Xenotransplantation of cryopreserved composite organs on the rabbit

Nowadays, It is easy to define optimal conditions (cryoprotective agent, speed and steps of freezing, speed of warming) for the cryopreservation of a homogeneous cell population or a one cell-layer tissue. Meanwhile, It is still hard to obtain cryopreservation of composite organs. Each tissue has its own requirements and its own reactivity to the cryopreservation process. The challenge consists of, on the one hand, to select the ideal combination of cryoprotective agents that can fit the needs of the different tissues, and the definition of adequate technical parameters, on the other hand. All the experimental trials have studied the survival rate of non-vascularized cryopreserved tissues. The aim of our experimental work is to demonstrate the feasibility of cryopreserving a composite organ with its nutrient vessels “artery and veins” in order after thawing to revitalize it by reestablishing the blood irrigation by microsurgical vascular anastomosis. We report our experimental results on the cryopreservation of composite organs - amputated digits - xenotransplanted in the rabbit. Digital segments were cryopreserved, then revitalized after warming using vascular microsurgical techniques. Preliminary results are encouraging and may pave the way in the future to the microvascular allotransplantation of cryopreserved composite organs.     

Xenotransplantation of cryopreserved composite organs on the rabbit

Nowadays, It is easy to define optimal conditions (cryoprotective agent, speed and steps of freezing, speed of warming) for the cryopreservation of a homogeneous cell population or a one cell-layer tissue. Meanwhile, It is still hard to obtain cryopreservation of composite organs. Each tissue has its own requirements and its own reactivity to the cryopreservation process. The challenge consists of, on the one hand, to select the ideal combination of cryoprotective agents that can fit the needs of the different tissues, and the definition of adequate technical parameters, on the other hand. All the experimental trials have studied the survival rate of non-vascularized cryopreserved tissues. The aim of our experimental work is to demonstrate the feasibility of cryopreserving a composite organ with its nutrient vessels “artery and veins” in order after thawing to revitalize it by reestablishing the blood irrigation by microsurgical vascular anastomosis. We report our experimental results on the cryopreservation of composite organs—amputated digits—xenotransplanted in the rabbit. Digital segments were cryopreserved, then revitalized after warming using vascular microsurgical techniques. Preliminary results are encouraging and may pave the way in the future to the microvascular allotransplantation of cryopreserved composite organs.     

Cell cultures from cryopreserved human lung tissue.

To assess gene induction in primary human fibroblasts, we have developed a method for cryopreservation of lung biopsies in liquid nitrogen. Fresh biopsies (n = 10) were chopped into 5 x 5 mm pieces and transferred into an ice-cold freezing medium. Biopsies were kept on ice for 15 min, followed by further cooling of the tissue to -70 degrees C. With this method, lung biopsies were preserved for more than 1 year before they were used for generating cell cultures. There was no significant difference in the biological responsiveness of fibroblasts generated from immediately cultured lung biopsies compared with those from cryopreserved tissue. The doubling rate of fibroblasts from fresh tissue was 23.6 +/- 1.1 hr; compared to 23.5 +/- 1.5 hr for fibroblasts generated from cryopreserved tissue. PDGF-BB enhanced de novo synthesis of DNA 100 times, in both the immediately cultured fibroblasts and those generated from cryopreserved biopsies. Macrophages, dendritic cells and endothelial cells could also be recovered from cryopreserved lung tissue. This method permits long-term storage of lung tissue and the possibility of establishing primary cell lines from the same tissue at later times without appreciable changes in their cellular biological characteristics.     

