Sweet potato (Ipomoea batatas L.) shoot tips grown in vitro were successfully cryopreserved by encapsulationvitrification. Encapsulated explants are very easily manipulated, due to the relatively large size of the alginate beads, and a large number of samples can be treated simultaneously. In this study, the effects of sucrose preculture, cryoprotectant preculture, and post-warm recovery media on regrowth, following liquid nitrogen (LN) exposure, were investigated to establish an efficient encapsulation-vitrification protocol for sweet potato. Shoot tips of plants grown in vitro were precultured in 0.3 M sucrose for 2 d before encapsulation. Encapsulated shoot tips were pre-incubated in liquid MS (Murashige and Skoog) medium containing 0.5 M sucrose for 16 h, before preculturing in sucrose-enriched medium (0.7 M sucrose) for 8 h. Shoot tips were osmoprotected with 35% plant vitrification solution 3 (PVS3) for 3 h, before being dehydrated with PVS3 for 2 h at 25°C. The encapsulated and dehydrated shoot tips were transferred to 2 mL cryotubes, suspended in 0.5 mL PVS3, and plunged directly into liquid N. High levels of shoot formation were obtained for the cv. Yeulmi (65.7%) and Yeonwhangmi (80.3%). The regrowth rates of cryopreserved samples in Yeulmi (78.9%) and Yeonwhangmi (91.3%), following culture on ammonium-free MS medium for 5 d, were much higher than those cultured on standard MS medium (65.7% and 80.3%, respectively). This encapsulation-vitrification is a promising method for the long-term preservation of sweet potato.

Twenty apple germplasm accessions from the Korean Genebank were successfully cryopreserved using two-step freezing to back up genetic resources maintained by field collections. This study examined the morphological and genetic stability of cryopreserved dormant apple buds that were stored in liquid nitrogen, and then rewarmed and regrown. Whole plants were regenerated directly from dormant buds through budding without an intermediary callus phase. The cryopreserved buds produced high levels of shoot formation (76.2-100%), similar to those of noncryopreserved buds (91.3-100%), with no observed differences between cryopreserved and noncryopreserved materials. Three of the twenty cryopreserved apple germplasm accessions were used to assess morphological and genetic stability. No differences in morphological characteristics including shoot length, leaf shape, leaf width/length ratio, and root length were observed between controls (fresh control and noncryopreserved) and cryopreserved plantlets. The genetic stability of regenerants (before and after cryopreservation) was investigated using inter simple sequence repeat (ISSR) markers. The ISSR markers produced 253 bands using four primers, ISSR 810, SSR 835, ISSR 864, and ISSR 899. These markers showed monomorphic banding patterns and revealed no polymorphism between the mother plant and regenerants before and after cryopreservation, suggesting that cryopreservation using two-step freezing does not affect the genetic stability of apple germplasm. These results show that two-step freezing cryopreservation is a practical method for long-term storage of apple germplasms.

Cryopreservation allows for the advances of the reproductive technique and livestock industry. However, cryopreservation inevitably causes various types of stress, such as cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. Although cryoprotectant agent (CPA) is added to protect spermatozoa from freezing damage during cryopreservation, it has intrinsic toxicity that can affect components of the sperm membrane. Moreover, the addition of CPA induces osmotic stress and excessive reactive oxygen species (ROS) generation, resulting in disruption of mitochondrial membrane potential, alteration of membrane permeability, and damage of sperm surface proteins. To identify the effects of CPA to spermatozoa, we analyzed the sperm movement, capacitation status, and viability using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, we performed two-dimensional electrophoresis to find protein markers related CPA addition in cryo processes. CPA addition reduced sperm motility (%), viability (%), and non-capacitated spermatozoa, whereas acrosome-reacted spermatozoa increased significantly (p<0.05). Following addition of CPA, a total of ten proteins were altered their expression (eight increased, two decreased) (>3 fold, p<0.05). Among these, four differentially expressed proteins were related to several canonical pathways, such as the ephrinR-actin, ROS metabolism, actin cytoskeleton assembly, actin cytoskeleton regulation, and respiratory chain and oxidative phosphorylation pathway (p<0.05). The present study suggests that CPA significantly alters the functions and proteome content of spermatozoa. Additionally, we anticipated that the differentially expressed proteins might consider as biomarker of CPA-induced stress.

