• Plant Resources
• /
• 제7권2호
• /
• pp.87-92
• /
• 2004

Five different poplar hybrids were tested for rescuing embryo to elongate in vitro plantiets after hybridization. Ovaries and ovules were cultured on Woody Plant Medium (WPM) supplemented with cytokinins, 6-benzylamine (BA) and zeatin. Multiple shoots were initiated from half section of capsule with immature embryos after 21 days from pollination and tiny shoots were formed after the expansion of cotyledons in ovule cultures. Germinating response was better in intraspecific hybrids $(6.53\pm1.66)$ than interspecific crosses $(0.93\pm0.54)$ from half section of capsules on WPM medium. In general, zeatin was better than BA in inducing multiple shoots from isolated ovules. The highest average number $(19.40\pm4.53)$ of shoots was produced from immature ovules of 21 days post-pollination of WPM medium supplemented with 5.0 mg/L zeatin. The highest percentage of germination was 93% from the half section of in vitro ovary cultures. Soil acclimation was successfully conducted in cell tray containing artificially mixed soil with 96% survival rate.

• 한국수정란이식학회지
• /
• 제9권1호
• /
• pp.73-83
• /
• 1994

The objective of this study was to develop an effective in vitro production system capable of obtaining more porcine embryos from immature oocytes. These experiments were thus conducted to examine the effect of oocytes type and maturation time on the in vitro maturation(IVM) and fertilization(IVF) of oocytes and the in vitro development (IVD)of IVF embryos. 1. The degree of oocyte maturation based on cumulus expansion index(GEI) did not differ for A- and B-typed oocytes but the index of oocyte type C was lower(P<0.05) than that of other oocyte types. 2. When the oocytes of type A and B were matured for 36, 42 and 48hrs, the GEl was not different between the 36- and 42-h maturation but the GEl after 48hrs was greatly lower(P<0.05) than that of other maturation times. 3. The highest cleavage rate(48.6%) of IVF oocytes was obtained from A typed oocytes and 42-h maturation but the developmental potential based on cleavage index was the highest when B-typed oocytes were matured for 42hrs.

• 한국수정란이식학회지
• /
• 제23권3호
• /
• pp.223-227
• /
• 2008

The cryopreservation of Hanwoo embryos has become an integral part of assisted reproduction in animal. The objective of this study was to assess the effect of The objectives of this study were: (1) to evaluate the influence of bovine embryo developmental stage on in vitro embryo development after freezing, (2) to study the efficiency compared with conventional freezed embryos at different embryo source. For conventional slow-freezing, day 7 or 8 expanded blastocysts were collected. The standard freezing medium was 1.8 M ethylene glycol (EG). Embryos were equilibrated in 1.8 Methylene glycol(EG) with 0.1 M sucrose in Dulbecco's phosphate-buffered saline (D-PBS) supplemented with 0.5% bovine serum albumin. Embryos were then loaded individually into 0.25 ml-straw and placed directly into cooling chamber of programmable freezer precooled to $-7^{\circ}C$, after 2 min, the straw was seeded, maintained at $-7^{\circ}C$ for 8 min, and then cooled to $-35^{\circ}C$ at $0.3^{\circ}C$/min, plunged and stored in liquid nitrogen for at least 3 days. For thawing, the straw containing embryos were warmed in air for 10 see and exposed to $37^{\circ}C$ water for 20 sec. Straws were then removed from $37^{\circ}C$ water. Rates of blastocyst survive and hatched were evaluated at 12 to 48h post-warming. The re-expansion and hatched rates of morula embryos were significantly lower than those obtained for blastocysts and expansion blastocysts (31.6%, 10.5% vs, 68.9%, 22.2% vs, 73.7%, 53.6%, respectively). No differences in re-expansion rates were found between in vivo and in vitro blastocysts. whereas hatched rates was significantly higher (51.2%) in vivo compared with in vitro embryos (18.6%). in conclusion, demonstrate that conventional freezing can be used successfully in cryopreservation of in vitro and in vivo bovine embryos, and that it might be considered for use in commercial programs and embryo preservation.

• Journal of Periodontal and Implant Science
• /
• 제48권3호
• /
• pp.152-163
• /
• 2018

