2)The results of multiple-dose toxicity study showed that FDP had no significant effect on the hematocrit and red blood cell pressure in rats,and there was no significant difference between the experimental group and the control group(P >0.05).After the administration of 7D and 14 d,the white blood cell count of the experimental group was significantly different from that of the control group(P <0.05),but there was no significant difference between the red cell count and the red blood cell pressure and the control group(P>0.05).The results of BUN,Cr,ALT and UA in the experimental group were not significantly different from those in the control group(P >0.05).After the administration of 7D and 14 d,the rat AST in the experimental group was higher than the control group,the difference was statistically significant(P <0.01),and there was no significant difference between the control group and the control group(P >0.05)after the administration of 21 d and 28 d.ConclusionPlatelet optimization protection solution is composed of +2%DMSO+ second messenger +2mmol/LVitB6+75% protein protectant,which can protect platelets structure and function well.The platelet freeze-drying special bag can be used for the preservation of platelets in 100 mL.The freeze-drying process of the platelets is good;the frozen dried platelets have good cell promotion.Proliferation,and no obvious side effects.