• Title/Summary/Keyword: Minimum volume cooling method

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A New Protocol for Effective Cryopreservation of Human Embryonic Stem Cells by a Minimum Volume Cooling Method

  • Kim, Eun-Young;Lee, Keum-Sil;Shin, Hyun-Ah;Park, Sae-Young;Yoon, Ji-Yeon;Kil, Kwang-Soo;Lee, Young-Jae;Kim, Nam-Hyung;Chung, Kil-Saeng
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • 2003.10a
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    • pp.98-98
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    • 2003
  • Recently, human embryonic stem (hES) cells have become very important resources for ES cell basic research, cell replacement therapy, and other medical applications; thus, efficient cryopreservation methods for these cells are needed. This study examined whether a newly developed minimum volume cooling (MVC) vitrification method, which was tested through cryopreservation of sensitive bovine oocytes, can be used for freezing hES cells. Feeder-free cultured hES cell (MB03) colonies were mechanically dissected into several small clumps following enzymatic treatment. We compared the freezing efficiency of a slow-cooling method using a cryo-module (0.4-0.6C/min, 20-30 clumps/vial) and MVC vitrification using a modified 0.5-ml French mini-straw designated as a MVC straw (>$20,000{\circ}C$/min, 10 clumps/straw) After thawing, in vitro survival of hES cell clumps was higher for MVC-vitrified cells (80.8%, 97/120) than for slow-cooled cells (38.2%, 39/102). Further, the proliferation rate of surviving MVC-vitrified cells was similar to that of control hES cells from 2 weeks after thawing. In addition, vitrified-thawed hES cells demonstrated a normal karyotype, were positively immunostained for surface marker antibodies (AP, SSEA-4 and TRA-1-60) and the Oct-4 antibody, and could differentiate into all three embryonic germ layer cells in vitro. This result demonstrates that hES cell clumps can be successfully cryopreserved by a newly developed MVC vitrification method without loss of human cell characteristics.

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A New Efficient Cryopreservation of Human Embryonic Stem Cells by a Minimum Volume Cooling Method (Minimum Volume Cooling 방법을 이용한 효율높은 인간배아줄기세포동결)

  • Kim, Eun-Young;Park, Sae-Young;Yoon, Ji-Yeon;Ghil, Gwang-Su;Lee, Chang-Hyun;Lee, Gun-Soup;Tae, Jin-Cheol;Kim, Nam-Hyung;Lee, Won-Don;Chung, Kil-Saeng;Park, Se-Pill;Lim, Jin-Ho
    • Clinical and Experimental Reproductive Medicine
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    • v.31 no.1
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    • pp.41-50
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    • 2004
  • 연구 목적: 본 연구는 인간배아줄기세포 동결에 minimum volume cooling (MVC) 초자화 동결방법이 유용하게 이용될 수 있는지의 여부를 조사하고자 실시하였다. 연구재료 및 방법: 인간배아줄기세포 콜로니는 0.05% collagenase 처리와 기계적 처리에 의해 작은 clumps로 자른 다음 동결 방법에 따른 효율을 비교 검토하고자, i) 대략 40-50개의 clumps를 10% DMSO가 들어있는 동결액에 $5{\sim}10$분간 처리하여 1ml cryo-vial에 넣고 slow-cooling용 cryo-module에 장착하고 -80C에서 overnight한 후 $LN_2$에 침지하여 완만동결을 실시하거나 ii) 10% ethylene glycol (EG)이 들어 있는 동결액에서 5$\sim$10분 처리하고 30% EG과 0.5 mol sucrose가 들어 있는 동결액에서 30초간 처리하여 본 연구를 위해 개발된 MVC straw에 10 clumps씩 적재한 다음 $4{\sim}5$ MVC straw를 $LN_2$가 들어있는 cryo-vial에 넣어 MVC 초자화동결을 실시하였다. 융해 후 생존율을 조사하였고 배아줄기세포의 특성을 유지하고 있는지의 여부를 조사하였다. 결 과: 융해 후, 인간배아줄기세포의 생존율은 완만동결을 실시했던 군 (20.0%) 보다 MVC 초자화동결을 실시했던 군에서 (76.0%) 유의하게 높게 나타났다. 또한 회복과정에서 완만동결을 실시했던 군에서는 세포성장이 매우 더디고 안 좋은 반면, MVC 초자화동결을 실시했던 군은 배아줄기세포의 증식이 동결을 실시하지 않은 세포와 같은 상태로 2주 이후부터 빠르게 전환되고 회복되는 것을 확인할 수 있었다. 이와 더불어 MVC 초자화동결-융해 후 회복된 배아줄기세포에서 정상 핵형, alkaline phosphatase acitivity, SSEA-4와 TRA-1-60 염색 및 Oct-4 발현을 확인하였으며 체외분화의 특성도 확인하였다. 결 론: 새로이 개발된 MVC 초자화동결을 이용하면 인간배아줄기세포는 고유의 특성을 잃지 않고 성공적으로 동결될 수 있다.