Spermatogonial stem cells (SSCs) possess the unique capacity of self-renewal and differentiation and thereby can transmit genetic information to the next generation. Combination with techniques such as isolation, culture, preservation, and transplantation of the SSC has facilitated the efficient method for production of transgenic animals, and preservation of livestock and endangered species. The purpose of this study was to genetically modify enriched populations of pre-pubertal germ cells using lentiviral transduction and to develop an efficient in vitro culture system and cryopreservation technique for bovine SSCs. To maximize the efficiency of genetic modification of bovine SSCs, effective enrichment techniques need to be developed. Selection of bovine SSCs using a combination of laminin and gelatin was resulted in a 8-fold enrichment. Selected cells were then transduced using a lentiviral vector containing the transgene for the enhanced green fluorescent protein. Transduction efficiency was 17%. Next, to enhance the efficiency of proliferation for in vitro culture, the effects of various culture conditions and growth factors on bovine cell proliferation were evaluated. Based on the results, we developed the optimal culture conditions [2× rat sertum free medium (rSFM) containing 0.1% FBS together with GDNF, GFRα1, bFGF, EGF, LIF, and CSF-1] for maintaining bovine SSCs over 3 months without any alteration of stem cell characteristics and functions. Also, to develop an effective cryopreservation technique for bovine SSCs, the effects of different freezing methods and various cryoprotective agents were tested. The recovery rate, and proliferation capacity of bovine SSCs were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, and proliferation capacity of germ cells compared to control. As a results, cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs. Collectively, these findings can serve as a model for comprehensively understanding the biology of SSCs and the factors that regulate male fertility. Furthermore, results of this study will be integral for the continued refinement of techniques to manipulate bovine SSCs.

인삼 개갑종자의 초저온보존 효과 및 최적조건을 구명하기 위하여 건조 조건과 수분함량 및 액체질소 보존 전 동결처리와 보존 후 해동처리에 따른 초저온보존 효과를 조사하였다. 건조처리는 25℃ air flow 챔버(RH 10～12%)와 15℃ 건조실(RH 22～25%)을 이용하여 실시하였고 보존 전 동결은 –24℃에서 30일간 처리하였으며 보존 후 해동은 4℃에서 24시간, 40℃에서 2분 처리구를 각각 비교하였다. 그 결과 15℃에서 종자수분 8-12%로 건조된 종자가 보존 전 처리 없이 액체질소에 보존된 후 40℃에서 해동되었을 때 발아율이 가장 높은 것으로 나타났다. 그러나 종자수분이 2.2% 이하일 때 건조가 지속되면 발아율은 감소하는 것으로 나타났으며, 4% 이하에 이르면 보존 전후 처리효과는 나타나지 않는 것으로 조사되었다. 반면 25℃에서 건조된 종자는 상대적으로 높은 발아율을 유지하였다. 건조 및 초저온보존에 의한 인삼 개갑 종자의 유근 및 자엽 생장율을 조사한 결과, GA배지 치상 10일 후 무처리한 대조구에서 첫 유근이 출현하였으며 3주 후에는 자엽발달이 시작되었다. 30일째에는 자엽생장율이 유근발달과 함께 유의하게 증가하는 것으로 나타났다. 치상 30일까지 초저온보존에 의한 생장율은 같은 양상을 보이면서 대조구보다 현저히 낮게 나타났으나 40일째에는 양상은 같았으나 회복율이 대조구보다 증가세를 보였다. 또한 보존 전 동결처리 없었던 종자는 동결처리한 종자보다 모든 처리구에서 높은 유근 및 자엽 생장율을 보였다.

Study question: What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? Summary answer: The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. What is known already: Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified–.warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. Study design, size, duration: A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. Participants/materials, setting, methods: The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of a-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified–.warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. Main results and the role of chance: The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4–.58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6–.92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. Limitations, reasons for caution: The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. Wider implications of the findings: We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.

