Cryopreservation of intact human articular cartilage.

Damaged articular cartilage (AC) impairs joint function and many treatment techniques are being investigated to determine their long term results. Successful cryopreservation of AC can provide a reliable source of intact matrix with viable chondrocytes to maintain the cartilage over long periods of time. This study investigated the application of an established cryopreservation protocol to determine the recovery of intact chondrocytes from human AC. Ten millimeter diameter osteochondral dowels were harvested from two human donors. The cryopreservation protocol was performed and the samples were rapidly warmed from varying experimental holding temperatures (-10, -20, -30, -40 degrees C), with and without plunging into liquid nitrogen, using 1 M dimethyl sulfoxide as cryoprotectant. The cartilage was stained with membrane integrity dyes and viewed under fluorescence microscopy. The percent of intact chondrocytes was compared to fresh controls. Low recovery of intact chondrocytes was recorded from all temperature levels with and without cryoprotectant. The results of this experiment demonstrated that the cryopreservation procedure used to achieve moderate success with intact sheep AC was not successful with intact human AC and further investigation is required.     

精子冻融技术及其研究进展

精子冻融是辅助生殖技术的基础,冻融可能造成精子结构损伤和活力功能丧失,因此优化冻融方法十分必要。提高冷冻前精液质量、进行精子优选、添加合适的冷冻保护剂、选择适宜的冷冻复苏方法等均可有效地减少冻融损伤,有利于保存男性生育力。本文对精子冻融的基本原理、方法改良、针对特殊质量精液的冷冻方法及冷冻后精子质量评估进行了综述。     

Cryopreservation of human semen

A review is given of the techniques for the cryopreservation of human semen, including the preparation of cryoprotective media, the use of ampoules, straws, and pellets, and freezing and thawing techniques. The use of cryopreserved semen for therapeutic artificial insemination by donor is described. The advantages of cryopreserved semen over fresh donor semen mostly lie in the ability to exclude infections before use and the extra convenience, in spite of the lower success rate and increased cost. The recovery of sperm motility on thawing is described, as are other methods for assessing the degree of damage to the spermatozoa by the freezing procedure. The success rates reported by large semen banks are summarized.     

脂肪组织的体外储存

自体脂肪移植已有上百年的历史，由于其来源丰富，取材方便，操作简单等优点，度甚为流行，但移植后吸收率较高这一弊端，限制了这一技术的应用与发展。20世纪80年代中期，有学者发现采用较小的颗粒状脂肪组织注射移植，其成活率明显提高，1986年Illouz提出了脂肪颗粒注射移植理论。随着这一理论的提出及近年来脂肪抽吸术的发展，再次掀起了脂肪移植的高潮。     

肝细胞的冷冻保存

肝细胞移植是目前治疗终末期肝病的有效措施,其包括肝细胞的分离、培养、冷冻保存等步骤.肝细胞的冷冻保存是其中重要的环节之一,决定了肝细胞复苏后的存活率.本文将对肝细胞的冻存问题作以下综述.     

未成熟卵母细胞冻存

随着人类辅助生殖技术的发展,人未成熟卵母细胞的冷冻技术因其特有优势得到了较多的关注,研究数据支持未成熟卵母细胞冻存是保存女性生育力的一种有临床应用前景的方式。冷冻和复融过程会对卵母细胞造成一系列形态结构、细胞代谢、生理生化及发育潜能的影响。作为一个较新的方向,未成熟卵母细胞的冷冻技术缺乏标准方案,很多相关研究成果尚存在争议。本文着重未成熟卵母细胞冷冻技术的意义、优势、影响因素等方面的新观点予以综述。     

Successful Cryopreservation of Microvenous Allografts

Vein grafts are used in approximately 20% of microsurgical cases. Although autogenous veins currently form the major source, they are associated with increased operating time and donor site scars. Cryopreserved allograft veins may serve as an alternative source. To our knowledge, cryopreservation of veins (1 mm or less) has not been reported. In this article we have described the process of cryopreserving rat veins (less than 1 mm in diameter) and their preliminary use as interpositional vein grafts.     