Cryopreservation of cartilage cell and tissue for biobanking

Preservation of cell and tissue samples from endangered species is a part of biodiversity conservation strategy. Therefore, setting up proper cell and tissue cryopreservation methods is very important as these tissue samples and cells could be used to reintroduce the lost genes into the breeding pool by nuclear transfer. In this study, we investigated the effect of vitrification and slow freezing on cartilage cell and tissue viability for biobanking. Firstly, primary adult cartilage cells (ACCs) and fetal cartilage cells (FCC) were cryopreserved by vitrification and slow freezing. Cells were vitrified after a two-step equilibration in a solution composed of ethylene glycol (EG), Ficoll and sucrose. For slow freezing three different cooling rates (0.5, 1 and 2 °C/min) were tested in straws. Secondly, the tissues taken from articular cartilage were cryopreserved by vitrification and slow freezing (1 °C/min). The results revealed no significant difference between the viability ratios, proliferative activity and GAG synthesis of cartilage cells which were cryopreserved by using vitrification or slow freezing methods. Despite the significant decrease in the viability ratio of freeze–thawed cartilage tissues, cryopreservation did not prevent the establishment of primary cell cultures from cartilage tissues. The results revealed that the vitrification method could be recommended to cryopreserve cartilage tissue and cells from bovine to be used as alternative cell donor sources in nuclear transfer studies for biobanking as a part of biodiversity conservation strategy. Moreover, cartilage cell suspensions were successfully cryopreserved in straws by using a controlled-rate freezing machine in the present study.     

Residual Antibiotics in Decontaminated Human Cardiovascular Tissues Intended for Transplantation and Risk of Falsely Negative Microbiological Analyses

We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.     

Preliminary report of novel technique for cryopreservation--vacuum-assisted cryoprotectant infiltration

To date, cryopreservation of large soft tissues has not been successfully achieved because of limitation of cryoprotective agent (CPA) infiltration into the tissue. This study aimed to investigate the effects of a vacuum on the tissue-infiltration of a CPA. An instant pickle-maker was modified for use as a vacuum apparatus, and glycerol was selected as the CPA. Twenty-six rats were used, and their thighs were divided into three treatment groups. Group 1: fresh control; Group 2: cryopreserved control, i.e., immersed in the CPA for 1h under atmospheric pressure and cryopreserved; Group 3: vacuum-assisted CPA infiltration, i.e., immersed in the CPA under negative pressure (20, 40 and 60 cmHg, for durations of 10, 20 and 30 min at each) and cryopreserved. The Groups 2 and 3 specimens were thawed after 3 weeks of cryopreservation at -80 °C and histologically examined, in comparison with Group 1. Skin: in Groups 2 and 3, the skin was well preserved. Muscle: in Group 2, both extracellular and intracellular ice crystal formation was widely distributed throughout the muscle tissue. In Group 3, under an adequate vacuum, the muscle tissue was well preserved, with no ice crystal formation. However, when the treatment was conducted under excessive vacuum conditions, the muscle tissue showed focal necrosis. Blood vessels: in Group 3, both the arteries and veins were well preserved up to the tunica intima. The method described in this paper may be a useful technique for achieving cryopreservation of large soft tissues.     

Optimal conditions for heart cell cryopreservation for transplantation

Cultured myocyte transplantation into an infarcted myocardium has been shown to improve contractile function. Cryopreservation of cultured muscle cells or heart tissue will be important for the technology to be practical. This study, using fetal cardiomyocytes, evaluated the optimal conditions for muscle cell cryopreservation. Study 1: Fetal rat cardiomyocytes were isolated and cultured. The freshly isolated and passage 1, 2, 3 and 4 cells were cryopreserved in a solution containing 70% IMDM, 20% FBS and 10% DMSO and stored in –196°C for 1, 2, 4, 8, 12 and 24 weeks. The cells were thawed and cultured. Cell number and contractility were evaluated at 0, 2, 4, 6, 8 and 10 days of culture. Study 2: Rat myocardium was cryopreserved in sizes of 0.2, 2 and 6 mm³ for 1 week. The tissue was thawed and cells were isolated. Cell growth and contractility were evaluated. (1) Cardiomyocytes grew and contracted after cryopreservation. Storage time did not affect cell survival rate, beating cell numbers and beating rates. Increasing cell passage prior to cryopreservation decreased the percentage of beating cells. (2) Cells isolated from cryopreserved tissue grew in vitro and contracted normally. Cell yield decreased with increased cryopreserved tissue size. Fetal rat cardiomyocytes survived and functioned after in vitro cryopreservation. Viable cells can be isolated from cryopreserved myocardium and cultured. Cryopreservation of small pieces of myocardium is preferred for maximal cell yields.     

