Magnetic cryopreservation for dental pulp stem cells

Magnetic cryopreservation has been successfully used for tooth banking with satisfactory implantation outcomes, suggesting that the method preserves human periodontal ligament cells and dental pulp stem cells (DPSCs). Therefore, magnetic cryopreservation may be applied for the preservation of DPSCs; however, this method has not been evaluated yet. A reliable cryopreservation method for live-cell preservation is important for the clinical applications of regenerative medicine. The conventional slow-freezing procedure with 10% dimethylsulfoxide (DMSO) may not be appropriate for stem cell-based therapies because DMSO is cytotoxic. The objective of this study was to investigate whether magnetic cryopreservation can be applied for DPSC cryopreservation. Cells isolated from human dental pulp were subjected to magnetic cryopreservation. Postthawing cell viability, adhesion, proliferation, expression of markers for mesenchymal stem cells (MSCs), differentiation ability of magnetically cryopreserved DPSCs and DNA stability were compared to those of cells subjected to the conventional slow-freezing method. The results indicated that a serum-free cryopreservation medium (SFM) containing 3% DMSO is optimal for magnetic cryopreservation. Post-thaw magnetically cryopreserved DPSCs express MSC markers, and perform osteogenesis and adipogenesis after induction similarly to fresh MSCs. No significant DNA damage was found in magnetically cryopreserved DPSCs. Magnetic cryopreservation is thus a reliable and effective method for storage of DPSCs. The smaller amount of DMSO required in SFM for cryopreservation is beneficial for the clinical applications of post-thaw cells in regenerative medicine.     

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。     

An efficient method for the cryopreservation of fetal human liver hematopoeitic progenitor cells

The use of human hematopoietic progenitor cells (HPC) for transplantation requires efficient recovery methods and cryopreservation procedures. The purpose of this study was to determine cryopreservation techniques for fetal human liver (FHL) CD34(+) cells. We assessed FHL HPC recovery efficiency after freezing and thawing by viability testing, fluorescence-activated cell sorting analysis, and colony-forming ability under different conditions. We also determined optimal cell freezing concentrations and the effect of rate-controlled freezing on cell recovery. Lastly, cell recovery after varying freezing time periods was examined. Our results indicated that optimal cell recovery occurs when: A) cryopreservation medium consists of either 5% dimethylsulphoxide (DMSO) or 10% DMSO in combination with either 20% fetal bovine serum (FBS) or 70% FBS and when Iscove's modified Dulbecco's medium consists of not more than 10% DMSO; B) a rate-controlled freezing device container is used; C) CD34(+) cells are frozen at a concentration of 1 x 10(6)/ml, and D) a thawing temperature of 37 degrees C is used. These observations indicate that cryopreservation of FHL HPC is possible for up to 18 months in optimal conditions without losing hematopoietic activity.     

Cryopreservation does not affect proliferation and multipotency of murine neural precursor cells

Stem cell research offers unique opportunities for developing new medical therapies for devastating diseases and a new way to explore fundamental questions of biology. Establishing an efficient freezing protocol for neural precursor cells (NPCs) is of great importance for advances in cell-based therapies. We used fluorescence-activated cell sorter-based cell death/survival analysis and Western blot analysis of proliferation markers (proliferating cell nuclear antigen) and prosurvival proteins (Bcl-2) to study the effect of a variety of cryoprotective agents on fetal mouse forebrain NPCs. Neurospheres frozen at -70 degrees C or in liquid nitrogen in a rate-controlled manner and thawed after 5 days retained viability of 60%-70% measured 24 hours after thawing. However, 1 week after thawing, viability dropped to 50%-60%. Using a clonogenic sphere formation assay, we showed that recovery rate of frozen NPCs was approximately 26% and did not significantly differ between dimethyl sulfoxide (DMSO)- and glycerol-supplemented samples. Application of the caspase inhibitor zVAD-fmk during freezing or in the first week after thawing resulted in protection of cryopreserved neurospheres after thawing but not during the freezing process, indicating that apoptosis limits recovery of NPCs. Cell survival was not reduced in cells that were enzymatically separated before cryopreservation. Optimal protection of NPCs was achieved when 10% DMSO alone or in a combination with 10% fetal calf serum (FCS) was used. However, 10% glycerol alone was equally effective. Using these protocols, NPCs retained their multipotency and differentiated into both glial (GFAP-positive) and neuronal (Tuj1-positive) cells. Percentage of Tuj1-positive cells in 5% and 10% DMSO, in 10% DMSO + 10% FCS, and in 10% glycerol remained at the same level as before freezing and varied from 5%-7%. We conclude that cryopreservation (up to 1 month at -70 degrees C and up to 1 year in liquid nitrogen) does not markedly alter the rate of proliferation and multipotency of murine neural precursor cells.     

Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells

Human bone marrow-derived mesenchymal cells contain mesenchymal stem cells (MSCs), which are well known for their osteo/chondrogenic potential and can be used for bone reconstruction. This article reports the viability of cryopreserved human mesenchymal cells and a comparison of the osteogenic potential between noncryopreserved and cryopreserved human mesenchymal cells with MSC-like characteristics, derived from the bone marrow of 28 subjects. The viability of cryopreserved mesenchymal cells was approximately 90% regardless of the storage term (0.3 to 37 months). It is clear by fluorescence-activated cell sorter analysis that the cell surface antigens of both noncryopreserved and cryopreserved mesenchymal cells were negative for hematopoietic cell markers such as CD14, CD34, CD45, and HLA-DR but positive for mesenchymal characteristics such as CD29 and CD105. To monitor the osteogenic potential of the cells, such as alkaline phosphatase (ALP) activity and in vitro mineralization, a subculture was conducted in the presence of dexamethasone, ascorbic acid, and glycerophosphate. No difference in osteogenic potential was found between cells with or without cryopreservation treatment. In addition, cells undergoing long-term cryopreservation (about 3 years) maintained high osteogenic potential. In conclusion, cryopreserved as well as noncryopreserved human mesenchymal cells could be applied for bone regeneration in orthopedics.     

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1×10¹¹ L^-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1×10¹¹ L^-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。     

Cryopreservation of rat islets of Langerhans by vitrification

Cryopreservation could be a possible means of addressing the shortage of islets of Langerhans. We investigated the effects of EDT324 solution on the vitrification of isolated rat islets of Langerhans. Rat pancreatic islets were cryopreserved in 10% DMSO by a slow-rate freezing method or were cryopreserved in EDT324 solution by vitrification. The cryopreserved islets were compared in terms of viability, stimulation index and metabolic function after transplantation. After cryopreservation, the viability and stimulation of islets stored in EDT324 were 92.4% and 6.4, respectively, and were higher than islets stored by slow freezing (72.5% and 1.5, respectively). Streptozotocin-induced diabetic rats were transplanted with islets cryopreserved in EDT324, which corrected diabetes and achieved euglycemia within 2?days after transplantation. These results indicate that EDT324 allows successful cryopreservation of rat islets for long-term storage as an alternative solution to traditionally used solutions, such as 10% DMSO. Transplantation of cryopreserved islets into diabetic rats can achieve euglycemia.     

1H NMR spectroscopy as a tool to evaluate key metabolic functions of primary porcine hepatocytes after cryopreservation

Proton NMR spectroscopy of biological fluids has produced interesting results lately. We used the technique to investigate the effects of cryopreservation on primary porcine hepatocytes as successful cryopreservation of primary porcine hepatocytes is of importance to the development of bioartificial liver support systems. After isolation 10(8) hepatocytes were cryopreserved for 1 week in Williams E/10% DMSO, either by quick freezing (-5 to -30 degrees C/min), slow freezing (-0.3 to -3 degrees C/min) or stepwise freezing protocols on cell suspensions and confluent cell plates. Plating efficiency was assessed by percentage LDH release. Metabolic functions of cryopreserved hepatocytes at 24 h post-thawing were compared with those of fresh hepatocyte cultures at 48 h. 1H nuclear magnetic resonance spectroscopy of the culture medium post-incubation, using the presaturation technique, assessed the following: glucose metabolism, transamination and glutamine synthesis and succinate synthesis. Freshly isolated cells had a viability of 82 +/- 4.3% and a plating efficiency of 87 +/- 3.8%. All cryopreservation protocols resulted in significantly reduced viability and plating efficiency. No significant differences were observed between different cryopreservation media or protocols. When comparing cryopreserved with freshly isolated cells, we observed that metabolism of acetyl-CoA precursors was significantly impaired in cryopreserved cells. Lactate and pyruvate production was also significantly less, although glucose consumption was similar. No differences were observed in gluconeogenic amino acid metabolism, transamination and urea synthesis. 1H NMR spectroscopy can be used to provide information about metabolic activity and functions of cultured primary cells.     

