• KSBB Journal
• /
• v.21 no.2
• /
• pp.110-117
• /
• 2006

During routine maintenance, animal cell lines are commonly cryopreserved in growth medium containing serum with 10% DMSO. But, in case of bioprocess under the serum-free conditions, including cultivation of cell lines and producing of pharmaceuticals, the cryopreservation should be executed without serum to prevent a cross-contamination. This experiments were performed to investigate the effects of the serum-free cryopreservation on the CHO cells. To improve the survival rates of the cryopreserved CHO cells in serum-free condition, first, the effects of permeable and non-permeable additives for substitute serum on cell viability were investigated. The combination of 10% DMSO and 0.03 M raffinose in MEM-${\alpha}$ without serum indicated 76% of cell viability. However, it did not reach the survival rates(more than 95%) of the conventional cryopreservation. In the second, to evaluate the cryopreservative ability of the serum-free medium(SFM) we compared viability of the CHO cells cryopreserved in the SFMs(Sigma C5467, C4726, and C1707, JBI SF486 and PF486), the cryoprotectant(Genenmed CAN-1000) and the MEM-${\alpha}$ with serum. All solution contained 10% DMSO. As a result of the comparison, cryopreserved cells in the SFMs showed over 95% of viability and appeared predominant viability better than cryoprotectant CAN-1000. Finally, we assessed the stability of the CHO cells in the long-term cryopreservation(LTC) using SFM. Every three months, the cryopreserved CHO cells were thawed to estimate the cell viability and the recovery rates. Then, real-time RT-PCR analyzed the inserted CHO DHFR gene. All results for the LTC appeared the same stability as the serum containing cryopreservation. In the conclusion, it could be seen that the LTC in the SFM can substitute for serum using methods in the bioprocess proceeded by CHO cells for more than 18 months.

• Journal of Wetlands Research
• /
• v.25 no.4
• /
• pp.257-266
• /
• 2023

Amphibian populations are declining globally, pushing many species to the brink of extinction. To promote biodiversity and sustainable management, countries are actively researching amphibian reproductive ecology. Sperm cryopreservation is a crucial assisted reproductive technology that aids in preserving the genetic diversity of amphibians. However, because amphibian sperm cells are sensitive to osmotic stress, the optimal cryopreservation method therefore differs from species to species. This literature review offers an overview of the significance of developing cryopreservation techniques for amphibian conservation and highlights the need to create optimal cryopreservation methods and the introduction of long-term monitoring (e.g., fertilization success and offspring reproduction) to advance cryopreservation technology development. This review can be used as basic research data for amphibian conservation methods.

• Journal of Sericultural and Entomological Science
• /
• v.37 no.2
• /
• pp.186-187
• /
• 1995

동결보존에서 가장 재생율이 높은 품종은 조직배양에서 발근력이 뛰어난 것으로 알려져 있는 신광뽕으로 최고 92.0%의 재생율을 보였으나, 개량, 용천 및 검설뽕에서는 재생을 보이지 않았다. 또, 건조과정을 거치지 않은 것이나, 건조과정과 예비동결 과정을 거치지 않고, 동결방어제만을 사용한 것의 재생은 전혀 확인할 수 없었다. 금후, 동결보존한 동아의 재생에서 돌연변이 유무는 RAPD로 확인될 수 있을 것이다.

• Journal of Life Science
• /
• v.24 no.11
• /
• pp.1252-1257
• /
• 2014

While dimethyl sulfoxide (DMSO) is the most commonly used cryoprotectant agent in the cryopreservation of cultured mammalian cells, it has been reported to cause differentiation of some cell lines by DNA methylation and associated histone modifications. To avoid the side effects of DMSO in cryopreservation, other agents might be more appropriate for maintaining the stable differentiation of cultured cell phenotypes through cryopreservation. All cryoprotectants should be highly soluble in water and display low cell toxicity. Cryoprotective agents have been shown to be effective in animal sperm preservation, and eight types of amides were examined in the cryopreservation of cultured mouse endothelial cells. Among the amides examined, acetamide and lactamide were effective cryoprotectants for cultured mammalian cells. The most effective concentration of lactamide, 1.5 M, had an even lower cryoprotective ability than 1M DMSO. Because successful cryopreservation of cultured cells is hampered by osmotic stress, the adequate ionic concentration was determined by diluting phosphate-buffered saline (PBS) in the 1.5M lactamide solution. The most effective concentration was $0.4{\times}PBS$, which minimized osmotic stress during the cryopreservation of cultured cells. As the addition of high molecular weight materials in cryopreservation media improves the viability of cells, the effects of bovine serum albumin (BSA), hydroxyethyl-starch (HES), and dextran were examined. The best combination of lactamide-based media for cryopreservation was found to be 1.5 M lactamide in $0.4{\times}PBS$ with 1% BSA.