Transplantation of articular cartilage following a step-cooling cryopreservation protocol

Using a step-cooling cryopreservation protocol that held the tissue 60 min at -4 degrees C, 30 min at -8 degrees C, and 10 min at -40 degrees C before plunging into liquid nitrogen, we were able to get a substantial improvement in the magnitude and pattern of chondrocyte recovery following cryopreservation, achieving postthaw recoveries of 62 +/- 13%. These results are consistent with the hypothesis that ice growth within articular cartilage is planar, but they provide no direct support for that hypothesis. Transplanting (step-cooled) cryopreserved osteochondral allografts into adult Suffolk/Romanoff crossbred sheep for periods of 3 months and 1 year further tested the efficacy of the cryopreservation protocol. Unfortunately, the cryoinjury sustained by the chondrocytes during cryopreservation, although apparently nonlethal immediately after thawing in many cases, was not innocuous in the long term. The presence of large clusters of chondrocytes at 1 year after transplantation illustrates that cryoinjury not detectable with a membrane integrity assay can still have far-reaching effects on transplanted tissue.     

Cryopreservation of composite tissues and transplantation: preliminary studies

Despite advances in cryobiology, the reliable cryopreservation of complex tissues has not yet been achieved. This study evaluates the viability of cryopreserved composite flaps and demonstrates the feasibility of their transplantation. Epigastric flaps were harvested from male Lewis rats. 1.5 M dimethyl sulfoxide (Me₂SO) was used as the initial cryoprotectant agent (CPA). Samples were frozen at controlled rate to −140 °C and transferred to liquid nitrogen for at least two weeks. Hematoxylin and eosin (H/E) staining, MTT tetrazolium salt assay, and factor VIII immunostaining were used to evaluate the overall histology, epithelial viability, and vascular endothelial integrity, respectively, of cryopreserved flaps. For the in vivo phase, flaps were isotransplanted to 35 recipient animals, divided into three groups: fresh (n = 10), perfused (n = 8), and cryopreserved (n = 17). Blood vessel patency was assessed via Doppler at 1, 7, and 60 days post-transplantation. For in vitro studies, cryopreserved samples (10/10) retained normal cell architecture and vascular endothelial integrity upon H/E and factor VIII staining. The viability index of cryopreserved composite flap skin (n = 10) was 11.17 ± 2.01, which was not significantly different from fresh controls (n = 10, 12.15 ± 1.32). All transplanted flaps in the fresh and perfusion groups survived with healthy color and hair growth at 60 days after operation. Survival in the cryopreserved group ranged from 2 to 60 days, with a mean of 12 days. These results demonstrate that the long term survival of cryopreserved composite tissue transplants is possible. Further studies are needed to refine protocols for the reliable cryopreservation of composite parts.     

Viability of pulp stromal cells in cryopreserved deciduous teeth

The cryopreservation of exfoliated deciduous teeth and harvesting of stem cells from them as required would reduce the costs and efforts associated with banking stem cells from primary teeth. The aim of this study was determine whether the viability of pulp stromal cells from deciduous teeth was influenced by the cryopreservation process itself or the period of cryopreservation. In total, 126 deciduous teeth were divided into three groups: (1) fresh, (2) cryopreserved for <3 months (cryo<3), and (3) cryopreserved for 3–9 months (cryo3–9). The viability of the pulp tissues was compared among the three groups by evaluating the outgrowth from pulp tissues and cell activity within those pulp tissues. In addition, the terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) assay was performed to compare cell apoptosis within fresh pulp tissue and pulp tissue that had been cryopreserved for 4 months. The outgrowth from and cell activity within the pulp tissues did not differ significantly between the fresh and cryo<3 pulp tissues. However, these parameters were significantly reduced in the cryo3–9 pulp tissue. In TUNEL assay, 4-month cryopreserved pulp tissues has more apoptotic cells than fresh group. In conclusion, it is possible to acquire pulp stromal cells from cryopreserved deciduous teeth. However, as the period of cryopreservation becomes longer, it is difficult to get pulp cells due to reduced cell viability.     