An efficient, economical slow-freezing method for large-scale human embryonic stem cell banking

Human embryonic stem cells (hESCs) are one of the most interesting cell types for tissue engineering, cell therapy, basic scientific research, and drug screening. Fast advancement in these areas requires the availability of large amounts of safe and well-characterized hESCs from hESC banks. Therefore, optimized freezing protocols, allowing the cryopreservation of large amounts of hESC without direct contact with liquid nitrogen, need to be established. In this study, 6 different cryoprotector combinations [dimethylsulfoxide (DMSO), ethylene glycol, and hydroxyethylstarch (HES)] combined with 2 different application methods were screened with the VUB01 cell line, to establish a new slow-freezing protocol with high recovery rates and a good expansion capacity. Our best conditions were confirmed in 4 other hESC lines: H1, H9, 181, and UGent2. To our knowledge, this is the first time that HES is evaluated as a cryoprotector for hESCs. The use of 5% DMSO+5% HES combined with a new detachment protocol leads to efficient hESC cryopreservation. This protocol involves treating the hESC colonies with cell dissociation solution, a mild dissociation solution uncommonly used for hESC culture. A recovery ratio ranging from 45.5% to 168.2% was obtained, and these were significantly different from the other tested conditions (Student's t-test, P<0.05). The cryopreserved hESCs were morphologically comparable to control cells, exhibited a good expansion profile, were positive for pluripotent expression markers, and could still differentiate into the 3 germ layers. This new protocol allows efficient and economical hESC cryopreservation, ideal for hESC banking.     

Cryopreservation of human embryonic stem cells without the use of a programmable freezer

BACKGROUND: An effective freezing-thawing technique is crucial for the clinical application of human embryonic stem (ES) cells. The aim of this study was to find an optimal cryopreservation protocol for human ES cells using slow freezing-rapid thawing without a programmable freezer. METHODS: The human ES cell line, SNUhES-3, was cultured on an STO feeder layer in gelatin-coated tissue culture dishes. All cryopreservation steps were performed using a simple commercial freezing container. The survival rate of cryopreserved-thawed human ES cells was estimated by counting colony numbers under a stereomicroscope. Initially, we compared the survival rates of cryopreserved human ES cells using three cryoprotectants: dimethylsulphoxide (DMSO), ethylene glycol (EG) and glycerol. In this experiment, 5% DMSO/95% fetal bovine serum (FBS) (vol/vol) showed the highest survival rate. We next tested the impact of various concentrations of FBS (95, 50 and 5%) with 5% DMSO, and then examined the effects of adding EG or glycerol to 5% DMSO + optimal FBS. RESULTS: No significant difference in survival rate was observed between 95 and 50% FBS in the presence of 5% DMSO. A significant improvement in survival rate was obtained by adding 10% EG to 5% DMSO+50% FBS. After thawing, surviving cells were found to maintain the inherent characteristics of human ES cells. CONCLUSION: 5% DMSO+50% FBS+10% EG may be an optimal cryoprotectant for the slow freezing-rapid thawing of human ES cells.     

