Antigenicity of cryopreserved arterial allografts: comparison with fresh and glutaraldehyde treated grafts

The use of cryopreserved aortic allografts in cardiovascular surgery is widespread and has resulted in excellent outcomes. However, it is controversial whether cryopreservation suppresses the antigenicity of tissue. We designed experimental models to study whether the cryopreservation process alters antigenicity in comparison with that found in fresh and glutaraldehyde treated tissues. Fresh, cryopreserved, and glutaraldehyde treated thoracic aorta from Brown Norway rats were subcutaneously implanted into Lewis rats. Inflammatory cells infiltrating around the grafts were measured on days 7, 14, 28, and 56 after implantation. The glutaraldehyde treated grafts showed significantly less infiltration than the fresh or cryopreserved grafts (p < 0.005). No significant difference was detected between the fresh and cryopreserved grafts. Another study examined the effect of modifications of the aortic allograft on subsequent allogeneic skin graft antigenicity. Subcutaneous implantation of fresh, cryopreserved, and glutaraldehyde treated aortic grafts from Brown Norway into Lewis rats resulted in subsequent skin graft rejection at 4.4+/-0.7, 5.1+/-0.8, and 6.6+/-2.1 days, respectively. There was no significant difference between the fresh and cryopreserved groups; whereas skin grafts in the glutaraldehyde group survived longer than those in the cryopreserved group. These results indicate that cryopreservation had no significant influence on antigenic suppression of arterial allografts.     

Guidance for removal of fetal bovine serum from cryopreserved heart valve processing

Bovine serum is commonly used in cryopreservation of allogeneic heart valves; however, bovine serum carries a risk of product adulteration by contamination with bovine-derived infectious agents. In this study, we compared fresh and cryopreserved porcine valves that were processed by 1 of 4 cryopreservation formulations, 3 of which were serum-free and 1 that utilized bovine serum with 1.4 M dimethylsulfoxide. In the first serum-free group, bovine serum was simply removed from the cryopreservation formulation. The second serum-free formulation had a higher cryoprotectant concentration, i.e. 2 M dimethylsulfoxide, in combination with a serum-free solution. A colloid, dextran 40, was added to the third serum-free group with 2 M dimethylsulfoxide due to theoretical concerns that removal of serum might increase the incidence of tissue cracking. Upon rewarming, the valves were inspected and subjected to a battery of tests. Gross pathology revealed conduit cracking in 1 of 98 frozen heart valves. Viability data for the cryopreserved groups versus the fresh group demonstrated a loss of viability in half of the comparisons (p < 0.05). No significant differences were observed between any of the cryopreserved groups, with or without bovine serum. Neither routine histology, autofluorescence-based multiphoton imaging nor semiquantitative second-harmonic generation microscopy of extracellular matrix components revealed any statistically significant differences. Biomechanics analyses also revealed no significant differences. Our results demonstrate that bovine serum can be safely removed from heart valve processing and that a colloid to prevent cracking was not required. This study provides guidance for the assessment of changes in cryopreservation procedures for tissues.     

Preclinical evaluation of ice-free cryopreserved arteries: structural integrity and hemocompatibility

Objective: Arterial allografts are routinely employed for reconstruction of infected prosthetic grafts. Usually, banked cryopreserved arteries are used; however, existing conventional freezing cryopreservation techniques applied to arteries are expensive. In contrast, a new ice-free cryopreservation technique results in processing, storage and shipping methods that are technically simpler and potentially less costly. The objective of this study was to determine whether or not ice-free cryopreservation causes tissue changes that might preclude clinical use. Methods: Conventionally frozen cryopreserved porcine arteries were compared with ice-free cryopreserved arteries and untreated fresh controls using morphological (light, scanning electron and laser scanning microscopy), viability (alamarBlue assay) and hemocompatibility methods (blood cell adhesion, thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, β-thromboglobulin and terminal complement complex SC5b-9). Results: No statistically significant structural or hemocompatibility differences between ice-free cryopreserved and frozen tissues were detectable. There were no quantitative differences observed for either autofluorescence (elastin) or second harmonic generation (collagen) measured by laser scanning microscopy. Cell viability in ice-free cryopreserved arteries was significantly reduced compared to fresh and frozen tissues (p < 0.05). Conclusions: The formation of ice in aortic artery preservation did not make a difference in histology, structure or thrombogenicity, but significantly increased viability compared with a preservation method that precludes ice formation. Reduced cell viability should not reduce in vivo performance. Therefore, ice-free cryopreservation is a potentially safe and cost-effective technique for the cryopreservation of blood vessel allografts.     

