玻璃化保存肌腱的生物力学观察

目的探讨玻璃化保存法对兔肌腱力学性能的影响。方法玻璃化保存组，6条新鲜胫前肌肌腱，以18．64％二甲基亚砜＋13.37％乙酰胺＋9．17％1．2丙二醇＋浓度0．10mmol／L海藻糖＋10％小牛血清为玻璃化冷冻保护剂．将新西兰纯种大白兔肌腱采用三步法预处理．-196℃液氮保存14d；“两步法”深低温冷冻保存组，6条新鲜胫前肌肌腱，以15％二甲基亚砜＋10％小牛血清作为冷冻保护剂，“两步法”处理后-196℃液氮冷冻保存14d；对照组．6条新鲜新西兰纯种大白兔胫前肌肌腱。分别进行肌腱拉伸实验．检测肌腱破坏载荷峰值、最大载荷拉伸位移及杨氏弹性模量。结果肌腱破坏荷载峰值：新鲜肌腱组与玻璃化保存组及“两步法”深低温冷冻保存组间差异均有统计学意义（P=0．002），玻璃化保存组与“两步法”深低温冷冻保存组无统计学意义（P=0．256）；最大载荷拉伸位移：新鲜肌腱组与玻璃化保存组及“两步法”深低温冷冻保存组3组间均无统计学意义（P=0．065）；杨氏弹性模量：新鲜肌腱组与玻璃化保存组及“两步法”深低温冷冻保存组间差异均有统计学意义（P=0．006）．玻璃化保存组与“两步法”深低温冷冻保存组差异无统计学意义（P=0．577）。结论玻璃化保存法保存肌腱具有良好的发展前景。     

组织工程研究与开发中的冻存技术

综述了有关组织工程种子细胞、支架材料和工程化组织的冷冻保存问题。对冻存技术在组织工程研究和开发中的重要作用进行了阐述。指出低温保存可能是长时间保存有活性的组织工程产品的有效方法之一。组织工程种子细胞采用低温冷冻保存 ;组织工程支架材料用冷冻干燥保存 ;工程化组织的保存采用玻璃化冷冻保存方法。     

不同保存方式下嗅鞘细胞的活性

背景：获取和寻找适合的保存方式对嗅鞘细胞实验和临床应用有重要的意义。 目的：探索合适的嗅鞘细胞低温保存方式。 方法：取对数期生长的嗅鞘细胞,冻存1,3,6个月进行复苏。 结果与结论：MTT比色法及锥虫蓝染色显示5%二甲基亚砜-6%羟乙基淀粉处理的细胞活性最高,其次是10%二甲基亚砜处理的细胞,5%二甲基亚砜的保护作用最差。冰箱降温或程控降温仪降温方式及不同的冻存时间对嗅鞘细胞活性影响不明显。因此,推荐用5%二甲基亚砜-6%羟乙基淀粉作为嗅鞘细胞冻存低温保护剂。     

SD大鼠肌腱冷冻维持温度选择的实验研究

目的：探讨不同维持温度冷冻预处理超深低温保存对肌腱超微结构的影响，为同种异体肌腱保存移植提供依据。方法：实验组设计维持温度为-45℃、-50℃、-55℃、-60℃、-65℃、和-70℃组，以15％二甲基亚砜为低温保护剂，对SD大鼠肌腱进行上述温度冷冻预处理，液氮中保存1周，HE染色组织学观察和透射电镜肌腱超微结构观察。结果：电镜下-45℃组胶原纤维排列较稀疏；-60℃组多数腱细胞胞浆水肿，核膜不完整；-65℃组胞浆内细胞器破坏；-70℃组部分腱细胞坏死脱落。-50℃组和-55℃组与对照组之间差异不明显，肌腱组织超微结构得到良好保持。结论：肌腱组织冷冻预处理的维持温度在-50℃～-55℃之间，-50℃是肌腱组织冷冻预处理的最佳维持温度值，可获得具有大部分生物活性的肌腱。     