Quantitative,functional, morphological and ultrastructural recovery of platelets as predictor for cryopreservation

Cryopreservation of platelets is of great interest, since it could extend the shelf life of therapeutic platelet concentrates and facilitate stockpiling and inventory control in blood banking. Despite the use of many cryopreservation procedures the optimal cryopreservation procedure is not defined yet. We have compared the cryopreservation of human platelets by various protocols employing controlled-rate and non-controlled-rate freezing procedures in combination with different concentrations of DMSO (6% and 10%) or 5% DMSO + 6% HES combination. After storage for 1 to 3 months, samples were thawed and analyzed. Measurements included cell recovery, platelet viability according to hypotonic shock response (HSR), platelet aggregation with ADP, morphological and ultrastructural properties of defrozen platelets. Our findings show that the application of our original procedure for controlled-rate freezing consisting of six cooling steps (cooling rate 1 degree C/min) with compensation of released heat of fusion (cooling rate 2 degrees C/min) has significantly influenced the quality of thawed platelets. At the same time, a concentration of 6% DMSO proved to be the most effective. In summary, cryopreservation of human platelets using controlled-rate freezing procedure in combination with lower (6%) DMSO concentration resulted in less damage from freezing and higher recovered function of platelets.     

Effect of platelet-rich plasma on the in vitro proliferation and osteogenic differentiation of human mesenchymal stem cells on distinct calcium phosphate scaffolds: the specific surface area makes a difference

The in vitro effect of platelet-rich plasma (PRP) on cell loading, proliferation, and osteogenic differentiation of human mesenchymal stem cells (MSC) is assessed on distinct resorbable and synthetic calcium phosphate scaffolds. A high specific surface area scaffold composed of calcium-deficient hydroxyapatite (CDHA; 48m2/g) is compared with one made out of beta-tricalcium phosphate (beta-TCP; surface area <0.5 m2/g). Fivefold concentrated fresh PRP is applied to scaffolds loaded with 2 x 10(5) MSC (n = 5). These constructs are kept in a medium with osteogenic supplements for 3 weeks. The addition of PRP leads to a higher cell loading efficiency of MSC on CDHA (p = 0.0001), that reaches the values of beta-TCP. Proliferation over 21 days is improved by PRP both on CDHA (p = 0.0001) and beta-TCP (p = 0.014) compared to MSC/calcium phosphate composites. Without the addition of PRP, CDHA has a lower cell loading efficiency (p= 0.0001) and proliferation (p= 0.001) than beta-TCP. The ALP activity is higher in the MSC/ceramics groups than in the monolayer controls (p<0.05). The addition of PRP does not significantly affect ALP activity. However, ALP activity varies considerably within the cell donors and different PRP-pools (p = 0.001), while the cell numbers do not vary within these two parameters. PRP generates a positive effect on the loading efficiency of MSC on the high specific surface scaffold CDHA that thereby reaches the loading efficiency of beta-TCP. PRP improved proliferation, but its osteogenic properties on both calcium phosphate scaffolds are weak.     

A Cryopreservation Method of Human Peripheral Blood Mononuclear Cells for Efficient Production of Dendritic Cells

The establishment of a cryopreservation method for unstimulated fresh peripheral blood mononuclear cells (PBMC) with nearly 100% viability would greatly contribute to the conduct of various immunological experiments. The cells most sensitive to freezing and thawing procedure seem to be dendritic cells (DC) and their precursors, which are of the most potent antigen-presenting cells. The authors investigated and established a method of cryopreserving fresh PBMC from which DC were recovered and differentiated efficiently by using recombinant (r) GM-CSF and rIL-4. PBMC frozen in the presence of 12% dimethylsulfoxide and 25–30% fetal calf serum recovered DC as efficiently as freshly obtained PBMC. Established DC could also be cryopreserved in the presence of 12% DMSO with their viability maintained at more than 90%. The 12% DMSO freezing solutions were superior to both the 10% DMSO solution and the previously reported DC freezing medium (2 m or 15.4% DMSO). The DC obtained from the cryopreserved PBMC expressed HLA-DR, HLA-DQ, CD80 and CD86 antigens, and stimulated allogenic PBMC to an extent almost identical to that obtained from fresh PBMC. These findings indicate that the conditioned medium utilized here enables safe cryopreservation of DC and DC precursors in PBMC.     

