Holovati Jelena L, Gyongyossy-Issa Maria I C, Acker Jason P
Canadian Blood Services, Research and Development, 8249-114 Street, Edmonton, Canada AB T6G 2R8; Department of Laboratory Medicine and Pathology, University of Alberta, Canada.
Canadian Blood Services, Research and Development, 8249-114 Street, Edmonton, Canada AB T6G 2R8; Department of Pathology, University of British Columbia, Canada.
Cryobiology. 2009 Feb;58(1):75-83. doi: 10.1016/j.cryobiol.2008.11.002. Epub 2008 Nov 14.
We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10 mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3M trehalose or liposome solution, then cooled with slow (0.95+/-0.02 degrees C/min), medium (73+/-3 degrees C/min) and fast (265+/-12 degrees C/min) cooling rates and storage in liquid nitrogen, followed by a 37 degrees C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66%+/-5% vs 29%+/-4%) compared to control RBCs (c-RBC, p=0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p=0.039 and p=0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p=0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p=0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.
我们正在研究使用脂质体（一种合成的微观囊泡）将海藻糖细胞内递送至哺乳动物细胞。本研究聚焦于含海藻糖的脂质体对人红细胞冷冻保存后恢复情况及膜质量的改善作用。采用挤压法合成了由二棕榈酰磷脂酰胆碱（DPPC）、磷脂酰丝氨酸（PS）和胆固醇（60:30:10摩尔%）组成的脂质双层单室脂质体。经脂质体处理的红细胞（l-RBCs）重悬于生理盐水、0.3M海藻糖或脂质体溶液中，然后分别以慢（0.95±0.02℃/分钟）、中（73±3℃/分钟）和快（265±12℃/分钟）的冷却速率冷却并储存于液氮中，随后在37℃解冻。解冻后红细胞的质量通过回收率、红细胞形态、PS和CD47表达进行评估。脂质体处理对红细胞膜无不良影响。与对照红细胞（c-RBC，p=0.003）相比，解冻后l-RBCs的回收率显著更高（66%±5%对29%±4%）。与慢冷却速率相比，中冷却速率和高冷却速率导致的细胞回收率显著更高（分别为p=0.039和p=0.041）。对于所有三种冷却速率，在脂质体溶液和海藻糖溶液中冷冻的l-RBCs的回收率显著高于在NaCl溶液中冷冻的l-RBCs（p=0.021）。流式细胞术和形态学评估表明，脂质体处理可改善解冻后的膜质量。水相核心含海藻糖的脂质体处理的红细胞与水相核心含生理盐水的脂质体处理的红细胞解冻后的回收率无统计学显著差异（p=0.114）。脂质体处理显著改善了低温暴露后红细胞的回收率和膜完整性。