Cryopreservation of Composite Tissue Transplants

Composite tissue allotransplantation holds great promise for upper extremity reconstruction but is limited by donor part availability. Cryopreservation may increase the availability of donor parts and even reduce antigenicity. The purpose of the study was to evaluate the viability of cryopreserved composite tissues and to demonstrate the feasibility of microvascular isotransplantation of cryopreserved composite flaps. Twenty epigastric flaps were harvested from Lewis rats. Ten flaps were analyzed fresh. Ten flaps were perfused with dimethyl sulfoxide (DMSO)/trehelose cryoprotectant agent (CPA), frozen by controlled cooling to −140°C, and stored for 2 weeks. Flaps were evaluated by factor VIII endothelial staining and MTT tetrazolium salt assay. For the in vivo phase, 30 flaps were harvested. Ten were transplanted fresh to isogenetic recipient animals, ten were perfused with CPA and transplanted, and ten were cryopreserved for 2 weeks, thawed, and transplanted. All cryopreserved samples displayed intact vascular endothelia on factor VIII staining. On MTT analysis, the epithelial viability index for the cryopreserved samples was not significantly different from fresh controls (p = 0.12). All freshly transplanted flaps (10/10) were viable at 60 days. Nine of ten flaps in the perfused/transplanted group were viable at 60 days. Survival of cryopreserved/transplanted flaps ranged from 5 to 60 days. The skin and vascular endothelial components of composite tissue flaps appear to retain their viability after cryopreservation. The in vivo studies demonstrate that the long-term survival of cryopreserved composite tissue transplants is feasible and support an indirect injury, rather than direct injury from freezing or cryoprotectant agents, as the mechanism of flap loss. Presented at the Annual Meeting of the American Association for Hand Surgery, Rio Grande, Puerto Rico, January, 2007.     

Cryopreservation of cultured skin cells.

Cryopreservation of human skin keratinocytes and fibroblast cells has been evaluated using three cryoprotectants at various concentrations, three cooling rates and two warming rates. Optimal combinations of factors were determined. Selection for tolerance to cryopreservation was not detected after repeated freeze/thaw cycles. A beneficial effect of serum was shown to be cooling rate dependent.     

Cryopreservation of osteo-chondral grafts in rabbits

To study the Cryopreservation of osteoarticular allografts, a lateral femoral condyle of the rabbit was transplanted fresh, after uncontrolled freezing to-80°C with 4 weeks preservation, and after freezing 1°C per min to-100°C in 10 per cent dimethylsulphoxide medium with 4-week storage in liquid nitrogen. Autografts were used as controls. After 3 months, the incorporation of the grafted bone was good in all technically successful cases. The NADH₂ diaphorase activity and ³⁵S sulphate uptake indicated well-functioning chondrocytes in all autografts. In the allografts, areas lacking enzyme activity and lacking ³⁵S uptake were observed in cartilages with otherwise normally functioning chondrocytes. No differences were found between the three allograft groups. We conclude that freezing permits reasonably good short-term bank preservation of cartilage. We found no difference between conventional freezing and controlled slow freezing with preservative.     

Cryopreservation of osteo-chondral grafts in rabbits

To study the cryopreservation of osteoarticular allografts, a lateral femoral condyle of the rabbit was transplanted fresh, after uncontrolled freezing to -80 degrees C with 4 weeks preservation, and after freezing 1 degree C per min to -100 degrees C in 10 per cent dimethylsulphoxide medium with 4-week storage in liquid nitrogen. Autografts were used as controls. After 3 months, the incorporation of the grafted bone was good in all technically successful cases. The NADH2 diaphorase activity and 35S sulphate uptake indicated well-functioning chondrocytes in all autografts. In the allografts, areas lacking enzyme activity and lacking 35S uptake were observed in cartilages with otherwise normally functioning chondrocytes. No differences were found between the three allograft groups. We conclude that freezing permits reasonably good short-term bank preservation of cartilage. We found no difference between conventional freezing and controlled slow freezing with preservative.     

Cryopreservation prevents arterial allograft dilation

Historically, immune-mediated degradation and subsequent aneurysm formation have limited the usefulness of cryopreserved arterial allografts. This study tested the hypothesis that modern cryopreserved arterial allografts are protected from immune-mediated dilation. Abdominal aortas were harvested from anesthetized rats (Lewis and Brown-Norway) for immediate implantation or cryopreservation. Subsequently, Lewis rats underwent infrarenal aortic replacement with either an acutely harvested or a cryopreserved graft. There were four experimental groups: (1) acutely harvested isografts (Iso; n = 6), (2) cryopreserved isografts (C-Iso; n = 6), (3) cryopreserved allografts (C-Allo; n = 6), and (4) acutely harvested allografts (Allo; n = 6). All grafts were explanted at 8 weeks. A video camera and edge detection software were used to measure systolic and diastolic in vivo graft diameter (d). Measurement of arterial blood pressure (p) allowed calculation of compliance (Dd/Dp). Tail-cuff plethysmography was used to assess graft patency at 1 week. Graft diameter and blood pressure measurements were repeated at harvest. All harvested grafts were examined histologically. Our results showed that cryopreservation prevented immune-mediated dilation in arterial allografts in our 8-week rat implant model. Furthermore, the compliance of the cryopreserved grafts and was similar to that of controls. Further investigation is needed to delineate the exact mechanism of these potential cliniclly significant findings.     

