冻存人外周血单个核细胞的活率影响因素探究

1. 引言

1.1 研究背景

1.1.1 外周血单个核细胞的重要性和应用领域

外周血单个核细胞（PBMC）是免疫系统的重要组成部分，对人体的免疫反应至关重要。
PBMC在癌症治疗、器官移植和疫苗研发等领域具有广泛的应用价值。
随着科学技术的发展，对PBMC的需求日益增加，因此对PBMC的保存和运输提出了更高的要求。

1.1.2 冻存技术在PBMC保存中的应用

冻存技术是一种有效的PBMC保存方法，可以长时间保存PBMC的活性和功能。
冻存技术的关键在于选择合适的冻存保护剂、储存时间和温度条件。
目前，关于冻存PBMC的最佳条件仍存在争议，因此有必要对影响冻存效果的因素进行深入研究。

1.2 研究目的与意义

研究冻存PBMC的活率影响因素，为优化冻存条件提供实验依据。
提高冻存PBMC的活率和功能，满足临床和科研的需求。
推动冻存技术在PBMC保存中的应用，为相关领域的发展提供技术支持。

2. 材料与方法

2.1 实验材料

全血样本：采集自健康志愿者。
抗凝剂：EDTA。
冻存保护剂：CS10、CS5、90%FBS+10%DMSO。
实验室仪器：离心机、程序降温箱、液氮罐等。

2.2 实验方法

2.2.1 PBMC的分离

采集全血样本，加入EDTA抗凝剂，置于2-8°C环境中。
采用密度梯度离心法分离PBMC，洗涤后备用。

2.2.2 冻存方法

A组：不同储存时间对PBMC活率的影响。
B组：不同冻存保护剂对PBMC活率的影响。
C组：不同冻存温度条件对PBMC活率的影响。

2.2.3 冻存效果评价

活率检测：采用台盼蓝染色法检测冻存前后PBMC的活率。
回收率检测：计算冻存前后PBMC的数量变化，评估回收率。

3. 结果与分析

3.1 储存时间对PBMC活率的影响

A1组、A2组和A3组在冻存前的活率无显著差异。
复苏后，A1组与A2组、A2组与A3组的活率无显著差异；A1组与A3组有显著差异。
A1组、A2组和A3组的活细胞回收率无显著差异。

3.2 冻存保护剂对PBMC活率的影响

B1组、B2组和B3组在冻存前的活率无显著差异。
复苏后，B1组、B2组和B3组的活率有显著差异。
B1组与B2组、B2组与B3组的活细胞回收率有显著差异；B1组与B3组无显著差异。

3.3 冻存温度条件对PBMC活率的影响

C1组和C2组在冻存前的活率无显著差异。
复苏后，C1组和C2组的活率有显著差异。
C1组和C2组的活细胞回收率有显著差异。

3.4 结果分析

储存时间对PBMC活率的影响较小，但长时间储存会导致活细胞回收率降低。
冻存保护剂的选择对PBMC活率和回收率有显著影响，应根据具体需求选择合适的保护剂。
冻存温度条件对PBMC活率和回收率有显著影响，应选择适宜的温度条件进行冻存。

4. 讨论

4.1 冻存PBMC的影响因素

储存时间：适当缩短储存时间可以提高PBMC的活率和回收率。
冻存保护剂：选择合适的冻存保护剂可以有效保护细胞免受冻存过程中的损伤。
冻存温度条件：适宜的温度条件可以减少冰晶的形成，降低对细胞的损伤。

4.2 冻存技术的优化

通过优化冻存条件，可以提高冻存PBMC的活率和功能，满足临床和科研的需求。
进一步研究冻存过程中的分子机制，以更有效地保护细胞免受冻存损伤。
探索新型冻存技术，如玻璃化冻存等，以进一步提高冻存效果。

5. 结论

选择较短的储存时间、合适的冻存保护剂以及恰当的冻存温度条件能有效提高冻存后的活率和回收率。

关键词：外周血单个核细胞；冻存；活率；复苏；回收率

An investigation of the factors influencing the viability of frozen human peripheral blood single nucleated cells

Abstract

Objective: To investigate the factors affecting the viability of single nucleated cells from frozen stored peripheral blood.

Methods: In this paper, we will explore the three aspects of storage time after single blood collection, the choice of cryoprotectant and storage temperature conditions, and conduct experiments in groups as follows: 1. The effect of storage time after single blood collection on the viability of isolated PBMC (Group A): storage for 24h (Group A1), storage for 48h (Group A2), storage for 72h (Group A3).2. The cryoprotectant chosen for freezing storage protective agent on the viability of resuscitated PBMC (Group B): CS10 (Group B1), CS5 (Group B2), 90% FBS+10% DMSO (Group B3).3. The effect of temperature conditions of PBMC cryopreservation on the viability of resuscitated PBMC (Group C): (1) PBMC, placed in the programmed cooler, then cryogenically frozen at -80℃ for one night, and then transferred the next day to the liquid nitrogen reservoir (C1). (2) PBMC, placed in a cooling box, then refrigerated at 2-8℃for 10 min, then transferred to -20℃for 1 h, then transferred to -80℃for 1 h, and then transferred to liquid nitrogen tank storage on the 2nd day (C2).

Results: 1. Group A pre-freezing survival rate: Group A1, Group A2, and Group A3 were compared, and there was no significant difference. Viability after resuscitation in group A: there was no significant difference between group A1 relative to group A2 and group A2 relative to group A3; there was a significant difference between group A1 relative to group A3. Recovery rate of viable cells in group A: no significant difference between group A1, group A2 and group A3. viability before freezing in group B: no significant difference between group B1, group B2 and group B3. 2. Post-recovery viability of group B: group B1, group B2 and group B3 were compared, and there was a significant difference. Recovery rate of viable cells in group B: there is a significant difference between group B1 and group B2; there is no significant difference between group B2 and group B3; there is no significant difference between group B1 and group B3. viability of group C before freezing and storage: there is no significant difference between group C1 and group C2 when comparing the two groups. 3. The viability of group C after recovery: group C1 and group C2 were compared, and there was a significant difference. Recovery rate of live cells in group C: group C1 and group C2 were compared with each other, and there was a significant difference.

Conclusion: Selection of shorter storage time, suitable cryoprotants, and proper cryopreservation temperature conditions can effectively improve the viability and recovery rate after cryopreservation.

Keywords: Peripheral Blood Single Nucleated Cells; Frozen Storage; Viability; Recovery; Recovery Rate