Vitrification method is widely used in oocyte cryopreservation for IVF but the birth rates are lower than that of the fresh oocyte. One of the known main reasons is structural instability of meiotic spindle and chromosome systems of mature oocyte. To get the best way for keeping competence of matured oocytes, we studied the best conditions for vitrification focused on equilibration times. The mature oocytes were underwent vitrification with current popular method and analyzed the survival rates, microtubule stability and DNA integrity. The survival rates of recovered oocyte are almost same between groups and are more than 93%. The structural configuration of meiotic spindle was well kept in 10 min equilibration group and the stability rate was almost same with that of control. The chromosomal breakdown was observed in all experimental groups, but the chromosomal stability was higher in 10 min equilibration group than the other groups. The 10 min equilibration group showed best condition compared with the other groups. Based on these results, the equilibration time is one of the key factors in successful keeping for competence of mature oocyte. Although, more fine analysis about the effects of physical stress on oocyte during vitrification is needed to define the optimal condition, it is suggested that the optimal equilibration time to get competent oocyte in mouse is 10 min. Information acquired this study may provide insight into intracellular structural events occurring in human oocytes after vitrification and application for cryopreservation of human oocyte.

Biological resources including proteins, cells, and tissues were confronted with both safe and stable preservation for practical use in biotechnological industry. Particularly, cell therapy for regenerative engineering is needed to restricted regulation and accurate preservation. Therefore, this study was investigated improved conditions of mesenchymal stem cells from human umbilical cord (hUCs) or aspirated adipose tissues (hATs) for clinical cell banks. Both cells were isolated according to standard operation procedure of Hurim BioCell Inc. and analyzed the inherent characteristics in passage 4. To compare the ability of experimental groups after cryopreservation, proliferation ability using calculated values and cytomorphological patterns of each experimental step were analyzed. Also proteins such as ice-binding protein or caspase inhibitor were applied to add the preservation medium of hUCs or hATs. Result of preservation solution with 20% serum was considered a positive group. Recovery rate and expansion results showed specific dosage and cell type-dependent differences in the experimental group. Chromosomal stability and multipotency of hUCs or hATs were expressed stable pattern after cryopreservation using advanced medium. As a result, these additives could be substituted for xenogenic sources in banking of hUCs or hATs.

Cryopreservation has been known as an efficient method for long-term preservation of clonally propagated plants, and several cryopreservation methods have been developed. Among them, a droplet-vitrification method for potato using axillary shoot tips in vitro has been established previously. In this study, we have optimized the procedure in which explants were submitted to a step-wise pre-culture in liquid sucrose-enriched medium (0.3 and 0.7 M for 7 and 17 h, respectively). The pre-cultured explants were dehydrated with PVS3 (w/v, 50% glycerol + 50% sucrose) for 90 min or modified PVS2 vitrification solution (w/v, 37.5% glycerol + 15% DMSO + 15.0% ethylene glycol + 22.5% sucrose) for 30 min. This two dehydration solutions produced post-cryopreservation regeneration percentages of 57.2% and 80.9%, respectively. We also compared a new post-culture medium (0.1 mg L ･ -1 GA3, 0.1 mg L ･ -1 kinetin) with the conventional one (0.15 mg L ･ -1 IAA, 0.2 mg L ･ -1 zeatin, 0.05 mg L ･ -1 GA3); the shooting initiation rates were 80.9% and 43.5%, respectively. The results suggest that the modified droplet-vitrification protocol described in this study is more effective, easier to implement, and more economical than the droplet-vitrification protocols currently used for potato.

담수에서 사육한 감성돔 Acanthopagrus schlegeli(담수감성돔) 냉동/해동 정자의 생존율이 가장 높았던 결빙억제제(cryprotective agent, CPA)는 dimethyl sulfoxide(DMSO)였으며, 다음으로 glycerol, ethylene glycol(EG) 그리고 methanol 순이었다. 해동 후 정자운동성은 glycerol, DMSO, EG, methanol 순으로 높았으며, 이상의 결과들은 해수에서 사육한 감성돔(

Human eyelid adipose-derived stem cells (hEAs) and amniotic mesenchymal stem cells (hAMs) are very valuable sources for the cell therapeutics. Both types of cells have a great proliferating ability in vitro and a multipotency to differentiate into adipocytes, osteoblasts and chondrocytes. In the present study, we evaluated their stem cell characteristics after long-time cryopreservation for 6, 12 and 24 months. When frozen-thawed cells were cultivated in vitro, their cumulative cell number and doubling time were similar to freshly prepared cells. Also they expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM, and immune-related genes of HLA-ABC and 2M. Following differentiation culture in appropriate culture media for 2-3 weeks, both types of cells exhibited well differentiation into adipocyte, osteoblast, and chondrocyte, as revealed by adipogenic, osteogenic or chondrogenic-specific staining and related genes, respectively. In conclusion, even after long-term storage hEAs and hAMs could maintain their stem cell characteristics, suggesting that they might be suitable for clinical application based on stem cell therapy.