Preservation of mitochondrial functional integrity in mitochondria isolated from small cryopreserved mouse brain areas

Studies of mitochondrial bioenergetics in brain pathophysiology are often precluded by the need to isolate mitochondria immediately after tissue dissection from a large number of brain biopsies for comparative studies. Here we present a procedure of cryopreservation of small brain areas from which mitochondrial enriched fractions (crude mitochondria) with high oxidative phosphorylation efficiency can be isolated. Small mouse brain areas were frozen and stored in a solution containing glycerol as cryoprotectant. Crude mitochondria were isolated by differential centrifugation from both cryopreserved and freshly explanted brain samples and were compared with respect to their ability to generate membrane potential and produce ATP. Intactness of outer and inner mitochondrial membranes was verified by polarographic ascorbate and cytochrome c tests and spectrophotometric assay of citrate synthase activity. Preservation of structural integrity and oxidative phosphorylation efficiency was successfully obtained in crude mitochondria isolated from different areas of cryopreserved mouse brain samples. Long-term cryopreservation of small brain areas from which intact and phosphorylating mitochondria can be isolated for the study of mitochondrial bioenergetics will significantly expand the study of mitochondrial defects in neurological pathologies, allowing large comparative studies and favoring interlaboratory and interdisciplinary analyses.     

Infectivity of cryopreserved Giardia cysts for Mongolian gerbils (Meriones unguiculatus)

Although Giardia species trophozoites have been cryopreserved successfully, no report on the successful cryopreservation of cysts could be found. Using infectivity to Mongolian gerbils (Meriones unguiculatus) as a measure of cyst viability, we tested the viability of 4 strains of Giardia cysts that had been cryopreserved for 1-67 wk. Cysts were frozen in either Keister's medium or physiological saline, both containing 5% or 7.5% dimethylsulfoxide as the cryoprotectant. Viability of cryopreserved cysts was dependent upon the number of cysts inoculated, the length of time cysts were held at 4 C before cryopreservation, and the cryopreserving medium. Infection was established in gerbils by inoculating them with cysts that had been cryopreserved for up to 67 wk, cysts that had been held for less than 30 days before cryopreservation, and cysts frozen in Keister's medium. Saline appears to be unsuitable as a freezing medium for the cryopreservation of Giardia cysts.     

Secondary follicle growth and oocyte maturation by culture in alginate hydrogel following cryopreservation of the ovary or individual follicles

An option for fertility preservation for women facing a cancer diagnosis involves the cryopreservation of ovarian tissue for later re‐transplantation or in vitro culture, with in vitro culture preferred to avoid reintroduction of the cancer. Small, immature follicles survive the freeze‐thaw process, and can be matured through in follicle maturation (IFM) that involves an initial growth of the follicle and subsequent maturation of the oocyte. The ovarian tissue can be cryopreserved in two forms: (i) cortical strips consisting of follicles and surrounding stroma (Cryo‐Ov) or (ii) individually isolated follicles (Cryo‐In). The aim of this study was to assess the follicle growth and oocyte maturation for follicles that were cryopreserved either as strips or individually using a slow‐freezing cryopreservation method. The two follicle groups, together with non‐cryopreserved control follicles, were grown in an alginate‐based three‐dimensional culture system for 12 days. The overall survival, size increase and antrum formation rates were comparable among the three groups. At day 12 of culture, Androstenedione levels were decreased in the Cryo‐Ov group relative to the other two, and the ratio of progesterone to estradiol was increased in the two cryopreserved groups relative to the control. Both Gja1 (known as connexin 43) and Gja4 (known as connexin 37) mRNA expression were decreased at day 6 in the cryopreserved groups relative to controls, and by day 12, Gja1 was similar for all three groups. Moreover, Cryo‐In resulted in lower GVBD rate indicating some impaired oocyte development. Overall, the present study demonstrated that mouse preantral follicles, either within ovarian tissues or individually isolated, could be successfully cryopreserved by the slow‐freezing method, as evidenced by post‐thaw follicle development and steroidgenesis, oocyte maturation and molecular markers for oocyte and/or granulosa cells connection. Biotechnol. Bioeng. 2009;103: 378–386. © 2009 Wiley Periodicals, Inc.     

