超低温冰箱转液氮阶梯降温法与传统程序降温法保存造血干细胞的比较

背景：在造血干细胞整个冷冻保存过程中,受到降温速率、储存温度深浅、冷冻保护剂组合等因素影响,各国学者在提倡选择何种保存方法上面存在不同的主张。目的：比较-80℃低温冰箱转液氮阶梯降温法与传统程序降温法保存外周造血干细胞的效果。方法：将采集的造血干细胞分两组,第一组细胞浓度为1×1011L-1,加入10%二甲基亚砜,放入程序冷冻仪内程序设置为室温至-4℃按1℃/min的速率降温,按35℃/min快速降至-45℃,再以15℃/min升至-21℃,然后以5℃/min降至-90℃,取出冷冻管置-196℃液氮罐内。第二组细胞浓度为1×1011L-1,加入5%二甲基亚砜、3%羟乙基淀粉、4%人血白蛋白,将冷冻管置-80℃冰箱过夜后取出,置-196℃液氮罐内的气相,再过夜后置液氮罐液相内。结果与结论：两组冷冻方法保存细胞的锥虫蓝拒染率、回收率、凋亡率和死亡率差异无显著性意义（P〉0.05）。结果显示用5%二甲基亚砜、4%白蛋白、3%羟乙基淀粉组成的冷冻保护剂,通过-80℃低温冰箱转液氮阶梯降温法冷冻保存造血干细胞与传统10%二甲基亚砜作为冷冻保护剂用程序降温的方法取得一样效果,操作简便易于临床应用。     

不同冷冻保护剂对低温冰箱转液氮阶梯降温冷冻保存造血干细胞效果的影响

背景:非程序降温-80 ℃低温冰箱保存方便快捷,程序降温-196 ℃液氮保存可靠长久,将两者合二为一简化流程已成功用于临床.目的:观察不同冷冻保护剂对-80 ℃低温冰箱转液氮阶梯降温冷冻保存造血干细胞效果的影响.方法:分设10%二甲亚砜组、5%二甲亚砜联合3%羟乙基淀粉组、5%二甲亚砜联合0.25 mol/L海藻糖组、5%二甲亚砜联合3%羟乙基淀粉及0.25 mol/L海藻糖组.采用 -80 ℃低温冰箱转液氮阶梯降温法对单采外周造血干细胞进行冷冻保存,通过透射电镜观察细胞超微结构变化,流式细胞仪观察Annexin-V、PI、Caspase-3水平.结果与结论:4组冷冻保存细胞的存活率、凋亡率和死亡率差异均无显著性意义(P > 0.05).透射电镜下各组细胞超微结构变化差异不明显.单个核细胞群落冷冻保存后存活率在90%以上,含成熟细胞较多的CD45+细胞群落凋亡发生率可达50%左右.造血干祖细胞群落中,早期细胞较晚期细胞更能耐受冷冻损伤.提示在基础冷冻保护剂二甲亚砜的基础上,加入羟乙基淀粉和海藻糖并未显示出对冷冻保存效果的增强作用.     

不同冷冻保护剂对低温冰箱转液氮阶梯降温冷冻保存造血干细胞效果的影响

背景：非程序降温-80℃低温冰箱保存方便快捷,程序降温-196℃液氮保存可靠长久,将两者合二为一简化流程已成功用于临床。目的：观察不同冷冻保护剂对-80℃低温冰箱转液氮阶梯降温冷冻保存造血干细胞效果的影响。方法：分设10%二甲亚砜组、5%二甲亚砜联合3%羟乙基淀粉组、5%二甲亚砜联合0.25mol/L海藻糖组、5%二甲亚砜联合3%羟乙基淀粉及0.25mol/L海藻糖组。采用-80℃低温冰箱转液氮阶梯降温法对单采外周造血干细胞进行冷冻保存,通过透射电镜观察细胞超微结构变化,流式细胞仪观察Annexin-V、PI、Caspase-3水平。结果与结论：4组冷冻保存细胞的存活率、凋亡率和死亡率差异均无显著性意义（P〉0.05）。透射电镜下各组细胞超微结构变化差异不明显。单个核细胞群落冷冻保存后存活率在90%以上,含成熟细胞较多的CD45＋细胞群落凋亡发生率可达50%左右。造血干祖细胞群落中,早期细胞较晚期细胞更能耐受冷冻损伤。提示在基础冷冻保护剂二甲亚砜的基础上,加入羟乙基淀粉和海藻糖并未显示出对冷冻保存效果的增强作用。     