돼지 중간엽 줄기세포를 Dimethyl sulfoxide(DMSO), Ethylene glycol(EG), 그리고 DMSO/EG을 이용하여 세포동결을 유도한 후 적절한 동결보호제를 알아보았다. 2개월 이내 돼지 골수에서 중간엽 줄기세포를 분리하여 colony 형성 및 alkaline phosphatase(AP) 활성을 확인하고, 지방 세포로의 분화 유도에 의한 줄기세포의 능력을 확인하였다. 이들 중간엽 줄기세포의 완만 동결을 위해, DMEM에 각각 10

Mesenchymal stem cells (MSC) are of great interest for cell-based therapies and tissue engineering approaches, as these cells are capable for extensive self-renewal and display a multilineage differentiation potential. Clinical application of these cells for degenerative and age-related diseases has been accumulating. However, preparation of MSC before the onset of the diseases, it needs to develop the cryopreservation method. Most cryopreservation methods include fetal bovine serum (FBS) which is essential for effective cryopreservation. Yet it should not be used clinically because of the potential risk of infection. In the present study, we investigated whether human serum albumin (HSA), human serum (HS), and knockout serum replacement (KSR) can be used as an alternative of FBS for cryopreservation of human adipose derived stem cells (hADSC). Cells cryopreserved with 9% HSA showed much higher viability after thawing compared with cells frozen with 5% or 1% HSA. Cells cryopreserved with 90% HS or KSR exhibited greater viability than cells frozen with 25% and 5% HS or KSR, respectively. Viability of cells frozen with 9% HSA, 90% HS or 90% KSR was comparable to that with 90% FBS. Morphology and proliferation ability of these cells were not affected by cryopreservation when compared the freshly obtained cells. Cryopreserved hADSC expressed transcription factor genes including Oct3/4, Nanog, Nestin and Sox2, which are related to the self-renewal of stem cells. Flow cytometric analyses showed that both fresh and cryopreserved hADSC were positive for the antigens of HLA-ABC, CD44, CD73, CD90, and CD105, CD166, and negative for HLA-DR, CD31, and CD34. Similar to fresh cells, cryopreserved hADSC could differentiate into mesodermal lineages, adipogenic, osteogenic, or chondrogenic cells. These results suggest that 9% HSA, 90% HS or 90% KSR can be used to replace FBS during successful cryopreservation of hADSC.

The human eyelid adipose-derived stem cells (HEACs) are known as a candidate source for stem cell-based therapy. HEACs possess the ability to proliferate in vitro and multipotency to differentiate into adipogenic, osteogenic and chondrogenic cells. To be used later than the time of collection, a long-term storage is needed. In this study, we investigated stem cell characteristics after cryopreservation of HEACs for 6 months and 1 year in liquid nitrogen. Frozen-thawed stem cells have shown that cumulative cell and doubling numbers were similar to those of fresh HEACs. After thawing, HEACs expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM. They also consistently expressed transcripts of the immune-related genes of HLA-ABC and β2M. To induce mesodermal differentiation, cell were cultivated in adipogenic, osteogenic or chondrogenic medium for 2～3 weeks. After each differentiation culture, HEACs expressed adipocyte-, osteocyte- and chondrocytespecific genes. They were also stained with Oil red O, von Kossa, or alcian blue, revealing adipogenic, osteogenic, or chondrogenic character, respectively. The results suggest that long-term storage up to 1 year do not affect their biological properties, HEACs may be suitable for clinical application on cell-based therapies.