Quick Judgments of Properties of Fine Aggregate to Use the Electric Arc Furnace Oxidizing Slag

  • Lee, Hyung-Min;Lee, Han-Seung;Choi, Jae-Seok
    • Journal of the Korea Institute of Building Construction
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    • v.11 no.5
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    • pp.442-451
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    • 2011
  • Blast furnace slag is recycled as a high value-added material, while steel slag is difficult to recycle or is recycled as a low-grade filler material due to its expansive characteristics. Its property is caused by the high content of free lime and instable steel oxides. Recently, an innovative and rapid cooling method for melting steel slag has been developed in Korea, which reduces free lime content to a minimum level and increases the stability of steel oxides. However, researches on the long-term stability are not sufficient so far. Therefore, this study, focusing on the electric arc furnace oxidizing slag in the steel slag, aims to investigate the properties of the steel slag aggregate, its long-term volume stability and the engineering strength of mortar, and using it as a fine aggregate. This study result indicated that it was possible for it to be used as concrete aggregate because the volume change of the steel slag appeared to be stable.

Relationship between Total Body Fat and S/V Ratio and Body Cooling for Two Hours at $15^{\circ}C$ (한냉에 노출된 인체의 냉각과 총지방량 및 S/V 비율 사이의 관계)

  • Chung, Kwan-Ogg;Nam, Kee-Yong
    • The Korean Journal of Physiology
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    • v.3 no.1
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    • pp.19-28
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    • 1969
  • Skin temperatures on 10 sites and rectal temperature at every 10 minutes, oxygen consumption at every 20 minutes were measured on 18 male subjects (ages between 14 and 47 years) after exposure to cold air at $15^{\circ}C$ for two hours in a climatic room. Total body fat measured by means of a skinfold method and ratio of body surface area (S) to body volume (V), S/V, were utilized as basis of observations. Surface area was calculated after DuBois equation and body volume was calculated by our original formula. In influencing on the heat loss from the body core to the cold environment, % fat showed inverse relations, whereas, S/V ratio showed direct relations. Thus these two factors acted antagonistically on the body heat loss. Local skin temperatures showed negative correlations with skinfold thickness on the same site, nemaly, on chest, r=-.567; on back, r=-.507; and on upper arm, r=-.353. The other 7 skin sites showed low correlations with % fat. Minimum mean weighted skin temperature (MWST) showed a negative correlation (r=-.443) with % fat, and showed no correlation with S/V ratio. Oxygen consumption in the cold air at $15^{\circ}C$ increased from the first measurement at 20 minutes after exposure and maintained the same increasing trend up to 120 minutes. ${\Delta}T_R$ was greater in tile lean subjects who showed a greater % change in oxygen consumption. The antagonistic actions of % fat and S/V ratio on the heat loss were manifested by observations as follows: minimum rectal temperature was higher In fat subjects (r=.600) and lower in subjects with a greater S/V ratio (=-.582), ${\Delta}T_R$ was smaller in fat subjects (r=-.738) and greater in subjects with a greater S/V ratio (r=.618). Temperature difference between body core and skin surface (minimum rectal temperature minus minimum MWST) showed a positive correlation with % fat (r=.600) and a negative correlation with S/V ratio (r=-.881). Decrease in the mean body temperature and heat debt, respectively, showed negative correlations with % fat and positive correlations with S/V ratio.