人骨软骨组织体外冷冻保存的效果

背景：异体骨软骨移植是治疗关节软骨缺损的有效方法,然而由于移植物体外有效保存时间短,限制了临床应用以及移植物的利用效率。 目的：观察梯度降温冷冻保存同种异体关节软骨的保存效果。 方法：将关节软骨块经体积分数10%二甲基亚砜预处理后,应用程序冷冻仪以-1 ℃/min行梯度降温至-4 ℃,-10 ℃,     -20 ℃,-40 ℃各30 min,最后至-80 ℃冷冻保存。 结果与结论：保存1,3,6个月及1年后检测发现,与新鲜组相比,冷冻后软骨细胞存活率、细胞活性以及浅、中、深层的蛋白多糖水平下降(P < 0.05）,且软骨细胞存活率、细胞活性以及浅、中、深层的蛋白多糖水平随冷冻保存时间延长而降低(P < 0.05）,提示冷冻保存可有效地延长骨软骨移植物存活时间。     

玻璃化深低温冷冻保存气管的实验研究

玻璃化深低温冷冻在降温过程中大大减少冰晶的形成，极大的减轻了冷冻保存过程中对组织、细胞的损伤，前景广阔。本文旨在研究玻璃化快速降温对气管组织结构的影响，实验分为玻璃化快速降温组、程控缓慢降温组、新鲜未冷冻组3组，通过HE染色、TUNEL染色、透射电镜及扫描电镜等检查，评价不同冷冻方法对气管软骨和纤毛结构影响。结果显示，玻璃化快速降温组的气管软骨板结构完整，黏膜上皮及其纤毛结构、软骨细胞形态保持良好，与传统程控缓慢降温相比，快速降温冷冻对软骨细胞损伤轻，有更高的软骨细胞存活率和纤毛上皮的覆盖率，获得较高的软骨复苏率，较好地保持形态结构完整，其保存效果优于传统缓慢降温的保存效果。     

血管低温保存的实验研究

血管组织的低温保存具有重要的药疗和实验研究价值。低温保存过程是离不开抗冻剂的 ;合理地选择和配制抗冻剂 ,并选取最佳降温与复温速率 ,对低温保存的成败是至关重要的。本文主要研究了在最佳降温复温过程中抗冻剂DMSO浓度变化对小牛主动脉保存后细胞活性和功能的影响。实验结果表明 :DMSO浓度为15 %时 ,血管保存效果最好 ,70 %的血管组织超微结构保存完好 ,血管对 10 -6Mnorepinephrine的收缩力达到新鲜血管的 80 %左右 ,对acetylcholine的舒张力达到 30～ 4 0 % ,动脉血管的其他功能保持完好。     

移植用微囊化细胞低温保存的研究

目的：探索移植用微囊化细胞低温保存的可行性。方法：以海藻酸钠-聚赖氨酸-海藻酸钠微胶囊（APA微胶囊）为研究体系，以转内皮抑素重组中国仓鼠卵巢细胞（rCHO）为模型细胞，观察低温保存过程中降温方式、预平衡方式、冷冻保护剂浓度等关键条件对微囊化细胞复苏活性的影响，以及低温保存后微囊膜对IgG的通透性及微囊内细胞的存活、增殖及产物分泌情况。结果：包埋功能细胞的APA微囊经一定时间的低温保存后，微囊膜仍可屏蔽IgG，并未改变其原有的免疫隔离特性，且囊内细胞具有较高的细胞活性，能够正常生存并保持增殖和产物分泌能力。结论：微囊化细胞进行低温保存是可行的。     