阶段降温冷冻保存淋巴瘤患者自体造血干细胞临床应用观察

目的探讨阶段降温对外周血干细胞活性和临床应用的影响。方法应用自体血浆40%,CPD20%、DMSO终浓度为10%作为冷冻保护剂冷冻自体造血干细胞,-86℃过夜,转到-196℃液氮中保存至使用前,冷冻前后计数活细胞数、CD34+细胞比例和细胞计数,并计算回收率;同时评价回输后干细胞植活时间。结果共对18名患者的31份自体干细胞进行了深低温冷冻保存,解冻后活细胞数为(75.47±15.57)%(58%-95%)、CD34细胞回收率80.28%±24.37%(62%-92%)。回输后的干细胞全部植活,其中白细胞植活的时间为(13.8±3.85)d(9-22d),血小板植活的时间为(22.4±4.79)d(11-25d)。结论应用的阶段降温法可能造成的细胞丢失,但是28份回输患者植活证明,对于不具备程序降温设备的情况采用阶段降温法保存造血干细胞是可行的。     

阶梯降温冷冻保存的自体外周血造血干细胞移植的临床研究

目的:观察低温冰箱转液氮阶梯降温冷冻保存的自体外周血造血干细胞移植治疗恶性血液病的效果,并分析造血干细胞移植后患者生存情况。方法:使用3%羟乙基淀粉、4%白蛋白和5%二甲亚砜(DMSO)组成的冷冻保护剂保护自体造血干细胞,后置于-80℃低温冰箱,再置-196℃液氮罐长期储存,然后用于自体移植。2002年1月至2016年12月98例恶性血液病患者,其中包括急性淋巴细胞白血病10例,非急性髓系白血病24例,多发性骨髓瘤11例,恶性淋巴瘤53例的自体移植。对患者造血恢复时间、总生存率(overall survival,OS)、无进展生存率(progression-free survival,PFS)进行了统计分析。结果:除1例因颅内出血死亡未成功植入外,余97例患者均获得造血重建。中性粒细胞计数≥0.5×10~9/L所需时间平均为9.24±1.89 d,连续3 d不输血小板且血小板≥20×10~9/L所需时间平均为11.04±1.84 d。中位生存时间为47.6 (1-80)个月。1、3和5年总生存率分别为(97.2±1.9)%、(84.2±4.6)%和(77.8±5.6)%。3和5年PFS分别为(74.4±5.1)%和(61.2±6.2)%。结论:通过低温冰箱转液氮阶梯降温法冷冻保存造血干细胞自体移植,可以达到和传统的程序降温冷冻保存法一样的临床移植效果,且移植后并发症发生率无增多。     