Purpose: To determine whether the swelling and mechanical properties of osmotic self-inflating expanders allow or not the induction of intraoral soft tissue expansion in dogs. Methods: Three different volumes (0.15, 0.25, and 0.42 mL; referred to respectively as the S, M, and L groups) of soft tissue expanders (STEs) consisting of a hydrogel core coated with a silicone-perforated membrane were investigated in vitro to assess their swelling behavior (volume swelling ratio) and mechanical properties (tensile strength, tensile strain). For in vivo investigations, the STEs were subperiosteally inserted for 4 weeks in dogs (n=5). Soft tissue expansion was clinically monitored. Histological analyses included the examination of alveolar bone underneath the expanders and thickness measurements of the surrounding fibrous capsule. Results: The volume swelling ratio of all STEs did not exceed 5.2. In tensile mode, the highest mean strain was registered for the L group ($98.03{\pm}0.3g/cm$), whereas the lowest mean value was obtained in the S group ($81.3{\pm}0.1g/cm$), which was a statistically significant difference (P<0.05). In addition, the S and L groups were significantly different in terms of tensile strength ($1.5{\pm}0.1g/cm$ for the S group and $2.2{\pm}0.1g/cm$ for the L group, P<0.05). Clinical monitoring showed successful dilatation of the soft tissues without signs of inflammation up to 28 days. The STEs remained volumetrically stable, with a mean diameter in vivo of 6.98 mm, close to the in vitro post-expansion findings (6.69 mm). Significant histological effects included highly vascularized collagen-rich fibrous encapsulation of the STEs, with a mean thickness of $0.67{\pm}0.12mm$. The bone reaction consisted of resorption underneath the STEs, while apposition was observed at their edges. Conclusions: The swelling and mechanical properties of the STEs enabled clinically successful soft tissue expansion. A tissue reaction consisting of fibrous capsule formation and bone loss were the main histological events.

• 한국가축번식학회지
• /
• 제21권4호
• /
• pp.355-362
• /
• 1997

This study was undertaken in an effort to product embryos through in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) after cryopreservation of immature and mature porcine oocytes. The experiments were conducted to investigate IVM rate of oocytes frozen with 3 different cryoprotectants and to examine IVF and IVC of frozen-thawed oocytes. The CEI(cumulus cells expansion index) after IVM of frozen-thawed immature oocytes was higher in oocytes frozen with PG＋PEG(propylene glycol plus polyethylene glycol) than those frozen with single cryoprotectant and this index was almost 90% of unfrozen oocyte's index(2.39 vs. 2.66). The IVF rate of all frozen oocytes was very low(68% of unfrozen oocytes) and the IVF rate of frozen immature oocytes was slightly higher than that of frozen mature oocytes(39.0% vs. 34.4%), but polyspermic penetration was higher in frozen immature oocytes(21.9% vs. 19.1%). The cleavage rate after IVF of frozen-thawed oocytes was 9.3% for frozen mature oocytes and 11.3% for frozen immature oocytes and this rate was significantly lower(P<0.05) than that of control(60.7%). The development to 8-cell stage was greatly lower in frozen mature oocytes than in frozen immature oocytes. The results indicate that the use of PG plus PEG as cryoprotectant may be very effective for vitrification of porcine oocytes and the frozen-thawed immature porcine oocytes can be used fro in vitro embryo production based on IVM, IVF and IVC system.

• 한국발생생물학회지:발생과생식
• /
• 제17권1호
• /
• pp.63-72
• /
• 2013

A specific inhibitor of RNA polymerase II, ${\alpha}$-amanitin is broadly used to block transcriptional activities in cells. Previous studies showed that ${\alpha}$-amanitin affects in vitro maturation of cumulus-oocyte-complex (COC). In this study, we evaluated the target of ${\alpha}$-amanitin, and whether it affects oocytes or cumulus cells (CCs), or both. We treated ${\alpha}$-amanitin with different time period during in vitro culture of denuded oocytes (DOs) or COCs in comparison, and observed the changes in morphology and maturation status. Although DOs did not show any change in morphology and maturation rates with ${\alpha}$-amanitin treatment, oocytes from COCs were arrested at metaphase I (MI) stage and CCs were more scattered than control groups. To discover causes of meiotic arrest and scattering of CCs, we focused on changes of cumulus expansion, gap junctions, and cellular metabolism which to be the important factors for the successful in vitro maturation of COCs. Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in ${\alpha}$-amanitin-treated CCs. However, these changes were not observed in oocytes. In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in ${\alpha}$-amanitin-treated CCs but not in oocytes. Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

• 한국가축번식학회지
• /
• 제10권2호
• /
• pp.211-217
• /
• 1986

This experiment was conducted to determine the effect of FSH on in vitro maturation and in vitro fertilizing ability of oocytes recovered from normal follicles of different sizes in superovulated rabbits. Follicular oocytes recovered were cultured in modified Ham's F12 medium containing 0, 0.1, 1.0 and 10$\mu\textrm{g}$ FSH/ml for 18 hours and investigated the degree of cumulus cells expansion and nuclear maturation, which were fertilized with in vivo capacitated rabbit sperm. 1. The number of normal follicles<1.5mm, 1.6 to 2.5mm and> 2.5mm in diameter at 16 to 18hrs after HCG administration was 4.8 (38.8%), 5.5(45.4%) and 3.3(15.8%), respectively. Average percent of oocytes recovered was 69.7% and larger follicles tended to have a higher percent, recovery rate than smaller follicles. 2. The degree of cumulus expansion in medium containing 0.1$\mu\textrm{g}$ FSH/ml was similar to that of control, but markedly decreased under the level of above 1$\mu\textrm{g}$ FSH/ml. The proportions of oocytes which reached the second meiotic metaphase were 57.1, 61.5, 43.8 and 45.0% in medium containing 0, 0.1, 1.0 and 10$\mu\textrm{g}$ FSH/ml, respectively. Oocytes from larger follicles showed a higher nuclear maturation than that from smaller follicles. 3. In vitro fertilization rate of oocytes matured under 1$\mu\textrm{g}$ FSH/ml was slightly, not significant, higher than that of others. 4. Progesterone level in follicular fluid was about 67 to 71ng/ml with no difference in follicular sizes and estradiol-17$\beta$ level was under 25pg/ml.