不同时段超低温保存对兔肌腱力学性能的影响

目的 :探讨不同时段超低温保存对兔肌腱组织生物力学性能的影响。方法 :以 15 %DMSO(二甲基亚砜 )为低温保护剂 ,采用两步冷冻保存步骤 ,对青紫兰兔肌腱进行维持温度 - 5 0℃的冷冻预处理 ,- 196℃液氮保存 18d、180d ,测试前用 4 0℃水浴复温 ,每组肌腱 8段 ,与新鲜肌腱对照进行拉伸实验。结果 :肌腱的破坏荷载在对照组、18d组、180d组分别为 (6 6 .0± 19.4 9)N、(6 5 .0± 7.0 7)N和 (5 4 .0± 11.4 )N ,各组肌腱的延伸率 (% )依次为 6 3.5± 17.3、38.6± 0 .98和 30 .6± 0 .4 7。结论 :经不同时段超低温冷冻保存的兔肌腱 ,拉伸实验的生物力学指标无明显差异 (P>0 .0 5 )。     

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。     

Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts

BACKGROUND: Successful cryopreservation of gonadal tissue is an important factor in guaranteeing the fertility preservation via germ cell or testicular tissue transplantation. The aim of this study was to evaluate the effects of cooling and cryopreservation on spermatogonial stem cell survival and function of immature non-human primate testicular tissue xenografted to nude mice. METHODS: Group 1 (control group) received subcutaneous grafts of fresh immature rhesus monkey testes. The treatment groups received grafts after 24 h cooling in ice-cold medium (Group 2), after 24 h of cryopreservation without cryoprotectant (Group 3), with ethylene glycol (Group 4: 1.4 M) or with dimethylsulphoxide (DMSO) (group 5: 1.4 M; group 6: 0.7 M), using cooling rates of 0.5 degrees C/min. The graft number, weight and histology were examined 3-5 months later. RESULTS: After xenografting, grafts from fresh and cooled tissue showed good survival and spermatogenic induction to spermatocytes. Cryopreservation in 1.4 M DMSO also allowed grafts to initiate spermatogenesis. In contrast, 0.7 M DMSO and ethylene glycol showed inferior protection. CONCLUSIONS: Our observations suggest that cryopreservation of immature primate testis is a feasible approach to maintain spermatogonial stem cells and may serve as a promising tool for fertility preservation of prepubertal boys. The possibility to delay the transplantation of cooled samples suggests an option for clinical centralization of testicular tissue cryopreservation.     

Effects of freezing-induced cell-fluid-matrix interactions on the cells and extracellular matrix of engineered tissues

The two most significant challenges for successful cryopreservation of engineered tissues (ETs) are preserving tissue functionality and controlling highly tissue-type dependent preservation outcomes. In order to address these challenges, freezing-induced cell-fluid-matrix interactions should be understood, which determine the post-thaw cell viability and extracellular matrix (ECM) microstructure. However, the current understanding of this tissue-level biophysical interaction is still limited. In this study, freezing-induced cell-fluid-matrix interactions and their impact on the cells and ECM microstructure of ETs were investigated using dermal equivalents as a model ET. The dermal equivalents were constructed by seeding human dermal fibroblasts in type I collagen matrices with varying cell seeding density and collagen concentration. While these dermal equivalents underwent an identical freeze/thaw condition, their spatiotemporal deformation during freezing, post-thaw ECM microstructure, and cellular level cryoresponse were characterized. The results showed that the extent and characteristics of freezing-induced deformation were significantly different among the experimental groups, and the ETs with denser ECM microstructure experienced a larger deformation. The magnitude of the deformation was well correlated to the post-thaw ECM structure, suggesting that the freezing-induced deformation is a good indicator of post-thaw ECM structure. A significant difference in the extent of cellular injury was also noted among the experimental groups, and it depended on the extent of freezing-induced deformation of the ETs and the initial cytoskeleton organization. These results suggest that the cells have been subjected to mechanical insult due to the freezing-induced deformation as well as thermal insult. These findings provide insight on tissue-type dependent cryopreservation outcomes, and can help to design and modify cryopreservation protocols for new types of tissues from a pre-developed cryopreservation protocol.     

