• Journal of Embryo Transfer
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• v.22 no.1
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• pp.21-26
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• 2007

본 연구는 mSOF(modified synthetic oviduct fluid medium) 배양액을 이용하여 $100{\mu}1$와 $10{\mu}1$ 배양 소적에서 일본 흑우의 수정란 생산 효율을 개선하기 위하여 수행하였다. 난구세포가 부착된 미성숙 난자는 각각 단독 배양조건($S;\;10{\mu}1$ 소적) 및 그룹 배양 조건 ($G;\;100{\mu}1$ 소적)에서 실시하였고 배양액은 TCM-199의 기본 배지에 10% FCS, 0.02IU/ml FSH와 $1{\mu}g/ml$ $estradiol-17{\beta}$를 첨가하여 사용하였다. 배반포 단계로 발육한 수정란은 1.5M ethylene glycol로 직접 이식법에 의한 동결 방법으로 동결을 실시하였고, 세포수는 융해 후 생존 수정란에 대해 조사하였다. 체외 배양 시간이 $16{\sim}17$시간 배양 조건에서 난자의 성숙율은 그룹 배양 조건$(27.1{\pm}16.8%)$보다는 단독 배양조건$(57.1{\pm}15.0%)$에서 성숙율이 높았다(p<0.05). 그러나 체외 배양 시간이 $18{\sim}19$ 시간과 $20{\sim}21$시간 배양시는 유사한 성숙율을 보였다. 난자의 체외 배양율은 체외 배양 시간의 증가에 의해 성숙도가 $86.3{\pm}9.9%$로 증가하였다. 접합체(zygote)의 분할율은 단독이나 그룹 배양 조건에서도 유사한 결과를 얻었다. 배반포 발달율은 배양 $7{\sim}8$일째에 조사한 결과 단독 배양 방법보다는 그룹 배양 방법에서 발달율이 높았으나, 분할된 접합체를 기준으로 한 경우 배반포 발달율$(S;\; 21.4{\pm}10.6%,\;G;\;39.0{\pm}13.1%)$에서는 유의적인 차이가 없었다. 단독 배양과 그룹 배양에서 $6.5{\pm}8$일 사이에 배반포로 발달된 수정란의 세포수 조사에서는 유사한 결과를 얻었다. 동결 융해 후 24시간 배양 후 배반포 생존율(5; 24.2%, G; 30.2%), 부화율(S: 20.9%, G: 12.7%) 및 생존 수정란수(S; 45.2%, G: 42.8%)에서도 배양 조건에 따른 유의적인 차는 없었다. 결론적으로 mSOF배양액을 이용하는 경우 미성숙 난자의 체외 성숙 유도 배양 시 단독이나 그룹 배양 시 배반포 발달율에서 그룹간에 유의적인 차가 인정되었다(p<0.01).

• KSBB Journal
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• v.22 no.5
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• pp.305-312
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• 2007

It is crucial to develop a miniaturized cultivation method for large and rapid screening of high-yielding mutants of monacolin-K, a powerful anti-hypercholesterolemic secondary metabolite biosynthesized by the fungal cells of Monascus ruber. In order to investigate as many strains as possible in a short time, a miniaturized fermentation method especially suitable for the cultivation of the filamentous Monascus mutants was developed using $50m{\ell}$ culture-tube ($7m{\ell}$ of working volume) instead of the traditional $250m{\ell}$ flask ($50m{\ell}$ of working volume). Generally, in filamentous fungal cell fermentations, morphologies in growth and production cultures should be maintained as thick filamentous and compact-pelleted (usually less than 1 mm in diameter) forms, respectively, for enhanced production of secondary metabolites in final production cultures. In this study, we intended to induce the respective optimal morphologies in the miniaturized culture system for the purpose of rapid screening of overproducers. Miniaturized growth culture system was successfully developed due to the mass production of spores in the statistically optimized solid medium. When large amounts of spores were inoculated into the growth cultures, and brown rice flour (20 g/L) was also supplemented to the growth medium, dense filamentous morphologies were successfully induced in the growth cultures performed with the 50 ml culture tubes. It was implied that the amounts of spores inoculated into the growth tube-cultures and the growth medium components should be the key factors for the induction of the filamentous forms in the growth fermentations. Furthermore, in order to statistically optimize production medium, multiple experiments based on Plackett-Burman design and response surface method (RSM) were carried out, resulting in more than 2 fold enhanced production of monacolin-K in the final production cultures with the optimized production medium. Notably, under the production culture conditions with the statistically optimized medium, optimal pellet sizes below 1 mm in diameter were reproducibly induced, in contrast to the thick and viscous filamentous morphologies observed in the previous production cultures.