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。     

Proteoglycan in porcine aortic tissue after cryopreservation.

This study was to investigate the effects of cryopreservation on proteoglycans of arterial conduit tissue. Proteoglycans from fresh and cryopreserved porcine aorta tissues were extracted with 4 M guanidine hydrochloride (Gdn-HCl) at 4 degrees C in the presence of protease inhibitors. From the tissue extracts, proteoglycans were isolated by cesium chloride (CsCl) isopycnic centrifugation and fractionated by gel filtration. Quantitative analysis of extracted proteoglycans revealed that the content of proteoglycans from cryopreserved tissue, measured as the amount of uronate and protein per unit weight of wet tissue, was similar to that from fresh tissue (0.44 +/- 0.300 versus 0.43 +/- 0.007 mg uronate/g wet tissue and 3.14 +/- 0.039 versus 2.64 +/- 0.015 mg protein/g wet tissue). Gel permeation column chromatography studies suggested that proteoglycans present in three CsCl fractions (I, II, and III) from cryopreserved tissue have approximately the same molecular weights as those from fresh tissue; Kav = 0.13 and 0.47 (I), 0.20 (II), and 0.43 (III) from cryopreserved tissue versus Kav = 0.13 and 0.50 (I), 0.23 (II), and 0.40 (III) from fresh tissue. These studies indicate that there is no significant alteration in the content and molecular size of proteoglycans in properly cryopreserved aortic tissue.     

Live births after autologous transplant of cryopreserved mouse ovaries

The fertility of mice after autologous transplantation of ovaries, before or after cryopreservation, was investigated in this study. Female mice were randomly assigned to either sham-operated (n = 14), ovariectomized (n = 11), fresh (n = 12) or cryopreserved (n = 11) ovarian transplant groups. Ovaries were cryopreserved in 1.4 M dimethyl sulphoxide (DMSO) by cooling to -55 degrees C at 0.5 degree C/min (ice nucleation at -7 degrees C), plunged in liquid nitrogen and then thawed at room temperature. Oestrous cyclicity was observed 7 days after sham operation or 15 days after fresh or cryopreserved ovarian transplant. Ovariectomized animals did not demonstrate oestrous cyclicity but were mated, and no pregnancies resulted. Live births were recorded from all sham-operated, all fresh transplant, and 8/11 (73%) cryopreserved transplant animals. Overall mean +/- SEM litter sizes from fresh (4.32 +/- 0.44) and cryopreserved (4.71 +/- 0.57) transplant groups were smaller (P < 0.05) than those of sham-operated animals (12.54 +/- 0.44), although the sizes were not significantly different (P > 0.05) from each other. Animals were mated at least four times, with four litters of live pups from 4/4 sham-operated, 1/10 fresh and 1/9 cryopreserved ovarian transplant animals. Litter sizes from pups of sham-operated and transplant animals were not significantly different from each other. Following autologous transplantation of mouse ovaries, before or after cryopreservation, offspring appeared normal, with high rates of fertility.      