细胞黏附基础与肌腱组织工程

细胞黏附是组织工程学中一个基础而非常重要的问题 ,纤连蛋白和整合素受体是细胞黏附中最重要的结构 ,同时成纤维细胞还通过表面受体直接与基质中的 I型胶原结合 ,另外成纤维细胞也可通过表面受体与纤层蛋白结合等而使细胞黏附。本文综合了近几年来国外的有关文献 ,详细阐述了纤连蛋白和整合素的结构及功能。介绍肌腱组织中与细胞黏附有关的结构。提出组织工程学产品保存过程中细胞与细胞外基质间黏附力的影响尚无文献报道 ,研究细胞与细胞外基质之间的黏附 ,寻找对其不影响或影响很小的保存方法是我们需要解决的一个重要课题。细胞黏附的深入研究对组织工程领域可望有良好的应用前景     

Cryopreservation of rhesus macaque embryonic stem cells

Studies using nonhuman primate embryonic stem (ES) cells will facilitate the translation of basic ES cell research to clinical use by providing a large animal model for in vivo testing. Unfortunately, nonhuman primate ES cells do not survive well following cryopreservation, a problem that limits the quantity and quality of these cells for research. More lines have to be established to fulfill demand, and thawed aliquots must go through more passages to generate adequate numbers. In addition, suboptimal cryopreservation can induce epigenetic changes and impose a selection bias for their outgrowth. Therefore, defining the optimal cryopreservation technique for nonhuman primate ES cells is critical for the further development of this research. To address this problem, we tested various cryoprotectants as well as cryopreservation procedures in an attempt to define a protocol that yields high viability with retention of ES cell phenotype and function. Here, we report a freezing protocol that preserves the intercellular attachments that are vital to primate ES cell function. We describe a slow, controlled-rate cooling protocol with ice crystal induction that increased the survival rate of ES cells from <22% to >90%. Preserved cells retained a normal karyotype and did not lose their ability to express markers of undifferentiated ES cells.     

How to optimize seeding and culturing of human osteoblast-like cells on various biomaterials

The optimization of seeding and culturing of human osteoblast-like cells on three collagen-based biomaterials (bovine, equine and calf collagen membrane) was studied by cell proliferation and cell colonization (scanning electron microscopy) analysis. Osteoblasts of five patients were seeded onto the three biomaterials and two different parameters were varied: the time intervals between initial seeding and adding culture medium (2 h 6 h. 12 h, 24 h) and the seeding concentration (1 x 10(5), 1 x 10(6), 2 x 10(6)cells/ml) of cells onto biomaterials. The results of the study demonstrated that the time interval between seeding osteoblasts and adding culture medium as well as the seeding concentration effects the cell proliferation and the cell colonization. The best proliferation rate was achieved by adding the culture medium 2 h after initial seeding and with a seeding density of 1 x 10(5) cells/ml. Moreover, all three biomaterials resulted in different proliferation rates. The best proliferation rate resulted with the bovine collagen membrane. In conclusion, the examined parameters are very important for the development of the tissue engineering techniques and in a larger perspective also for reconstructive surgery.     

Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes.

The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2 degrees C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by approximately 23 degrees C in mouse and only by approximately 6.5 degrees C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the incidence of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31(32%), 19/34 (56%) and 52/56 (93%) respectively. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5 degrees C.     

The effect of mechanical stimulation on the maturation of TDSCs-poly(L-lactide-co-e-caprolactone)/collagen scaffold constructs for tendon tissue engineering