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Establishment of Bovine Ovum Bank : I. Full Term Development of Vitrified Hanwoo (Korean Cattle) In Vitro Matured Oocytes by Minimum Volume Cooling (MVC) Method

  • 김은영;김덕임;이문걸;이종우;이금실;박세영;박은미;윤지연;허영태
    • Proceedings of the KSAR Conference
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    • 2001.03a
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    • pp.5-5
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    • 2001
  • This study was to test whether Hanwoo in vitro matured oocytes can be successfully cryopreserved by a new vitrification procedure using MVC method. For the vitrification, oocytes were pretreated in 10% ethylene glycol (EG10) for 5-10 min, exposed in EG30 for 30 sec, each oocytes were individually put on the inner wall of 0.25 $m\ell$ straw, and then straws were directly plunged into L$N_2$. Thawing was taken by 4-step procedures [1.0 Msucrose (MS), 0.5 MS, 0.25 MS, and 0.125 MS] at 37$^{\circ}C$. In vitro developmental capacity (survival, cleavage ($\geq$2-cell) and blastocyst rates) in vitrified group was no significant difference compared to that in other treatment groups (exposed; 100.0, 74.4, 32.3% and control; 100.0, 78.3, 36.3%): high mean percentage of oocytes (91.2%) was survived, 69.4% of them were cleaved and 27.9% of cleaved embryos were developed to blastocyst. Especially, after transfer of in vitro developed embryos in vitrified group, four of six recipient animals were found to pregnant and three of them were ongoing pregnant by manual palpation at 250 days after transfer. However, among them, two healthy female calves (23 and 25kg) were born. This result demonstrates that MVC method is very appropriate freezing method for the Hanwoo in vitro matured oocytes and that ovum bank can be maintained efficiently by MVC cryopreservation method.

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Analysis of Greenhouse Thermal Environment by Model Simulation (시뮬레이션 모형에 의한 온실의 열환경 분석)