骨髓细胞液氮保存21-25年后的细胞活力体外研究

为探讨适合临床应用的骨髓细胞保存方法,揭示细胞长期低温保存效果,将20人份骨髓加入DMSO-AuP(10%二甲亚砜、10%自体血浆)或DMSO-HES-HuA(5%DMSO、6%羟乙基淀粉、4%人血白蛋白)保护剂,分装2毫升/管,600-800管/人份,自控程序降温仪或-80℃低温冰箱预冻降温、液氮中保存21-25年。取冷冻骨髓细胞于38℃复温,检测细胞相关指标。结果表明:加保护剂的细胞标本在-80℃低温冰箱中为低速降温,-30℃前,与自控程序降温仪设定1℃/min的低降温速率接近。DMSO-HES-HuA与DMSO-AuP比较,红细胞形态畸形率分别为(3.5±1.5)%和(12.6±4.8)%;溶血率分别为(3.3±1.6)%和(23.1±5.1)%(p0.05);前者渗透性脆性不变,后者减低;红细胞、血小板、粒单系造血祖细胞、长期培养起始细胞回收率,前后者对比分别为(96.1±1.8)%、(70.0±9.5)%、(49.2±10.9)%、(54.2±13.8)%vs(76.3±5.6)%、(52.7±8.1)%、(43.5±12.3)%、(47.2±13.6)%。用上述保护剂配合上述降温法保存后,细胞在间充质干细胞培养上,生长特性良好。同一保护剂用上述两种降温法保存后,细胞各种回收效果差异不显著(p0.05)。结论:细胞在液氮中保存21-25年,其细胞形态和回收活力(率)良好;保存如骨髓这种细胞成分并非单一的标本时,用5%DMSO-6%HES-4%HuA为保护剂的-80℃冰箱预冻降温后液氮保存,方法简便、经济、易操作,适合临床应用。     

一种简便的外周血干细胞冷冻保存法

为了简化传统的细胞冷冻方法。即用10%二甲基亚砜（DMSO）作为冷冻保护剂并用程控降温仪-196℃液氮保存,研究了用5%DMSO,3%羟乙基淀粉（HES）和4%人血白蛋白（HSA）作为冷冻保护剂,而不用程控降温仪直接-80℃冰箱冷冻细胞的方法。对比了不同方法冷冻保存外周血干细胞（PBSCs)的细胞活性,单个核细胞数,CFU-GM和CD34^ 细胞回收率。结果发现,简便冷冻法可获得更高的单个核细胞（MNC）和CFU-GM回收率,无凝集现象;并且经长达24个月冷冻保存后仍可获得满意的CFU-GM和CD34^ 细胞回收率,37℃融冻和20℃室温融冻法之间各项指标无明显差别。细胞融冻后在室温下暴露于5%DMSO中1小时,CFU-GM回收率并不降低。而细胞活性从93.2%降至84.5%,所以,这种简便的细胞冷冻保存法简便易行。可替代传统的液氮保存法,对于没有程控降温仪的单位具有实用价值。而且还可减少病人DMSO输入量第一线决策。     

-80℃直接冻存人造血干细胞的实验研究和临床应用

以5％二甲基亚砜(DMSO)和6％羟乙基淀粉(HES)作为冷冻保护剂,非程控降温、-80℃冷冻保存人造血干细胞的简化方法用于骨髓移植(BMT)及外周干细胞移植(PBSCT)自80年代起已获成功。该方法冻存的造血干细胞活性与传统的10％DMSO作为冷冻保护剂、程控降温、液氮冷冻保存的相当。其主要优点为操作简便,费用低,便于普及。实验研究表明简化法冻存外周血干细胞5年后,BFU-E、CFU-GM的回收率无明显下降,细胞活性约为贮存前的80％。目前临床上推荐该方法用于短期(<1年)冻存造血干细胞。本文就其发展过程、作用机理、技术实施及应用现状作一介绍。     

骨髓、脐血、外周血干细胞-80℃保存研究

骨髓、脐血、外周血干细胞－８０℃保存研究２０００５１上海市血液中心范华骅马庆柏乃庆造血干细胞的体外长期保存一般采用１０％二甲基亚砜低温保护剂，程序降温后贮存在液氮中［１～３］，这种方法需要程序降温仪、液氮罐等价格昂贵的低温保存设备，且比较复杂、费时。...     