Human embryonic stem cells (hESCs) have the potential for use in regenerative medicine and in the field of basic research. Therefore, effective cryopreservation and storage of hESCs are important for preservation of newly established cell line for various purposes. Despite poor survival and slow recovery after thawing, the conventional slow freezing method is most commonly used for cryopreservation of hESCs due to its simplicity and ease of use for freezing a large number of hESCs appropriate to clinical applications. Here we controlled the clump size (Group Ⅰ; 400～450 ㎛, Group Ⅱ; 800～900 ㎛, and Group Ⅲ; 1500～1700 ㎛) of hESCs at 5 days after plating using a glass pipette during cryopreservation in order to obtain a larger amount of hESCs after thawing. Attachment rates differed significantly (P<0.05) in each of the three groups and the average of attachment rate of GroupⅡ was highest in SNUhES4 and H1. In particular, the attachment rate of Group Ⅱ in SNUhES3 showed a significant improvement with ROCK inhibitor Y-27632. These results indicate that clump size and cell-cell adhesions of GroupⅡ are appropriate for cryopreservation compared to the Group Ⅰ and Group Ⅲ. This method increased cell viability and reduced the recovery time leading to various experiments, and therefore has an advantage for use with hESCs like newly established in particular. We demonstrated that use of this effective cryopreservation method with control of the clump size of hESCs can effectively improve the attachment rate and survival of post-thaw hESCs with and without Y-27632.

줄기세포를 임상에 적용하기 위해서는 체외에서 증식 과정이 필수적이다. 그러나 배아줄기세포와는 달리 성체줄기세포는 체외에서 증식할 경우 일정시간이 지나면 줄기세포의 특성을 잃기 때문에 임상사용에 있어 제한점을 가지고 있다. 이러한 문제점을 극복하기 위해 줄기세포의 특성을 잃지 않게 세포를 보존하는 방법이 필요하며, 본 연구에서는 탯줄 유래 줄기세포를 동결 보존한 후 해동시켜 줄기세포의 특성을 분석하였다. 사람의 탯줄 유래 세포를 분리하여 체외에서 배양한 후 2번째 또는 3번째 계대의 세포를 25% FBS와 10% DMSO가 첨가된 냉동배양액에 넣어 196℃에서 동결보존한 후, 6개월 뒤에 해동시켜 세포의 성장 속도와 유전자 및 단백질 발현을 살펴보았다. 냉동 보존한 후 세포를 해동시킨 결과74%의 생존율을 보였으며, 이 세포를 체외에서 배양하였을 경우, 냉동보존하기 전의 세포와 유사하게 방추사 모양의 섬유아세포의 형태를 나타냈다. 또한, 성장 속도 역시 냉동보존하기 전의 세포와 똑같이 10번째 계대까지 배양되었으며, 42번분열 능력을 나타냈다. RT-PCR 결과, 냉동 전후 세포 모두에서 Oct-4, nanog, SCF, NCAM, nestin, GATA4, BMP4, HLA-1 유전자는 모두 발현하였으며, Brachyury와 HLA-DR은 발현하지 않았다. 면역세포 화학 염색 결과, 배아줄기세포 단백질로 알려진 SSEA-3, -4, Oct-4 그리고 중간엽줄기세포 단백질인 Thy-1은 모두 발현하였으며, vimentin, fibronectin, HLA-1, HCAM, ICAM 모두 발현하였다. 그러나 SSEA-4과 Thy-1, vimentin, fibronectin, HLA-1는 냉동보존한 후 배양된 탯줄유래 세포에서 발현량이 증가하는 양상을 보였으며, CD44와 CD54는 감소하는 양상을 나타냈다. 또한, 조직적합성복합체 항원인 HLA-DR은 냉동보존 전후 탯줄 유래 세포에서 모두 발현하지 않았다. 이와 같이 유전자와 단백질의 발현은 냉동보존하기 전후의 탯줄 유래 세포에서 큰 차이가 없었다. 냉동 보존된 탯줄 유래 세포는 세포의 분열능력과 유전자 및 단백질의 발현이 냉동 보존 전 세포와 유사한 것으로 나타났다. 이러한 결과는 냉동보존법이 임상적으로 세포 치료 시 적절한 세포의 수나 시간을 맞추는데 효과적인 방법이 될 수 있을 것으로 기대된다.

Reactive oxygen species(ROS)에 의한 산화적 손상은 냉동보존 과정과 체외 배양과정 중 세포 생존률 감소의 주된 요인 중 하나이며, 특히 줄기세포의 경우 냉동보존 후 쉽게 분화하거나 사멸하는 경향이 있음이 잘 알려져 있다. 따라서 본 연구는 체외 배양된 인간 조혈모 줄기세포의 냉동보존 시 선별된 항산화제를 처리하여 항산화제가 줄기세포의 생존 및 자동분화에 미치는 영향을 조사하고자 하였다. 해동 후 세포의 생존률은 -tocopherol