Mechanical stimulation plays an important role in the development and remodeling of tendons. Tendon-derived stem cells (TDSCs) are an attractive cell source for tendon injury and tendon tissue engineering. However, these cells have not yet been fully explored for tendon tissue engineering application, and there is also lack of understanding to the effect of mechanical stimulation on the maturation of TDSCs-scaffold construct for tendon tissue engineering. In this study, we assessed the efficacy of TDSCs in a poly(L-lactide-co-ε-caprolactone)/collagen (P(LLA-CL)/Col) scaffold under mechanical stimulation for tendon tissue engineering both in vitro and in vivo, and evaluated the utility of the transplanted TDSCs-scaffold construct to promote rabbit patellar tendon defect regeneration. TDSCs displayed good proliferation and positive expressed tendon-related extracellular matrix (ECM) genes and proteins under mechanical stimulation in vitro. After implanting into the nude mice, the fluorescence imaging indicated that TDSCs had long-term survival, and the macroscopic evaluation, histology and immunohistochemistry examinations showed high-quality neo-tendon formation under mechanical stimulation in vivo. Furthermore, the histology, immunohistochemistry, collagen content assay and biomechanical testing data indicated that dynamically cultured TDSCs-scaffold construct could significantly contributed to tendon regeneration in a rabbit patellar tendon window defect model. TDSCs have significant potential to be used as seeded cells in the development of tissue-engineered tendons, which can be successfully fabricated through seeding of TDSCs in a P(LLA-CL)/Col scaffold followed by mechanical stimulation.     

Cryopreservation of Mycobacterium tuberculosis Complex Cells

Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P < 0.05). Among the three defined cryopreservation media (PBS, 7H9, and 7H9 plus glycerol), there was no significant differential effect on viability (P = 0.06 to 0.87). Preincubation of thawed M. tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P = 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of “live” cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability.     

组织块法培养成年兔肌腱细胞

背景：体外分离培养肌腱细胞,熟悉其生物学特性是研究肌腱愈合机制、改善肌腱愈合内环境的前提和基础。 目的：采用组织块法体外培养成年兔肌腱细胞,观察其细胞形态、生长增殖情况以及Ⅰ型、Ⅲ型胶原蛋白的表达。 方法：无菌条件下取成年新西兰白兔双侧后肢趾屈肌腱,手术显微镜下剥离、去除肌腱外膜组织,剪成组织小块,0.25%胰蛋白酶和0.1%胶原酶Ⅰ消化10~15 min,离心去上清,将组织小块转移到培养瓶中,贴壁后加入1 mL培养液,待细胞游出贴壁生长时,再加培养液继续培养,每3 d换液1次,当细胞长到80%~90%融合时按1∶3传代。 结果与结论：一般10 d左右细胞会从组织块游出贴壁生长,此时细胞多呈星形或不规则形,随着时间延长细胞逐渐增多,呈梭形的成纤维细胞样。传代细胞刚接种时圆形,4~6 h开始贴壁并伸展为梭形,排列逐渐规则成群。从传代细胞的生长曲线可看出,前4 d细胞生长较慢,为潜伏期,第5天后进入快速增殖期,7 d以后进入平台期。免疫荧光法证实Ⅰ型胶原染色阳性,而Ⅲ型胶原染色呈阴性。结果表明采用组织块法可在体外成功培养成年兔肌腱细胞。     

Differences in the requirements for cryopreservation of porcine aortic smooth muscle and endothelial cells.

One of the basic requirements for the production of tissue-engineered constructs is an effective means of storing both the constructs and the cells that will be used to make them. This paper reports on the cryopreservation of porcine aortic smooth muscle and endothelial cells intended for the production of model vascular constructs. We first determined the cell volume, nonosmotic volume, and the permeability parameters for water and the cryoprotectant dimethyl sulfoxide (Me(2)SO) in these cells at 2-4 degrees and 22 degrees C. The following results were obtained: Table unavailable in HTML format. Using a cell culture assay, both cell types were shown to tolerate threefold changes in cell volume, in either direction, without significant injury. Although these data suggested that single-step methods for the introduction and removal of 10% w/w Me(2)SO should be effective, an additional mannitol dilution step was adopted in order to reduce the time required for removal of the Me(2)SO. Following cooling at 0.3, 1, or 10 degrees C/min and storage at less than -160 degrees C, the survival of porcine aortic smooth muscle cell suspensions, measured by a cell culture assay, was inversely related to cooling rate; at 0.3 degrees C/min, recovery was >80%. The survival rate for aortic endothelial cells was directly related to cooling rate over the range tested and was >80% at 10 degrees C/min.