枸杞多糖对胎儿卵巢组织冷冻保存的影响

背景：抗氧化剂的应用对于提高卵巢组织冻存后的活力起着很重要的作用。 目的：通过观察冷冻保存的胎儿卵巢组织异种移植到裸鼠肾被膜下的发育情况,探索枸杞多糖在卵巢组织冷冻保存中的作用。 方法：实验分为4组。①新鲜移植组：取材后的新鲜胎儿卵巢组织直接进行移植。②冻存对照组：冷冻保护液为基液。③β-巯基乙醇组：冷冻保护液为基液添加100 μmol/L β-巯基乙醇。④枸杞多糖组：冷冻保护液为基液添加 400 mg/L枸杞多糖。对冷冻复温后的胎儿卵巢皮质块进行祼鼠的肾被膜下移植,并于移植后12周取材。 结果与结论：各组在移植物的存活率上差异无显著性意义(P > 0.05)。在卵泡计数上冷冻对照组最低(P < 0.05)。在卵泡的存活率上,各组间差异均有显著性意义,其中以冷冻对照组最低,枸杞多糖组最高。β-巯基乙醇组和枸杞多糖组卵巢超微结构较冷冻对照组保存的好。提示400 mg/L的枸杞多糖和100 μmol/L的β-巯基乙醇有利于卵巢组织的冷冻保存,并可显著提高冷冻卵巢组织移植后的存活率。     

Impact of processing on surface structure of human cardiac valve allografts

Methods of processing and cryopreservation are believed to be the most important factors of long term clinical performance of biological heart valve prostheses. That is why we decided to cooperate in evaluating the impact of current AHV (allograft heartvalve) bank protocol on valve tissue morphology. AHV harvested from "heart-beating" cadaveric donors, considered as a fresh tissue, were compared with valve samples from non-heart beating donors, samples stored in saline, samples treated with antibiotic solution, and finally with cryopreserved valves, stored in liquid nitrogen for months. All samples were dissected, dried with hexamethyldisilazane (HMDS) method, gold-coated, studied and photographed in scanning electron microscope Tesla BS 301. Different superficial patterns were found on ventricular and vascular surfaces of "fresh" semilunar valves. We were able to detect early changes of endothelium after harvesting, denudation of endothelial covering during preservation with and without freezing. Our alternative method of drying samples by HMDS method proved to be suitable for thin membranes of human semilunar valves. Scanning electron microscopy seems to be helpful for morphological control of processing, cryopreservation and liquid nitrogen storage of AHV. We believe that further confrontation of morphological investigation with other methods helps us to develop more suitable protocol of handling AHV in heart valve banking.     

Measurements of the effects of decellularization on viscoelastic properties of tissues in ovine, baboon, and human heart valves

In the development of tissue-engineered heart valves based on allograft decellularized extracellular matrix scaffolds, the material properties of the implant should be ideally comparable to the native semilunar valves. This investigation of the viscoelastic properties of the three functional aortic/pulmonary valve tissues (leaflets, sinus wall, and great vessel wall) was undertaken to establish normative values for fresh samples of human valves and to compare these properties after various steps in creating scaffolds for subsequent bioreactor-based seeding protocols. Torsional wave methods were used to measure the viscoelastic properties. Since preclinical surgical implant validation studies require relevant animal models, the tests reported here also include results for three pairs of both ovine and baboon aortic and pulmonary valves. For human aortic valves, four cryopreserved valves were compared with four decellularized scaffolds. Because of organ and heart valve transplant scarcity for pulmonary valves, only three cryopreserved and two decellularized pulmonary valves were tested. Leaflets are relatively soft. Loss angles are similar for all tissue samples. Regardless of species, the decellularization process used in this study has little effect on viscoelastic properties.     

Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment

BACKGROUND: Banking of testicular tissue from pre-pubertal boys before gonadotoxic treatment is a crucial step in fertility preservation. We wanted to find optimal methods for cryopreservation of testicular tissue from pre-pubertal boys, modifying techniques developed for fetal and adult human testicular tissue cryopreservation. METHODS: Testicular tissue was collected from five pre-pubertal boys undergoing gonadotoxic treatment in a clinical programme. Two freezing protocols, originally developed for fetal and adult human testicular tissue, were applied for pre-pubertal testicular tissue cryopreservation. In both methods, 5% dimethyl sulphoxide (DMSO) was used as a cryoprotectant. The integrity of the tissue was investigated in non-frozen tissue cultured for 24 h and in cryopreserved-thawed tissue, using two different programmes. We also analysed frozen-thawed samples cultured for 24 h in comparison with untreated fresh fixed control tissue. Immunohistochemical analysis using anti-MAGE-A4, vimentin and CD34 monoclonal antibodies was performed in order to visualize and characterize the cryodamage of the different testicular cells and compartments. The structure of the tissue was evaluated using light microscopy. Qualitative control analysis was performed using transmission electron microscopy. RESULTS: No clear structural changes were observed in the fresh, fresh cultured and cryopreserved testicular tissue after using the protocol developed for adult testicular tissue. The programme earlier successfully used for human fetal testicular tissue cryopreservation caused more tissue damage. CONCLUSIONS: Pre-pubertal testicular tissue from boys facing gonadotoxic treatment survives cryopreservation, can be cryobanked and hopefully used for fertility preservation. Slow programmed freezing with DMSO as a cryoprotectant is efficient in maintaining the spermatogonia, Sertoli cells and stromal compartment during freezing, thawing and tissue culture.     

Viability and recovery of frozen-thawed human islets and in vivo quality control by xenotransplantation

Cryopreservation of islets of Langerhans offers advantages for the transplantation into diabetic patients. In this study two different methods of cryopreservation were compared with respect to islet viability and recovery after cryostorage. It was also investigated whether human islet survival in mice was affected by cryopreservation. Aliquots of human islets were cryopreserved conventionally or vitrified, respectively. After rapid thawing, islet viability and islet equivalent (IEQ) recovery rate were determined. Aliquots of freshly isolated or conventionally cryopreserved islets were transplanted beneath the kidney capsule of non-diabetic C57BL/6 mice. After three days renal insulin content was determined. Islet cell viability was 17.3±8.0% for vitrified and 51.8±3.0% for conventionally cryopreserved islets; the recovery rate was 84.8±12.2% and 92.8±12.4%, respectively. Insulin recovery after transplantation was 25.6±7.3% for fresh and 24.1±7.4% for cryopreserved islets. This study suggests that the conventional method of cryopreservation is superior to vitrification with respect to islet viability after thawing. We found no significant difference between fresh and cryopreserved islets with respect to insulin recovery after transplantation into mice.     

Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries

BACKGROUND: Ovarian tissue cryopreservation and transplantation can be used to restore fertility to sterile females. A question that warrants further investigation is whether the follicular content is affected by the freeze-thawing and grafting procedure, and if so, to what extent and by what mechanism. METHODS AND RESULTS: Intact newborn mouse ovaries were allografted under the kidney capsule or were cryopreserved by slow freezing with dimethylsulphoxide as the cryoprotectant prior to grafting. Estrogenic activity of ovariectomized recipient mice, as revealed by vaginal cytology, resumed after 11 days of transplantation. At 14 days after transplantation, ovarian grafts were recovered and processed histologically for follicle number counting. The follicular content of grafts of fresh ovaries was 58% of that from ovaries of age-matched 14 day old mice. In frozen-thawed ovarian grafts, the follicular content was only 9% lower than that of fresh grafted ovaries. Apoptosis of follicular cells was investigated by DNA nick end labelling. We observed a marked increase in the staining of fragmentation of DNA shortly after transplantation (2-12 h) of fresh newborn mouse ovaries. CONCLUSIONS: The results of the present study indicate that transplantation rather than cryopreservation accounts for the major and early loss of primordial follicles in grafted newborn mouse ovaries.     

