• Title/Summary/Keyword: Modified in-vitro method

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Reduction of Polyspermy in Porcine in vitro Fertilization by Modified Swim-UP Method

  • Park, C.H.;B.S. Koo;Kim, M.G.;J.I. Yun;H.Y Son;Lee, S.G.;Lee, C.K.
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • 2003.10a
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    • pp.110-110
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    • 2003
  • The high incidence of polyspermic fertilization is one of the major causes lowering the overall efficiency of porcine IVF. The common procedure for IVF involves the co-culture of both gametes in the medium drop, which increases sperm concentration and incidence of polyspermy. Therefore, the present study was carried out to increase the efficiency of porcine IVF by reducing polyspermy using a modified swim-up method. This method modifies conventional swim-up washing by placing oocytes directly at the time of washing. Sperm pellet was prepared in the tube and mature oocytes were placed on cell strainer with $70 \mu m$ pore size (Falcon 2350) at the top of the tube. After insemination, the oocytes were stained for examination. Also, the developmental potential of fertilized embryos was measured to evaluate for the feasibility of this method. While having similar penetration rates in both methods ($86.67 \pm 2.36% to 83.33 \pm 1.36%$), there was a significant reduction of polyspermy in modified swim-up method ($17.50 \pm 1.60%$) compare to the control ($44.1 \pm 3.70%$ (p<0.05). Subsequent culture showed higher rate of blastocyst formation in modified swim-up method (20.44$\pm$0.99%) than the control ($15.73 \pm 3.26%$) (P<0.05), even though there was no significant difference. These results suggest that, by controlling the number of spermatozoa reaching the oocytes, porcine oocytes might be protected from polyspermy in vitro. Also, the developmental potential of the fertilized embryos using this method could be improved by increasing the pool of spermatozoa with better quality. Further optimization of the procedure required to implicate this method in routine porcine IVF.

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Alternating Acquisition Technique for Quantification of in vitro Hyperpolarized [1-13C] Pyruvate Metabolism

  • Yang, Seungwook;Lee, Joonsung;Joe, Eunhae;Lee, Hansol;Song, Ho-Taek;Kim, Dong-Hyun
    • Investigative Magnetic Resonance Imaging
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    • v.20 no.1
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    • pp.53-60
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    • 2016
  • Purpose: To develop a technique for quantifying the $^{13}C$-metabolites by performing frequency-selective hyperpolarized $^{13}C$ magnetic resonance spectroscopy (MRS) in vitro which combines simple spectrally-selective excitation with spectrally interleaved acquisition. Methods: Numerical simulations were performed with varying noise level and $K_p$ values to compare the quantification accuracies of the proposed and the conventional methods. For in vitro experiments, a spectrally-selective excitation scheme was enabled by narrow-band radiofrequency (RF) excitation pulse implemented into a free-induction decay chemical shift imaging (FIDCSI) sequence. Experiments with LDH / NADH enzyme mixture were performed to validate the effectiveness of the proposed acquisition method. Also, a modified two-site exchange model was formulated for metabolism kinetics quantification with the proposed method. Results: From the simulation results, significant increase of the lactate peak signal to noise ratio (PSNR) was observed. Also, the quantified $K_p$ value from the dynamic curves were more accurate in the case of the proposed acquisition method compared to the conventional non-selective excitation scheme. In vitro experiment results were in good agreement with the simulation results, also displaying increased PSNR for lactate. Fitting results using the modified two-site exchange model also showed expected results in agreement with the simulations. Conclusion: A method for accurate quantification of hyperpolarized pyruvate and the downstream product focused on in vitro experiment was described. By using a narrow-band RF excitation pulse with alternating acquisition, different resonances were selectively excited with a different flip angle for increased PSNR while the hyperpolarized magnetization of the substrate can be minimally perturbed with a low flip angle. Baseline signals from neighboring resonances can be effectively suppressed to accurately quantify the metabolism kinetics.