不同组织冻存体系对牙周膜干细胞体外扩增的影响

背景：冻存是保证干细胞移植治疗的关键步骤之一。传统的冻存是将细胞直接置于冻存液中进行保存,然而冻存液中二甲基亚砜虽能减少细胞复苏过程中冰晶对细胞膜产生的机械性损伤,但同时又对细胞具有毒性作用,直接影响细胞生存状态,不利于临床移植治疗。 目的：寻找适宜牙周膜干细胞体外扩增的牙周周组织冻存的最佳方案。 方法：收集健康人牙,刮取牙周组织后将其平均分为3等份,随机分为新鲜组、5%二甲基亚砜组和10%二甲基亚砜组,后2组分别以体积分数5%和10%二甲基亚砜添加冻存1个月后提取牙周膜干细胞。新鲜组直接提取牙周膜干细胞。 结果与结论：5%二甲基亚砜组原代细胞游出组织团块所需时间和细胞收获量虽不及新鲜培养组,但却明显优于10%二甲基亚砜组(P < 0.05)。新鲜组、5%二甲基亚砜组和10%二甲基亚砜组第1代牙周膜干细胞克隆形成率、活细胞比率、第3代牙周膜干细胞BrdU细胞增殖能力、MTT细胞生长曲线和牙周膜干细胞表面标志物表达差异没有显著性意义(P > 0.05)。提示5%二甲基亚砜添加冻存体系不仅能比10%二甲基亚砜添加的普通冻存体系明显缩短牙周膜干细胞体外扩增所需时间,增加细胞收获量同时还能保持细胞基本生物学特性,降低二甲基亚砜的总体用量和其在反复冻存复苏细胞过程中对细胞造成的直接损伤,为未来更加安全的实施临床移植治疗提供了保障,是供体组织储存新的选择。     

Collection, cryopreservation, and characterization of human dental pulp-derived mesenchymal stem cells for banking and clinical use

Recent studies have shown that mesenchymal stem cells (MSC) with the potential for cell-mediated therapies and tissue engineering applications can be isolated from extracted dental tissues. Here, we investigated the collection, processing, and cryobiological characteristics of MSC from human teeth processed under current good tissue practices (cGTP). Viable dental pulp-derived MSC (DPSC) cultures were isolated from 31 of 40 teeth examined. Of eight DPSC cultures examined more thoroughly, all expressed appropriate cell surface markers and underwent osteogenic, adipogenic, and chondrogenic differentiation in appropriate differentiation medium, thus meeting criteria to be called MSC. Viable DPSC were obtained up to 120 h postextraction. Efficient recovery of DPSC from cryopreserved intact teeth and second-passage DPSC cultures was achieved. These studies indicate that DPSC isolation is feasible for at least 5 days after tooth extraction, and imply that processing immediately after extraction may not be required for successful banking of DPSC. Further, the recovery of viable DPSC after cryopreservation of intact teeth suggests that minimal processing may be needed for the banking of samples with no immediate plans for expansion and use. These initial studies will facilitate the development of future cGTP protocols for the clinical banking of MSC.     

Identification and reduction of cryoinjury in endothelial cells: a first step toward establishing a cell bank for vascular tissue engineering

We analyzed a cryopreservation protocol which improves long-term storage of endothelial cells (EC) for tissue engineering purposes. Human umbilical vein EC were frozen in a high-potassium solution containing 10% dimethyl sulfoxide using 3 different cooling rates. After a storage time in liquid nitrogen of 1, 4, or 12 months, samples were thawed and compared to fresh cells in terms of growth rates, anti-inflammatory, and anticoagulant functions. Independent of cooling rate and storage time, the retrieval after cryopreservation ranged between 60% and 80%. However, viability of the cells cryopreserved at 10 degrees C/min decreased significantly from 78 +/- 5% to 64 +/-3% with storage. Storage time of 4 months resulted in a decreased cell multiplication factor over 4 and 12 days in culture. The lag phases returned to normal in the next passage. Thawed cells showed increased metabolic activity, reduced expression of thrombomodulin, and unchanged basal expression of adhesion molecules. However, the tumor necrosis factor-induced expression of adhesion molecules was significantly increased after long-term storage. This effect was partially compensated after expansion of the cells, whereas the prostacyclin release increased. Expansion of cryopreserved/thawed EC resulted in highly proliferative cells with antithrombotic properties and a capacity for inflammatory reactions, which makes them suitable for vascular tissue engineering.     