• Journal of Marine Life Science
• /
• v.8 no.2
• /
• pp.115-120
• /
• 2023

This study aims to find out a suitable extender and cryoprotective agent (CPA) for cryopreservation and its optimum concentration in order to conduct planned artificial seed production of Korean bullhead Pseudobagrus fulvidraco and to preserve superior sperm. Experiments were designed to investigate the effects of the different combinations of three extenders (I: 300 mM glycose, II: Kurokura extender, III: Li extender), four cryoprotectants (dimethyl sulfoxide, ethylene glycol, methanol and glycerol) and four concentrations (5, 10, 15, 20%) on the cryopreservation of Korean bullhead sperm. Postthawed sperm survival rate and sperm activity index (SAI) were detected to evaluate the effects of sperm cryopreservation. The optimal combination of extender and CPA for cryopreservation of Korean bullhead sperm was extender III + 10 and 15% methanol, resulting in a survival rate and SAI of 66.9 ± 8.7, 67.3 ± 13.1% and 2.6 ± 0.4, 2.6 ± 0.5 respectively, which was higher than had been achieved with other extenders and CPAs.

• Clinical and Experimental Reproductive Medicine
• /
• v.37 no.4
• /
• pp.267-291
• /
• 2010

Life can be kept in suspended animation either before fertilization at oocyte stage or after fertilization at different stages of embryonic development for a variety of reasons. It not only has potential applications in fertility preservation and management in human but also has important roles in the preservation and management of animal genetic resources, low-cost international movement of selected genetics, and rapid dissemination of germplasm through assisted reproductive technologies (ART) and genetic engineering. Currently, slow-freezing and vitrification are the two approaches by which oocytes and embryos can be cryopreserved for long-term storage. Both of these methods have their own advantages and disadvantages but allow the cryopreservation of oocytes and embryos with comparable efficiency. Vitrification of oocyte and embryos, although proven successful just 13 years after slow-freezing, is generally considered an emerging technology and appears to slow gain acceptance in both animal and human ART despite having controversial storage and contamination issues. In this manuscript, we discuss the basic techniques of oocyt/embryo cryopreservation and review the current status and recent developments in vitrification.

• Korean Journal of Ichthyology
• /
• v.31 no.4
• /
• pp.195-200
• /
• 2019

An experiment was performed to obtain cryopreservation techniques of Pacific cod Gadus macrocephalus sperm. Milt were cryopreservation using five cryoprotectant demethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, methanol and propylene glycol (PG) with marine fish ringer's solution (MFRS) as diluent. Milt were cryopreserved each experimental methods like cryoprotectants (10% and 20%), equilibration time (3, 5 and 10 min) and freezing protocols (liquid nitrogen vapor above 3, 8 and 12 cm). Post-thaw sperm survival rate revealed the highest in 10% PG with optimum methods of equilibration time (3 min) and freezing protocol (liquid nitrogen vapor above 8 cm) about 21.3±1.8%. Hatching rate of fertilization eggs using fresh and cryopreserved sperm were no significantly different.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.84-84
• /
• 2003

많은 연구에서 돼지의 난포란 혹은 배아가 낮은 온도에 대해 민감한 것은 세포질 내에 함유된 지방구와 관련이 있다고 하였으며, 이런 세포질내의 지방구를 제거함으로 동결능력이 향상된다고 하였다. 이렇게 지방구가 제거된 돼지의 난포란과 배아를 효과적으로 동결보존하기 위해서는 새로운 개념의 동결보존 기술이 필요하다고 하겠다. 따라서, 본 연구는 미성숙 단계에서 지방이 제거된 돼지 난포란의 유리화 동결에 적합한 방법을 찾기 위하여 straw를 이용한 유리화 동결법과 electron microscopic grid(EM grid)를 사용한 유리화 동결법을 실시하여 효율적인 동결방법을 검토하였다.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.81-81
• /
• 2003

돼지 정액의 동결보존은 돼지 정자가 내동성이 약한 특성을 가지고 있어 아직 만족할만한 결과를 얻지 못하여 액상정액을 이용하여 인공수정을 실시하고 있는 실정이다. 돼지 정액의 동결보존을 효과적으로 이룩한다면 돼지 인공수정의 효율성을 증대시킬 수 있고, 우수한 종돈의 보호 및 국제적 돼지 품종의 교류를 증진시킬 수 있을 것이다. 본 실험은 돼지 정액 동결시 동결온도와 시간, 항산화제로 알려진 Taurine의 농도별 첨가, 동해보호제 및 융해조건이 돼지 정액의 동결 융해후 생존성에 미치는 영향을 검토하고자 수행되었다.

• Korean Journal of Ichthyology
• /
• v.19 no.2
• /
• pp.173-177
• /
• 2007

A series of experiments were conducted to compare the effects of various diluents and cryoprotectants on the motility and survival rate in cryopreservation of the red seabream, Pagrus major sperm. Sperm was efficiently cryopreserved using 300 mM glucose as a diluent. Two cryoprotectant, dimethyl sulfoxide (DMSO) and glycerol, were added to 300 mM glucose to formulate the extenders at concentrations between 5% and 30% by volume for freezing. The highest post-thawed sperm motility and survival rate were obtained with 10% DMSO.