Analysis of Labelling Efficiency According to Differences of Rotating Time in a Asan Medical Center (AMC) RBC Labelling Method (서울아산병원의 적혈구 표지 방법에서 교반 시간 차이에 따른 표지 효율의 분석)

  • Chung, Eun-Mi;Jung, Woo-Young;Ryu, Jae-Kwang;Shim, Dong-Oh;Lee, Yeong-Hee
    • The Korean Journal of Nuclear Medicine Technology
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    • v.14 no.1
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    • pp.90-93
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    • 2010
  • Purpose: In our nuclear medicine department, we suggested AMC RBC labeling method improved by modifying a part of existing modified in-vitro method to raise the efficiency of $^{99m}Tc$-RBC labeling. However, it needs to be more additional time and efforts than existing modified in-vitro method because the AMC RBC labeling method has to carry out the centrifugal separation process for 3~5 minutes. Therefore, in this study, we conducted researches to aim to maintain stable labeling effects and supplement a problem about additional time by reducing rotating time when labeling $^{99m}Tc$-RBC. Materials and Methods: This research has been conducted the object of 30 patients who examined study using $^{99m}Tc$-RBC and agreed to this research at our hospital from May 2009 to September 2009. We made 4 blood samples which consisted of ACD 1 cc along with 5 cc blood from each patient and used the AMC RBC labeling method. At this moment, each labeling efficiency was calculated by different rotating time 5 min, 10 min, 15 min, and 20 min and then we compared differences. Results: As a result, When comparing the $^{99m}Tc$-RBC labeling method efficiency by using the AMC RBC labeling method which differents from rotating time, each labeling efficiency were $92.3{\pm}5.0%$ in 5 min, $95.9{\pm}5.0%$ in 10 min, $97.4{\pm}4.9%$ in 15 min and $97.7{\pm}4.8%$ in 20 min. We analyzed differences of the labeling efficiency from change of rotating time by using an one-way ANOVA and verified that in Duncan method. There was relatively efficiency low in 5min rotating time and no statistically significant change in over. Conclusions: When comparing a existing method, the AMC RBC labeling method which goes through the centrifugal separation process again offers more favorable condition to combine RBC with $^{99m}TcO4^-$ by eliminating an plasma ingredient. When using the modified in-vitro method, we have almost 20 min to rotate to acquire stable labeling efficiency. But, when using the AMC RBC labeling method, we acquire labeling efficiency well what we want within only 10 min to rotate. Decrease of rotating time can complement the AMC RBC labeling method which goes through the centrifugal separation process again and also provide more rapid study such as G-I bleeding study due to fast labeling.

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Efficient Cryopreservation of In Vitro Grown Shoot Tips of Strawberry (Fragaria × ananassa Duch.) Germplasm Using Droplet-Vitrification

  • Bae, Jinjoo;Lee, Sun-Yi;Song, Jae-Young;Lee, Jung-Ro;Yoon, Munsup;Yi, Jung-Yoon;Kim, Haeng-Hoon;Lee, Young-Yi
    • Korean Journal of Plant Resources
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    • v.34 no.6
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    • pp.600-607
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    • 2021
  • Cryopreservation method using a droplet vitrification was applied to the thirty-one strawberry accessions of in vitro grown shoot tips. A protocol with 0.3 - 0.5 M preculture followed by C4 loading and B1 dehydration solutions efficiently implemented cryopreservation of twenty-six strawberry accessions. The highest regrowth rate was 85.8% for PHS0007 and others were ranged between 85.8% and 21.0%. A slightly modified protocol was applied to five accessions. With these two protocols, twenty-eight accessions obtained more than 40% regrowth rate. This study showed that the droplet vitrification method was able to practically implement cryopreservation of in vitro grown shoot tips of broad range of strawberry germplasm (105).

Predicting In Sacco Rumen Degradation Kinetics of Raw and Dry Roasted Faba Beans (Vicia faba) and Lupin Seeds (Lupinus albus) by Laboratory Techniques