Cryopreservation based on freezing protocols for the long-term storage of microencapsulated myoblasts

One important challenge in biomedicine is the ability to cryogenically preserve not only cells, but also tissue-engineered constructs. In the present paper, alginate-poly-l-lysine-alginate (APA) microcapsules containing erythropoietin (Epo)-secreting C₂C₁₂ myoblasts were elaborated, characterized and tested both in vitro and in vivo. Dimethylsulfoxide (DMSO) was selected as cryoprotectant to evaluate the maintenance of physiological activity of cryopreserved microencapsulated myoblasts employing procedures based on freezing protocols up to a 45-day cryopreservation period. High chemical resistance of the cryopreserved microcapsules was observed using 10% DMSO as cryoprotectant following a standard slow-cooling procedure. Although a 42% reduction in Epo release from the microencapsulated cells was observed in comparison with the non-cryopreserved group, the in vivo biocompatibility and functionality of the encapsulated cells subcutaneously implanted in Balb/c mice was corroborated by high and sustained hematocrit levels over 194 days and lacking immunosuppressive protocols. No major host reaction was observed. Based on the results obtained in our study, a slow-cooling protocol using 10% DMSO as cryoprotectant (confirmed for cryopreservation periods up to 45 days) might be considered a suitable therapeutic strategy if the long-term storage of microencapsulated cells, such as C₂C₁₂ myoblasts is pretended.     

果糖和二硫苏糖醇对骨髓间充质干细胞冻存后活性及多能性的影响

背景:冻存是保证干细胞临床应用的关键步骤之一,但现有冻存技术常导致细胞活性降低、多能性丧失及分化能力下降。目的:探究果糖及二硫苏糖醇是否有助于维持冻存后骨髓间充质干细胞多能性及成骨分化潜能。方法:分离培养SD大鼠骨髓间充质干细胞,在细胞冻存前分别用果糖(200μmol/L),二硫苏糖醇(500μmol/L)μmol/L)及果糖(200μmol/L)+二硫苏糖醇(500预处理1 h。冻存6个月后,复苏细胞并用倒置显微镜观察细胞形态,MTT实验检测细胞活性,定量PCR检测相关干性基因(Nanog,Oct4及Sox2)的表达,碱性磷酸酶活性测试及茜素红染色检测复苏骨髓间充质干细胞成骨分化能力。结果与结论:(1)复苏后各组细胞在形态上无明显差别;(2)果糖预处理及联合预处理有助于骨髓间充质干细胞活性维持;(3)二硫苏糖醇预处理可显著促进骨髓间充质干细胞多能性相关基因Nanog及Sox2的表达;(4)果糖、二硫苏糖醇及联合预处理皆有助于维持骨髓间充质干细胞成骨分化潜能,但以二硫苏糖醇及联合预处理组效果最佳;(5)结果表明,果糖预处理有助于维持冻存骨髓间充质干细胞活性,二硫苏糖醇有助于维持冻存骨髓间充质干细胞多能性及成骨分化能力。     

Flow-cytometric screening of platelet antibodies with previously frozen cells

In this study the flow-cytometric crossmatch results were compared between fresh cells and cells processed by various cryopreservation and storage methods. Platelets from healthy donors were incubated with 12 sera containing platelet reactive antibodies as well as with 62 control sera from blood donors. Direct comparisons were made between fresh platelets and platelets after freezing at -28 degrees C, -40 degrees C and -80 degrees C and in liquid nitrogen, using 6% DMSO as cryoprotectant. In addition, the effects of using controlled-rate freezing were evaluated. Finally we evaluated the application of the cryoprotectant ThromboSol. The best results were obtained after cryopreservation of the platelets with ThromboSol at -80 degrees C with controlled cooling rates. Using ThromboSol cryopreserved platelets, the sensitivity for the detection of incompatible platelets was 100% and the specificity was 97.1%, using the previous results obtained with flow-cytometry, MAIPA and LCT as a reference. CONCLUSION: Platelets can be frozen using ThromboSol as the cryoprotectant, with controlled rate freezing and storage at -80 degrees C for the screening of platelet antibodies and for flow-cytometric crossmatch procedures. This system yields a reproducible and logistically simple method for platelet crossmatching that yields results superior to fresh cells and can be easily incorporated into standard clinical laboratory practices.