海藻糖对低温保存皮肤的作用

目的 研究非渗透性低温保护剂海藻糖联合应用二甲基亚砜(DMSO)，用于人类皮肤的低温储存，并与皮厍常规应用的二甲基亚砜(DMSO)、丙二醇(propyleneglycol)、DMEM进行比较，以寻求更佳的皮肤冷冻保护剂。方法将新鲜成人皮肤分为5组，液氮冻存7、14d后复温，采用病理形态学、酶组织化学、组织代谢等方法，分别对海藻糖／二甲基亚砜、二甲基亚砜／丙二醇、二甲基亚砜／无血清角质细胞培养基(KSFM)、DMEM作为冷冻保护剂保存后的皮肤活力进行比较。结果O．5mol／L海藻糖／二甲基亚砜作为保护剂所冷冻的皮肤，复温后其活性明显高于其他保护荆组。结论海藻糖与二甲基亚砜联合应用是较佳的冷冻保护剂，皮库在常规应用保护剂时可以在渗透性保护剂中加入海藻糖。     

Cryopreservation and allogeneic transplantation of tendon tissue

The authors describe their methods of sterilization, cryopreservation and lyophilization of the tendinous tissue which provide sterility and preservation of the structure and the plastic properties. They present a technique of saturation of the plastic material with biologically active preparations selected with regard to the aims the character of the surgical intervention and new methods of alloplastic reconstruction of the tendons of the wrist. The long-term results of the treatment of 303 patients with inveterate injuries of the tendons and of the bursal and ligamentous apparatus who were given 432 cryopreserved allografts have been analysed. In 89.6% of the cases the results were good and satisfactory.     

Cryopreservation of freshly isolated porcine islet cells

INTRODUCTION: The use of xenogenic islet cells may be a possibility to overcome the shortage of human donor organs to treat diabetes. Microencapsulation seems to be a promising method for immunoprotection. Since isolation, purification, encapsulation, and transplantation of islet cells are labor intensive, cryopreservation has emerged as an attractive system of islet banking. The aim of this study was to determine the influence of three different freezing media (FM) on viability of freshly isolated porcine islet cells (FIPIC). METHODS: FIPIC were isolated using a modified Ricordi method and purification performed using a Lymphoprep density gradient. Viability of FIPIC prior to freezing and after thawing was determined using the MTT-based Cell Growth Determination Kit. Insulin production was detected using enzyme-linked immunosorbent assay. Three different FM containing dimethylsulfoxide (DMSO) or glycerol and sucrose were used for cryoprotection of FIPIC. RESULTS: Isolation and purification of FIPIC resulted in 95% +/- 1.3% viability and 97% +/- 1.4% purity. Cryopreservation with FM I (containing DMEM, FCS, DMSO) yielded 98.4% and FM III (containing DMEM, FCS, glycerol) 93.1% viability, whereas only 85.6% were alive when cryoprotection is performed with FM II (containing DMSO, BM). Glucose stimulation revealed a loss of 2.8% and 1.9% of insulin secretion per microgram DNA when working with FM I and FM III, but a decrease in glucose-dependent insulin secretion of 7.8% (P < .05) when FIPIC were stored in FM II. DISCUSSION: Low concentrations of DMSO or the use of glycerol and sucrose seem to be equivalent to cryopreserve FIPIC.     

脱细胞软骨支架材料修复兔关节软骨缺损

目的 观察异种异体脱细胞软骨支架材料(ACM)复合同种异体兔骨髓间充质干细胞(rBMSCs)修复兔股骨内髁关节软骨缺损的效果.方法 (1)密度梯度离心和差速贴壁法获得原代兔BMSCs,选择第3代BMSCs作为种子细胞;(2)利用冷冻干燥、胰酶消化和化学去垢剂等方法制备脱细胞软骨支架材料;(3)3个月龄新西兰兔股骨内髁制备直径4 mm,深3 mm砌关节软骨缺损模型,24只新西兰兔以2个时间段随机分为3组,Ⅰ ACM-BMSCs组:第3代BMSCs 1×106个/ml与ACM于37℃5%CO2饱和湿度复合48 h;Ⅱ ACM组;Ⅲ空白对照组.(4)移植6、12周后大体及组织学观察,免疫组织化学染色观察修复组织Ⅱ型胶原,Wakitani评分评估修复效果.结果 (1)大体观察及组织学观察:6和12周Ⅰ组再生组织与正常关节软骨面平齐,修复部位表面较平整,界限模糊,接近正常软骨.Ⅱ组修复组织表面不平整并有明显下陷,修复组织全层可见成纤维样细胞,深层可见极少数透明软骨样细胞.Ⅲ组未见明显修复,肉芽组织形成伴成纤维样细胞增生;(2)Wakitani组织学评分可见在不同的时间段I组和Ⅱ组均低于Ⅲ组,差异有统计学意义(P<0.05),Ⅰ组和Ⅱ组间组织学评分差异无统计学意义(P>0.05).(3)免疫组织化学:ACM-BMSCs组修复组织的细胞为软骨样细胞,可见柱状排列,周围软骨基质Ⅱ型胶原染色阳性.结论 以ACM为支架材料,同种异体BMSCs为种子细胞制备的组织工程化软骨对兔股骨内髁关节软骨缺损有修复作用,形成的新生软骨为透明软骨样组织.