—80℃直接冷冻保存人骨髓造血干细胞的实验研究

－８０℃直接冷冻保存人骨髓造血干细胞的实验研究贾东新宋永平陈绍倩王桂叶白任奎魏旭东王桂菊刘延方赵志平我们采用二甲基亚砜（ＤＭＳＯ）／羟乙基淀粉（ＨＥＳ）混合物作骨髓冷冻保护剂，－８０℃冰箱直接降温并在该温度下冻存１个月后，对骨髓造血干细胞活性进行评价...     

二甲基亚砜联合羟乙基淀粉的非程控降温法冻存脐血造血干细胞研究

为了探索有效低温保存脐血造血干／祖细胞的冷冻保护剂和技术，进行了二种方法试验。第一种方法联合使用二甲基亚砜(DMSO)和羟乙基淀粉(HES)(分子量120000)作为冷冻保护剂并用非程控降温冷冻技术；第二种方法只使用DMSO作为冷冻保护剂并用程控降温仪冷冻技术保存脐血。对二种方法的效果进行比较。结果表明：15份脐血冻存后细胞存活率、CFU—GM回收率，无论在使用非程控降温的第一种方法和使用程控降温仪的第二种方法均无显著性差异。183份脐血质量检测结果及21例临床移植结果也显示两种方法无显著性差异。结论：联合使用DMSO和HES作为冷冻保护剂并用非程控降温冷冻的方法对脐血造血干细胞有良好的保护作用，是一种简单易行、省时省力、适合于脐血造血干细胞库大批量冻存脐血的方法。     

Long‐term storage of peripheral blood stem cells frozen and stored with a conventional liquid nitrogen technique compared with cells frozen and stored in a mechanical freezer

BACKGROUND: Cryopreservation of hematopoietic progenitor cells using liquid nitrogen and controlled‐rate freezing requires complex equipment and highly trained staff and is expensive. We compared the liquid nitrogen method with methods using a combination of dimethyl sulfoxide (DMSO) and hydroxyethyl starch (HES) for cryopreservation followed by storage in mechanical freezers. STUDY DESIGN AND METHODS: Peripheral blood stem cells (PBSCs) were collected from normal donors by apheresis and allocated to one of four preservation and storage conditions: 1) 10% DMSO with freezing in liquid nitrogen and storage in liquid nitrogen, 2) 5% DMSO and 6% HES with freezing and storage in a ?80°C mechanical freezer, 3) 5% DMSO and 6% HES with freezing in a ?80°C mechanical freezer and storage in a ?135°C mechanical freezer, or 4) 5% DMSO and 6% HES with freezing and storage both in a 135°C mechanical freezer. Cells were stored for 5 years during which total nucleated cells (TNCs), cell viability, CD34+ cell content, and colony‐forming unit–granulocyte‐macrophage content were determined. RESULTS: There were some significant differences in the variables measured during freezing and the 5 years of storage compared to the values before freezing and storage; however, these differences were not consistent and do not favor one protocol over the others. Samples stored for 24 hours before cryopreservation showed a significant decrease in TNCs, but no other significant changes during the 5 years. CONCLUSION: In vitro measurements indicate that PBSCs can be successfully frozen and stored using a combination of DMSO and HES providing smaller amounts of DMSO and allowing simplified freezing and storage conditions.     

A simple controlled-rate freezing method without a rate-controlled programmed freezer provides optimal conditions for both large-scale and small-scale cryopreservation of umbilical cord blood cells