  • 서원명;윤용철
    • Journal of Bio-Environment Control
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    • v.5 no.2
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    • pp.215-235
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    • 1996
  • The thermal analysis by mathematical model simulation makes it possible to reasonably predict heating and/or cooling requirements of certain greenhouses located under various geographical and climatic environment. It is another advantages of model simulation technique to be able to make it possible to select appropriate heating system, to set up energy utilization strategy, to schedule seasonal crop pattern, as well as to determine new greenhouse ranges. In this study, the control pattern for greenhouse microclimate is categorized as cooling and heating. Dynamic model was adopted to simulate heating requirements and/or energy conservation effectiveness such as energy saving by night-time thermal curtain, estimation of Heating Degree-Hours(HDH), long time prediction of greenhouse thermal behavior, etc. On the other hand, the cooling effects of ventilation, shading, and pad ||||&|||| fan system were partly analyzed by static model. By the experimental work with small size model greenhouse of 1.2m$\times$2.4m, it was found that cooling the greenhouse by spraying cold water directly on greenhouse cover surface or by recirculating cold water through heat exchangers would be effective in greenhouse summer cooling. The mathematical model developed for greenhouse model simulation is highly applicable because it can reflects various climatic factors like temperature, humidity, beam and diffuse solar radiation, wind velocity, etc. This model was closely verified by various weather data obtained through long period greenhouse experiment. Most of the materials relating with greenhouse heating or cooling components were obtained from model greenhouse simulated mathematically by using typical year(1987) data of Jinju Gyeongnam. But some of the materials relating with greenhouse cooling was obtained by performing model experiments which include analyzing cooling effect of water sprayed directly on greenhouse roof surface. The results are summarized as follows : 1. The heating requirements of model greenhouse were highly related with the minimum temperature set for given greenhouse. The setting temperature at night-time is much more influential on heating energy requirement than that at day-time. Therefore It is highly recommended that night- time setting temperature should be carefully determined and controlled. 2. The HDH data obtained by conventional method were estimated on the basis of considerably long term average weather temperature together with the standard base temperature(usually 18.3$^{\circ}C$). This kind of data can merely be used as a relative comparison criteria about heating load, but is not applicable in the calculation of greenhouse heating requirements because of the limited consideration of climatic factors and inappropriate base temperature. By comparing the HDM data with the results of simulation, it is found that the heating system design by HDH data will probably overshoot the actual heating requirement. 3. The energy saving effect of night-time thermal curtain as well as estimated heating requirement is found to be sensitively related with weather condition: Thermal curtain adopted for simulation showed high effectiveness in energy saving which amounts to more than 50% of annual heating requirement. 4. The ventilation performances doting warm seasons are mainly influenced by air exchange rate even though there are some variations depending on greenhouse structural difference, weather and cropping conditions. For air exchanges above 1 volume per minute, the reduction rate of temperature rise on both types of considered greenhouse becomes modest with the additional increase of ventilation capacity. Therefore the desirable ventilation capacity is assumed to be 1 air change per minute, which is the recommended ventilation rate in common greenhouse. 5. In glass covered greenhouse with full production, under clear weather of 50% RH, and continuous 1 air change per minute, the temperature drop in 50% shaded greenhouse and pad & fan systemed greenhouse is 2.6$^{\circ}C$ and.6.1$^{\circ}C$ respectively. The temperature in control greenhouse under continuous air change at this time was 36.6$^{\circ}C$ which was 5.3$^{\circ}C$ above ambient temperature. As a result the greenhouse temperature can be maintained 3$^{\circ}C$ below ambient temperature. But when RH is 80%, it was impossible to drop greenhouse temperature below ambient temperature because possible temperature reduction by pad ||||&|||| fan system at this time is not more than 2.4$^{\circ}C$. 6. During 3 months of hot summer season if the greenhouse is assumed to be cooled only when greenhouse temperature rise above 27$^{\circ}C$, the relationship between RH of ambient air and greenhouse temperature drop($\Delta$T) was formulated as follows : $\Delta$T= -0.077RH+7.7 7. Time dependent cooling effects performed by operation of each or combination of ventilation, 50% shading, pad & fan of 80% efficiency, were continuously predicted for one typical summer day long. When the greenhouse was cooled only by 1 air change per minute, greenhouse air temperature was 5$^{\circ}C$ above outdoor temperature. Either method alone can not drop greenhouse air temperature below outdoor temperature even under the fully cropped situations. But when both systems were operated together, greenhouse air temperature can be controlled to about 2.0-2.3$^{\circ}C$ below ambient temperature. 8. When the cool water of 6.5-8.5$^{\circ}C$ was sprayed on greenhouse roof surface with the water flow rate of 1.3 liter/min per unit greenhouse floor area, greenhouse air temperature could be dropped down to 16.5-18.$0^{\circ}C$, whlch is about 1$0^{\circ}C$ below the ambient temperature of 26.5-28.$0^{\circ}C$ at that time. The most important thing in cooling greenhouse air effectively with water spray may be obtaining plenty of cool water source like ground water itself or cold water produced by heat-pump. Future work is focused on not only analyzing the feasibility of heat pump operation but also finding the relationships between greenhouse air temperature(T$_{g}$ ), spraying water temperature(T$_{w}$ ), water flow rate(Q), and ambient temperature(T$_{o}$).

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