Optimizing methods for human testicular tissue cryopreservation and spermatogonial stem cell isolation

Cryopreservation of testicular tissue before cancer therapy for fertility preservation in prepubertal boys with cancer is of great interest in reproductive medicine. Isolation of spermatogonial stem cells (SSCs) from cryopreserved tissues would be a suitable cell source to re-establish spermatogenesis after cancer therapy. We herein establish optimized protocols for cryopreservation of human testicular tissue and isolation of SSCs from cryopreserved tissue. We developed a freezing protocol that provided high testicular cell viability and supported structural integrity and tubular epithelium coherence similar to fresh tissue. Then, we established a protocol that allowed efficient isolation of functional SSCs from cryopreserved tissues. Isolated cells were found on the testicular basement membrane after xenotransplantation. Our results demonstrated the preservation of testicular tissue structure and high cell viability with efficient isolation of SSCs after testicular cryopreservation, which is promising for future therapeutic applications in fertility preservation.     

Influence of cryopreservation on the pulpal tissue of immature third molars in vitro

The purpose of this study was to evaluate in vitro the viability of isolated and non-isolated pulpal tissue of immature third molars after cryopreservation. This study was divided in three different experiments. Experiment 1: Pulpal tissue isolated from 19 third molars was divided in horizontal segments. Each segment was cultured separately in order to evaluate whether differences in growth capacity within the tissue could be found. Experiment 2: Pulpal tissue isolated from 27 third molars was divided in a mesial and a distal part. One part was cryopreserved before culturing, the other part was cultured immediately. Growth capacity of cryopreserved and non-cryopreserved tissue was evaluated and compared. Experiment 3: 43 third molars were cryopreserved. After thawing, the dimension of the apical foramen was measured and the pulp was isolated and segmented horizontally. The different parts were cultured and growth capacity was evaluated and compared. Results of experiment 1 and 2 showed no significant difference in growth capacity between fibroblasts originating from different pulpal segments of the same tooth without cryopreservation and between fibroblasts originating from cryopreserved and non-cryopreserved isolated pulpal tissue. In experiment 3 it was demonstrated that the dimension of the apical foramen and pulpal viability after cryopreservation are positively correlated. A minimum dimension of 9.42 mm2 enables the cryoprotective agent to penetrate sufficiently and to protect the pulpal tissue from apex to crown. This study proved that cryopreservation of human pulpal tissue is possible if the cryoprotective agent can reach the entire pulp.     

Generation of Live Piglets for the First Time Using Sperm Retrieved from Immature Testicular Tissue Cryopreserved and Grafted into Nude Mice

Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI), we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG), polyvinyl pyrrolidone (PVP) and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230−350 (day 0 =  grafting). Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230−350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will be applicable to many large animals, since pigs are phylogenetically distant from the murine recipients.     

改良血管套接法与血管断端吻合法对断指再植患者断指血运、断指成活以及运动功能的影响

目的:探讨改良血管套接法与血管断端吻合法对断指再植患者断指血运、断指成活以及运动功能的影响。方法:回顾性分析我院于2017年1月到2018年12月期间治疗的107例断指再植患者的临床资料。根据手术方法的不同将患者分为A组(n=55,共断指93指)和B组(n=52,共断指86指),A组给予血管断端吻合法,B组给予改良血管套接法,比较两组患者断指血运、断指成活、运动功能、生活质量及并发症。结果:B组毛细血管充盈时间、患指微循环检测值低于A组,氧饱和度、经皮氧分压则高于A组(P<0.05)。B组断指成活率、断指张力正常率及断指颜色正常率均高于A组(P<0.05)。B组术后1年社会关系领域、生理领域、心理领域以及环境领域的维度评分均高于A组(P<0.05)。B组的肌力、活动度、灵巧度、Carroll量表评分均高于A组(P<0.05)。两组并发症发生率对比未见统计学差异(P>0.05)。结论:与血管断端吻合法相比,断指再植患者采用改良血管套接法,在断指血运、断指成活、运动功能、生活质量方面的改善效果更佳,且不会增加并发症发生率。     