• Archives of Plastic Surgery
• /
• 제41권6호
• /
• pp.661-667
• /
• 2014

Background Tissue expansion is an effective and valuable technique for the reconstruction of large skin lesions and scars. This study aimed to evaluate the applicability and safety of a newly designed skin expanding bioreactor system for maximizing the graft area and minimizing the donor site area. Methods A computer-controlled biaxial skin bioreactor system was used to expand skin in two directions while the culture media was changed daily. The aim was to achieve an expansion speed that enabled the skin to reach twice its original area in two weeks or less. Skin expansion and subsequent grafting were performed for 10 patients, and each patient was followed for 6 months postoperatively for clinical evaluation. Scar evaluation was performed through visual assessment and by using photos. Results The average skin expansion rate was $10.54%{\pm}6.25%$; take rate, $88.89%{\pm}11.39%$; and contraction rate, $4.2%{\pm}2.28%$ after 6 months. Evaluation of the donor and recipient sites by medical specialists resulted in an average score of 3.5 (out of a potential maximum of 5) at 3 months, and 3.9 at 6 months. The average score for patient satisfaction of the donor site was 6.2 (out of a potential maximum of 10), and an average score of 5.2 was noted for the recipient site. Histological examination performed before and after the skin expansion revealed an increase in porosity of the dermal layer. Conclusions This study confirmed the safety and applicability of the in vitro skin bioreactor, and further studies are needed to develop methods for increasing the skin expansion rate.

• 한국수정란이식학회지
• /
• 제26권3호
• /
• pp.195-199
• /
• 2011

포유동물 수정란의 동결보존기술은 최근 기후 변화에 따른 생물종 다양성을 보존하기 위해서 중요하게 여겨지는 연구 분야이다. 따라서 멸실 위험에 처한 동물의 개량과 증식, 보존과 복원 및 생명공학의 분야에 이르기까지 응용 기술은 다양하게 이용되어진다. 본 연구에서는 한우 수정란의 동결 후 생존성 향상을 위해서 동결 방법에 따른 체내 외수정한의 내동성을 조사하였다. 완만동결에 따른 체내 외수정란의 동결 융해 후 수정란의 재확장률은 89.6%와 81.5% 그리고 72시간 배양하였을 때 부화된 수정란은 76.9%와 43.4%, 생존수정란의 총세포수는 136${\pm}$3.6개와 107${\pm}$3.8개의 결과를 보여 동결 융해 후 생존율에서는 차이를 보이지 않았으나, 수정란의 부화율과 세포수 조사에서는 체내수정란이 유의적으로(p<0.05) 높은 결과를 보였다. 체내수정란의 완만동결 후 배반포 회복률은 91.3% 그리고 12시간 이상 배양하였을 때 확장배반포까지의 발달률은 71.4%의 결과를 보였으며, 초자화 동경에서는 85.7%와 75.0%의 결과를 보였다. 결과적으로 본 연구에서 수행된 체내 외 수정란의 동결기술을 이용한다면 수정란이식 현장에서도 효과적인 활용이 가능할 것으로 사료된다.

• Reproductive and Developmental Biology
• /
• 제31권4호
• /
• pp.267-272
• /
• 2007

Human embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and have the capacity to differentiate into various types of cells in the body. Hence, these cells may potentially be an indefinite source of cells for cell therapy in various degenerative diseases including neuronal disorders. For clinical applications of human ES cells, directed differentiation of these cells would be necessary. The objective of this study is to develop the culture condition for the expansion of neural precursor cells derived from human ES cells. Human ES cells were able to differentiate into neural precursor cells upon a stepwise culture condition. Neural precursor cells were propagated up to 5000-fold in cell numbers over 12-week period of culture and evaluated for their characteristics. Expressions of sox1 and pax6 transcripts were dramatically up-regulated along the differentiation stages by RT-PCR analysis. In contrast, expressions of oct4 and nanog transcripts were completely disappeared in neural precursor cells. Expressions of nestin, pax6 and sox1 were also confirmed in neural precursor cells by immunocytochemical analysis. Upon differentiation, the expanded neural precursor cells differentiated into neurons, astrocytes, and oligodendrocytes. In immunocytochemical analysis, expressions of type III ${\beta}$-tubulin and MAP2ab were observed Presence of astrocytes and oligodendrocytes were also confirmed by expressions of GFAP and O4, respectively. Results of this study demonstrate the feasibility of long-term expansion of human ES cell-derived neural precursor cells in vitro, which can be a potential source of the cells for the treatment of neurodegenerative disorders.