BACKGROUND: Umbilical cord blood (CB) is being used as a source of alternative HPCs for transplantation with increasing frequency. The goal of CB banks for unrelated transplantation is to provide good quality-controlled CB units that can be transplanted for HPCs into the largest possible number of patients. STUDY DESIGN AND METHODS: Large CB samples in freezing bags wrapped with insulators and small samples in cryotubes placed into double styrene-foam boxes were cryopreserved at -85 degrees C without a rate-controlled freezing machine, followed by storage in the liquid phase of nitrogen. After thawing these cells, the viability and recovery of cells, as well as the recovery rate of HPCs such as CD34+ cells, CFU-GM, and total CFU were evaluated. RESULTS: Measurement of the freezing rate in CB bags and cryotubes demonstrated that this simple method for cryopreservation of CB cells provided optimal conditions for both large-scale and small-scale cryopreservation. Recovery of CB progenitor cells after cryopreservation was also shown to be potentially acceptable when evaluated with CD34+ cells, CFU-GM, and total CFU. These results were comparable to the method using a rate-controlled programmed freezer. CONCLUSIONS: A simple method for cryopreservation of CB cells without a rate-controlled programmed freezer could provide a sufficient-enough potential for the transplantability of HPCs after thawing.     

人脐带间充质干细胞不同冻存方法探讨

目的 观察不同冻存条件对人脐带间充质干细胞(HUC-MSC)冻存效果的影响.方法 将HUC-MSC传代培养增殖后,设立4个因素和3个水平,分为9个实验组,分别观察不同冻存条件下对各组HUC-MSC的存活率、回收率、免疫抗原CD34和CD44的影响.结果 第9组HUC-MSC解冻复苏后,其存活率、回收率以及免疫抗原CD44积分值均高于其他组(P<0.01),冻存前后其细胞形态无明显差别,细胞免疫抗原CD34均为阴性.结论以90%血清+10%二甲基亚砜(DMSO)作为冻存液,取106/mL以下浓度的HUC-MSC放入厚壁聚乙烯泡沫盒中,立即封存置-80 ℃冻存24 h,再投入液氮中冻存,为最佳冻存方法.     

A theoretically optimized method for cord blood stem cell cryopreservation

The objective of this study was to develop an optimal cryopreservation method for human umbilical cord blood hematopoietic progenitor cells as evidenced by improved retention of in vivo engraftment ability and multilineage differentiation. An extended understanding of the osmometric/permeability characteristics of cord blood stem cells was accomplished by measuring permeability of the cryoprotectant dimethyl sulfoxide (DMSO) at below-ambient temperatures (10 degrees and 3 degrees C). These data were combined with previously published osmotic and permeability data and the water-NaCl-DMSO phase diagram in conjunction with a mathematical model to determine an optimal initial DMSO concentration, cooling rate, and liquid nitrogen plunging temperature. Cells cryopreserved with the theoretically optimized procedure were then compared with cells frozen using standard methods for the ability to engraft in irradiated NOD/SCID mice. The optimal procedure was determined to include a 0.7 molal (approximately 5%) DMSO concentration at a cooling rate of 4 degrees C/min, and a plunging temperature of -44 degrees C. The optimized protocol resulted in significantly higher engraftment of human CD45(+) cells (17.2 +/- 1.6% vs. 8.4 +/- 1.6%), CD19(+) B lymphocytes (11.3 +/- 1.2% vs. 5.8 +/- 1.2%), and CD34(+) cells (1.9 +/- 0.09% vs. 0.6 +/- 0.09%) compared to cells frozen using a standard method. Engraftment of CD33(+) cells was not significantly different (4.0 +/- 0.3 vs. 3.2 +/- 0.6, respectively). This study demonstrated that the use of a theoretically determined optimal cryopreservation method is superior to standard methods for maintaining UCB PCBs with multilineage repopulation potential in NOD/SCID mice.     

脐血造血干细胞低温保存方法探讨

为使脐血有更广阔的应用前景及建立脐血造血干细胞(CBSC)库,笔者探讨了脐血低温保存的有效方法。结果显示:CBSC 4℃低温保存以2～3d为宜;液氮低温保存是一种有效的CBSC保存方法,回收率高,较长时间保存,回收率不受影响;-80℃冰箱保存法具有与液氮相同的低温保存效果;且简单、经济、方便和实用,无需程序降温仪,便于大规模开展应用和建立CBSC库;脐全血保存可以避免分离过程CBSC的丢失,保存效果不受影响。