兰州鲇精液超低温冷冻保存技术研究及细胞损伤检测

为保护兰州鲇(Silurus lanzhouensis Chen)种质资源,促进新品种选育,文章开展了兰州鲇精液超低温冻存抗冻剂筛选及程序保存技术研究,并通过精子解冻激活率、核DNA检测、扫描电镜和透射电镜检测技术对冻存效果和冻存损伤情况进行了评测。结果表明:兰州鲇精液以10%DMSO为抗冻剂, 4℃平衡20min,–80℃平衡30min后立即置于液氮超低温保存,并于40℃水浴解冻时精子冻存效果较佳,冻精激活率达(75.56±3.91)%。SCGE检测DNA损伤结果显示超低温冻存10d、20d及30d兰州鲇精液的彗星率和损伤系数无显著差异。扫描电镜检测显示兰州鲇精子明显分头部,中段及尾部3部分,属于单鞭毛型,无顶体,无侧鳍,中心粒相互垂直呈"T"形,鞭毛为典型的"9+2"微管结构。冻存结构损伤主要表现为膜损伤,细胞质膜与染色质膜发生破损、折皱、囊泡化或整体脱落,细胞核膜与质膜空隙加大,核发生变形,核质疏散,线粒体结构弥散,线粒体内容物外流,中心粒复合体易位,鞭毛外膜变形脱离,中段位置断裂,微管结构基本完好。研究筛选的超低温冷冻保存技术可长期有效保存兰州鲇精液,为兰州鲇种质资源保护及今后...     

Cryopreservation of ovarian tissues temporarily suppresses the proliferation of granulosa cells in mouse preantral follicles

Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles during in vitro culture, but the mechanism causing this impairment has not been brought to light. In order to elucidate the underlying mechanism of delayed follicular development, we evaluated the effects of cryopreservation on the proliferation of granulosa cells during culture of mouse preantral follicles, as a sufficient population of granulosa cells is critical for normal follicular development. Additionally the initial cell death of granulosa cells was estimated immediately after cryopreservation. The ovarian tissues obtained from 12-day-old female mice were cryopreservation by vitrification. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the expression of cell cycle regulators such as cyclin D2, CDK4, cyclin E and CDK2 in preantral follicles isolated from fresh and cryopreserved ovarian tissues that were cultured for 48 h. The viability of granulosa cells was evaluated by measuring the proportion of necrotic areas. The granulosa cell proliferation of the cryopreserved preantral follicles was decreased significantly compared to that of the fresh controls at 0 and 24 h after culture (P < 0.05), and this was increased to the control levels after 48 h of culture. The expressions of cyclin D2, Cdk 4, cyclin E and Cdk2 were also decreased in the cryopreserved ovarian tissues at 0 and 24 h after culture (P < 0.05), but they were increased to the control levels after 48 h of culture. The proportion of the necrotic area was significantly higher in cryopreserved preantral follicles compared to that of the fresh preantral follicles (P < 0.05). This suggests that cryopreservation of ovarian tissues may delay the preantral follicle development by temporary suppressing the granulosa cell proliferation through the cell cycle regulators (cyclin D2, Cdk4, cyclin E and Cdk2) and by granulosa cell death immediately after warming.     

Perfusion bioreactor-based cryopreservation of 3D human mesenchymal stromal cell tissue grafts

The generation of an off-the-shelf in vitro engineered living tissue graft will likely require cryopreservation. However, the efficient addition and removal of cryoprotective agents (CPA) to cells throughout the volume of a three-dimensional (3D) tissue graft remains a significant challenge. In this work, we assessed whether a perfusion bioreactor-based method could be used to improve the viability of cryopreserved mesenchymal stromal cell- (MSC) based tissue constructs as compared to using conventional diffusion-based methods. The bioreactor was first used to saturate 3D constructs with CPA under perfused flow. Following cryopreservation, the bioreactor was then also used for the efficient removal of the CPA from the 3D tissues. We demonstrate that addition and removal of CPA under perfused flow significantly increased the viability of MSC within cryopreserved 3D tissue constructs as compared to conventional diffusion-based methods.