  • Yu, P.;Egan, A.R.;Leury, B.J.
    • Asian-Australasian Journal of Animal Sciences
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    • v.13 no.10
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    • pp.1377-1387
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    • 2000
  • Two laboratory techniques: (1) an in vitro method with two procedures for measuring protein degradabilities and (2) an in vitro method with three procedures for measuring protein solubility, were investigated to determine which laboratory techniques could most accurately predict the quantity of rumen protein degradation kinetics of legume seeds after dry roasting under various conditions, in terms of (1) rumen protein disappearance ($D_j$, where j=0, 2, 4, 8, 12, 24 and 48 h incubation), (2) rumen protein effective degradability (EDCP), (3) the parameters describing rumen degradation characteristics (the soluble fraction: S, the potentially degradable fraction: D, undegradable fraction: U, lag time: T0 and the degradation rate: Kd) and (4) rumen bypass protein (BCP), which were determined by the method accepted internationally at present, in sacco nylon bag technique using the standardized Dutch method. Feeds evaluated were the raw and dry roasted whole faba (Vicia faba) beans (WFB) and whole lupin (Lupinus albus) seeds (WLS), each was dry roasted under various conditions (at 110, 130 or $150^{\circ}C$ for 15, 30 or 45 min). In vitro protein degradability ($D_1$_Auf and $D_{24}$_Auf) were determined using the modified Aufr re method by enzymatic hydrolysis for 1 h and 24 h using a protease extracted from Streptomyces griseus in a borate-phosphate buffer. In vitro protein solubility ($bf_1$_S, $bf_2$_S, $bf_3$_S) was measured in a borate-phosphate buffer with three different procedures. Results from laboratory techniques (in vitro) were correlated and linearly regressed with in sacco results. Of the three procedures of in vitro protein solubility evaluated, none of them could predict in sacco results with good precision. The highest Pearson correlation coefficient ($R^2$) was less than 0.50. Of two procedures of in vitro protein degradability studied, the $D_1$_Auf values were closely correlated with in sacco parameters: Kd, EDCP and %BCP with high R' values: 0.82, 0.85 and 0.85, respectively, and closely correlated with in sacco $D_j$ at 2, 4, 8 and 12 h rumen incubation with high $R^2$ values: 0.83, 0.91, 0.93 and 0.83, respectively. The $D_{24}$_Auf values could not predict in sacco results. The highest $R^2$ value was less then 0.40. These results indicated that in vitro protein solubility measured in borate-phosphate failed to identify differences in the rate and extent of protein degradation of legume seeds after dry roasting under various conditions and thus should not be used to predict rumen degradation, particularly for heat processed feedstuffs. But in vitro protein degradability using the modified Aufr re method by enzymatic hydrolysis for 1 h or possibly an intermediate time (>1 h and <24 h) is a promising laboratory procedure to detect effectiveness of dry roasting legume seeds on rumen protein degradation characteristics and could be used as a simple laboratory method to predict the rate and extent of protein degradation in the rumen in sacco with high accuracy. The equations to predict EDCP, Kd and BCP of dry roasted legume seeds (WLS and WFB) under various conditions are as follow: For both: EDCP (%)=-1.37+1.06*$D_1$_Auf ($R^2=0.85$, p<0.01). For both: Kd (%/h)=-21.81+0.49*$D_1$_Auf ($R^2=0.82$, p<0.01). For both: %BCP=103.37-1.07*$D_1$_Auf ($R^2=0.85$, p<0.01).

Anti-inflammatory Activity of Ketoprofen Soft Hydrogel (케토푸로펜 소프트 히드로겔의 항염증효과)

  • Lee, Eun-Kyung;Shin, Young-Hee;Lee, Chi-Ho
    • Journal of Pharmaceutical Investigation
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    • v.29 no.2
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    • pp.137-143
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    • 1999
  • Ketoprofen together with various permeation enhancers was incorporated into a novel soft hydrogel which is semi-solid in a container and to form a thin film within a few minutes after applying on the skin. The effect of various enhancers on the skin permeation of ketoprofen from a soft hydrogel was investigated using in vitro and in vivo method. In vitro rat skin permeation of ketoprofen from soft hydrogel was conducted using modified Keshary-Chien diffusion cells. In vivo ketoprofen absorption was also investigated in rats, and the results were compared with that of commercial products. Anti-inflammatory activities were determined using carrageenan-induced paw edema method and adjuvant-induced arthritis method in rats. The anti-inflammatory activity of ketoprofen soft hydrogel formulation with that of commercial products were compared. In vitro as well as in vivo studies showed that $HPE-101^{\circledR}$ was the most effective skin permeation enhancer among those used in this study. Addition of an adhesive (polyisobutylene) in the soft hydrogel decreased skin permeation of ketoprofen. Paw edema and anti-arthritis tests showed that soft hydrogel containing $HPE-101^{\circledR}$ was more effective than the commercial products, which was consistent with the in vivo absorption experiment results.