自制多脏器保存液对大鼠睾丸一氧化氮合酶的影响

背景：在睾丸移植过程中,低温保存和缺血可导致睾丸产生氧自由基而损伤睾丸组织。 目的：观察自制多脏器保存液对低温保存大鼠睾丸一氧化氮合酶的影响。 方法：采用自制多脏器保存液和UW液低温保存大鼠睾丸,分别于保存24,48,72 h时切取睾丸,测定睾丸组织内的总抗氧化能力和一氧化氮合酶活性。 结果与结论：自制多器官保存液低温保存各时点大鼠睾丸一氧化氮合酶活性和总抗氧化能力与UW液组比较差异均无显著性意义(P > 0.05)。表明自制多脏器保存液能明显减轻低温保存大鼠睾丸氧自由基损伤,其作用与经典的美国威斯康星大学UW保存液基本相当。     

冷冻保存异体软骨移植免疫排斥反应的研究状况

背景：目前多采用冷冻保存方法来降低异体软骨移植免疫排斥反应,但有关异体软骨的取材、冷冻保存方法、冷冻保存条件仍然需要深入的研究探讨。 目的：对于冷冻保存异体软骨移植后免疫排斥反应机制的研究进行回顾分析,并对不同保存方法的特点进行比较分析。 方法：由第一作者检索1990/2008 PubMed数据及万方数据库有关异体软骨移植后免疫排斥反应及冷冻保存方法对软骨移植影响等方面的相关文献。 结果与结论：异体软骨组织移植治疗关节软骨缺损治疗效果明显优于其他治疗方法。冷冻保存异体关节软骨保持了软骨组织的性状和生物学活性,而且可择期完成关节重建,并且有充裕的时间完成多项指标检测,防止供体携带细菌病毒和传染性疾病的传播,并且降低了软骨组织的抗原性,具有较大的临床应用价值。但冷冻保存的各个环节,如:低温保护剂的应用、降温和复温速度等方面还存在诸多问题,软骨移植后仍然会出现软骨吸收、退变等现象。随着冷冻生物学的不断进步,冷冻损伤机制的不断揭示,这些问题终将会解决,软骨组织冷冻保存技术会得到进一步的完善。     

Effects of cryopreservation and coculture with pancreatic ductal epithelial cells on insulin secretion from human pancreatic islets

Long-term storage methods, such as cryopreservation and long-term in vitro culture, hinder the therapeutical application of pancreatic islet transplantation, because they decrease islet viability. Pancreatic ductal epithelial cells (DEC) are putative stem cells for islets, which may secrete specific factors supporting islet growth and function. Hence, we studied the effect of coculture with DEC on the viability of fresh and cryopreserved human pancreatic islets. Islets and DEC were isolated from the pancreas of an organ donor, and part of them were cryopreserved. Fresh and cryopreserved-thawed islets were cultured alone or in the presence of DEC for 14 days at 33 degrees C or 37 degrees C. At day 1 and day 14 of culture, insulin secretion was stimulated by two sequential 45-min exposures to low and high glucose concentrations (3.3 and 16.7 mmol/l, respectively). Insulin concentrations were measured by radio-immunoassay, and the ratio between the insulin responses to high and low glucose was calculated (insulin stimulation index, ISI). After 14 days of culture, some fresh islets were processed for scanning electron microscopy (SEM). At day 14, ISI was markedly reduced in both fresh and cryopreserved islets with respect to 1 day cultures. Cryopreservation reduced ISI at day 1 and day 14, but in the latter case only when cultures were maintained at 37 degrees C. Coculture with DEC did not affect ISI of fresh islets at day 1, and enhanced it at day 14, but only at a culture temperature of 33 degrees C. Conversely, coculture raised ISI of cryopreserved islets at both day 1 and day 14, independently of the culture temperature. SEM showed that at day 14 of culture, the morphology of fresh islets displayed the best preservation when cocultured with DEC at 33 degrees C. Our findings confirm that both long-term culture and cryopreservation decrease viability of human pancreatic islets. Moreover, they indicate that coculture of islets with DEC at 33 degrees C represents a valuable tool to improve the survival and functional activity of islets, especially in the case of cryopreserved material.     