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Microprogation And Environment Conditions Affecting On Growth Of In Vitro And Ex Vitro Of A. Formosanus Hay

  • Ket, Nguyen-Van;Paek, Kee-Yoeup
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2002.11b
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    • pp.6-7
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    • 2002
  • The goal of this research was to develop the effectiveness of in vitro culture method for A. formosanus and study the environment in vitro conditions affecting on growth. The first series of experiments were examined to investigate the response of three different basal media, MS (Murashige and Skoog, 1962), Knudson (KC; Knudson, 1946) and modified hyponex on growth and multiplication during in vitro culture. Multiple shoot proliferation was induced in shoot tip explants on Hyponex (H3) media supplemented with BA (1 mg1$^{-1}$) or TDZ (1-2 mg1$^{-1}$). Addition of activated charcoal (1%) to the TDZ containing medium promoted rapid shoot tip proliferation (11.1 shoots per explant) but the same medium had an opposite effect resulting in poor proliferation in the nodal explants. However, the regenerated shoots had slow growth rate and failed to elongate. This problem was overcome by transferring the shoot clumps to a hormone free H3 media supplemented with 2% sucrose and 0.5% activated charcoal. Using bioreactor culture for scaling up was also shown the best way for multiple shoot induction and growth of this plant.(중략)

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Closed vitrification of mouse oocytes using the CryoLogic vitrification method: A modification that improves developmental competence

  • Jo, Jun Woo;Jee, Byung Chul;Suh, Chang Suk;Kim, Seok Hyun
    • Clinical and Experimental Reproductive Medicine
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    • v.40 no.4
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    • pp.148-154
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    • 2013
  • Objective: To compare the mouse oocyte vitrification outcomes of the CryoLogic vitrification method (CVM) and the conventional open method using a Cryotop. Two CVM methods (original CVM and modified CVM) were tested. Methods: Mature oocytes obtained from female BDF-1 mice were vitrified by two-step exposure to equilibrium and vitrification solutions. Three vitrification protocols were tested on three groups: the CVM-kit, modified CVM, and Cryotop groups. After exposure to the two solutions, the oocytes were vitrified. After warming, the oocytes were fertilized in vitro, and the embryo development was assessed. Blastomeres positive for caspase were counted using an in situ assay kit. The spindle morphology and chromosome configurations of warmed vitrified oocytes were also assessed. Results: The modified CVM and Cryotop groups showed similar developmental capacities, and similar proportions of cells with intact spindles and chromosome configurations. The modified CVM protocol was superior to the original CVM protocol for developmental competence and intact spindle preservation. However, the CVM group showed a relatively higher number of apoptotic cells in blastocysts. Conclusion: Closed vitrification using the modified CVM protocol may be used as an alternative to the conventional open method, but strategies to decrease apoptosis in the blastomere need to be investigated.

Invirto alternatives to photosensitization Test (광감작성 시험에서의 동물대체 시험법)

  • Lee, Ho;Nam, Ki-Taek;Koh, Jae-Sook;Park, Won-Jae
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.22 no.1
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    • pp.84-101
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    • 1996
  • To minimize the use of animals in toxicity testing, and to reduce the cost in vivo test, more rational test method was described which determines, in the same animal, photoxic and photoallergic potential of a substance, and is daptable to routine testing. The other purpose of this study was to investigate the usefulness of in vivo alternatives ; photostability and spectrophotometric carbonyl assay. In this modified photosensitization model, animal numbers and resting periods, the number and method of topical application were simplified. Two positive photoreactive agents, Benzocaine and 6-methyl coumarine, showed a similar photoallergic potential to that of Ichikawa's method. Two sunscreens, Octyl methoxy cinnamate, Butyl methoxyl dibenzoyl methane, hardly showed photoallergic potentials. The photostability test could be used in the step of prescreening of photosensitization potential because most of the photoreactive agents represented the reduction of more than 20% in the absorbance. And photoreactive agents have a high potential of photosensitization in the sddessment of spectrophotometric carbonyl level although two sunscreens have a low possibility of photosensitization. Therefore this method was assumed as a valuable in vivo alternatives in the respect even in the very low concentrations which phototoxicity test using almonella showed no phototoxic potential.

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