Ovary and Ovulation: Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients

Girls and young women who require ovariectomy or cancer therapymay consider having their own eggs, embryos or ovarian tissuestored (cryopreserved) for their own future use. Ovarian tissueis simple to collect and contains large numbers of germ cells.Transplantation of fresh and frozen- thawed ovarian tissue inhealthy sheep and mice has resulted in normal live young. Similartechniques may be effective in the human but it is unclear whetherovarian tissue cryopreservation and grafting is suitable forovaries from individuals with cancer or infections. If cancercells were present in an ovary at the time of collection andsurvived cryopreservation and grafting they could establishcancer in the recipient. We therefore performed ovarian cryopreservationand transplantation trials using a mouse lymphoma model. Thisestablished that the lymphoma was transmitted by grafts of bothfresh and frozen ovarian tissue. The normal healthy recipientmice died 9–43 days after receiving a small piece (1 mmof ovarian tissue from a donor with lymphoma. We conclude thatovarian tissue which is collected, cryopreserved and graftedwhile it contains cancer cells has the potential to spread thecancer to the graft recipient.     

Cryopreservation-induced acrosomal vesiculation in live spermatozoa from cynomolgus monkeys (Macaca fascicularis)

BACKGROUND: Cryopreserved spermatozoa are known to undergo accelerated capacitation and require a shorter incubation time for fertilization. However, details of their acrosomal membranes following cryopreservation remain unclear. METHODS: Percoll density gradient centrifugation was used to remove dead spermatozoa; thus >90% live spermatozoa were recovered after cryopreservation, and acrosomal status was compared among non-incubated and incubated fresh and cryopreserved spermatozoa. RESULTS: Transmission election microscopy (TEM) using microwave methods and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) staining revealed that 21.1 and 61.6% respectively of non-incubated, cryopreserved spermatozoa were intact, whereas 97.6% (TEM) or 91.9% (FITC-PSA) of non-incubated fresh spermatozoa were intact. TEM revealed that 28.8% of the cryopreserved spermatozoa were swollen, and probably included among those counted as intact by FITC-PSA staining. The non-incubated cryopreserved spermatozoa had fused plasma and outer acrosomal membranes, and 36.4% of them had vesiculation when observed by TEM. FITC-PSA staining indicated that 22% of the live spermatozoa were acrosome reacted. CONCLUSIONS: Acceleration of the acrosome reaction was evident by both TEM and FITC-PSA. Incubation of cryopreserved spermatozoa for 2 h accelerated vesiculation to a state similar to that of fresh spermatozoa that had been incubated for 8 h. These results reveal that in cryopreserved spermatozoa, the process of acrosome reaction begins before incubation.     

雌激素影响冻存肾脂肪囊来源脂肪间充质干细胞的成脂分化

背景：雌激素对脂肪干细胞向脂肪细胞诱导分化会有负向调节作用,但对长期深低温保存的肾脂肪囊来源脂肪间充质干细胞向脂肪细胞分化的效果尚未见报道。 目的：探讨雌激素对长期深低温保存的及新鲜提取的肾脂肪囊来源脂肪间充质干细胞成脂分化能力的影响。 方法：将长期深低温保存后复苏的及新鲜提取的第3代肾脂肪囊来源脂肪间充质干细胞分为4组进行成脂诱导分化,新鲜+雌激素组和冻存+雌激素组诱导时添加10-7 mol/L雌激素,新鲜组和冻存组不添加。成脂诱导分化14 d进行油红O染色及成脂量定量检测。 结果与结论：长期深低温保存后复苏的第3代肾脂肪囊来源脂肪间充质干细胞与新鲜提取的细胞形态和排列无差异;长期深低温保存后复苏的与新鲜提取的第3代肾脂肪囊来源脂肪间充质干细胞表面抗原分子CD29、CD44均呈阳性,CD31均呈阴性表达。成脂诱导后可观察到细胞内有脂滴形成,油红O 染色呈阳性。在成脂诱导分化14 d对各组进行成脂量比较,新鲜组与新鲜+雌激素组,冻存组与冻存+雌激素组的吸光度之间比较,差异有显著性意义,但新鲜组与冻存组之间差异无显著性意义。结果表明：小剂量雌激素可抑制长期深度低温保存后复苏的肾脂肪囊来源脂肪间充质干细胞的成脂分化;长期深低温保存后复苏的与新鲜提取的第3代肾脂肪囊来源脂肪间充质干细胞在成脂诱导方面无显著性差异。