• KSBB Journal
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• v.22 no.5
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• pp.297-304
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• 2007

For large and rapid screening of high-yielding mutants of lovastatin produced by filamentous fungal cells of Aspergillus terreus, one of the most important stage is to test as large amounts of mutated strains as possible. For this purpose, we intended to develop a miniaturized cultivation method using $7m{\ell}$ culture tube instead of traditional $250m{\ell}$ flask (working volume $50m{\ell}$). For obtaining large amounts of conidiospores to be used as inoculums for miniaturized cultures, 4 components i.e., glucose, sucrose, yeast extract and $KH_2PO_4$ were intensively investigated, which had been observed to show positive effect on enhancement of spore production through Plackett-Burman design experimet. When optimum concentrations of these components that were determined through application of response surface method (RSM) based on central composite design (CCD) were used, maximum spore numbers amounting to $1.9\times10^{10}$ spores/plate were obtained, resulting in approximately 190 fold increase as compared to the commonly used PDA sporulation medium. Using the miniaturized cultures, intensive strain development programs were carried out for screening of lovastatin high-yielding as well as highly reproducible mutants. It was observed that, for maximum production of lovastatin, the producers should be activated through 'PaB' adaptation process during the early solid culture stage. In addition, they should be proliferated in condensed filamentous forms in miniaturized growth cultures, so that optimum amounts of highly active cells could be transferred to the production culture-tube as reproducible inoculums. Under these highly controlled fermentation conditions, compact-pelleted morphology of optimum size (less than 1 mm in diameter) was successfully induced in the miniaturized production cultures, which proved essential for maximal utilization of the producers' physiology leading to significantly enhanced production of lovastatin. As a result of continuous screening in the miniaturized cultures, lovastatin production levels of the 81% of the daughter cells derived from the high-yielding producers turned out to be in the range of 80%$\sim$120% of the lovastatin production level of the parallel flask cultures. These results demonstrate that the miniaturized cultivation method developed in this study is efficient high throughput system for large and rapid screening of highly stable and productive strains.

• KSBB Journal
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• v.13 no.6
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• pp.681-686
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• 1998

Effect of glucose feeding strategy and initial concentration of glucose on Serratia marcescens ATCC 27117 in high cell density fed-batch fermentation was investigated. The final biomasses in batch, constant feeding, constant and exponentially feeding strategy at glucose starvation condition in fed-batch were 1.40, 5,07, 6,93 and 7.60 g/L at 40, 41, 24 and 40 hrs, respectively. Productivities of biomass were 0.035, 0.124, 0.289 and 0.190 g/L$.$h, respectively. As a result, constant feeding strategy at starvation condition was 1.5∼8.6 times higher than other strategies. The relationship between dissolved oxygen and glucose feeding times was good identified in exponential feeding strategy and constant feeding strategy at starvation condition. And high cell density cultivation was obtained when minimal media was used.

• Korean Aquaculture
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• v.12 no.1
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• pp.95-99
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• 2000

이번 호에서는 먹이생물 관련 양식 정보 시리즈 여섯 번째로 수산 증양식 분야에서 어린 유생의 먹이로서 이용되는 식물${\cdot}$동물 먹이생물의 배양 방법으로서 배량 배양으로 들어가기 전에 선결되어야 할 단일종 배양, 스트레스 배양 그리고 무균 배양에 대하여 기술한다.

• The Korean Journal of Zoology
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• v.10 no.1
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• pp.1-9
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• 1967

본 실험은 4 일간의 성주기가 뚜렷한 성체인 golden hamster로부터 미성숙인 난자를 적출하여 화학적배양액내에서 그의 성숙을 유도시키고 성주기에 따른 미성숙 난자의 성렬율을 관찰하는 것을 주요 목적으로 하여 행하여졌다. 한 개의 난소로부터 5-6개의 미성숙 난자를 적출하여 5% bovine serum albumin이 섞인 TC Medium 199에 넣어서$CO_2$-incubator를 이용하여 6 시간내지 24시간 동안 37$^{\circ}C$를 유지해가며, 배양액이 직접 공기와접촉하는 것을 막기 위해 유동성인 paraffin oil로 배양액을 덮고서 배양을 완수했다. 실험의 결과를 다음과 같이 완수했다. 1. Hamster의 난자의 성숙분렬을 유발시키는데 가장 적합한 배양액은 BMOC, Eagle's Medium , Waymouth's Medium 그리고 TC Medium 199의 네가지 가운데 TC Medium199이었다. 즉 이 배양액을 이용했을 때 난자가 높은 성렬도를 보여주었다. 2. 5% bovine serum alumn을 TC Medium 199에 섞어주었을 때 미성숙 난자가 성순분렬을 유기하는 율이 가장 높았다. 3. 발정기에 있는 난소로부터 얻은 난자가 가장 현저하게 높은 율로 성숙분렬을 보여주었다. 반대로 발정후기의 난자는 배양 시작 후 단시간 내에 대부분이 퇴화하였으며 발정기에 가까워 갈수록 퇴화율이 줄어들었다. 이것은 노포가 성숙분렬을 억제하는 물질을 생성하리라고 여겨지고 있긴 \ulcorner만, 동시에 이 노포는 또 한편 난자의 배란뒤에까지도 생명력을 유지할수 있는 능력을 발정기에 이르기까지의 기간동안 난자에게 부여하며, 난자는 이 능력을 배란전까지 축적하게 되며 이 때문에 노포로부터 유리되어 나온 난자가 장기간 그 생명력을 유지해나가는 것이라고 추정된다. 4. Paraffin oil 로 공기를 차단하여서 배양한 난자나 혹은 watch glass를 이용하여 공기와 접촉시킨 난자에서나 모든 비슷한 율로 성숙분렬을 보여주었다. 이로 보아 조작이 까다로운 paraffin oil을 이용하는 방법보다는 손쉽게 배양할 수 있는 watch glass 의 방법이 오히려 유용하다고 할 수 있다.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.6
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• pp.753-757
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• 1999

The growth of rotifer, Brachionus rotundiformis was evaluated at different culture conditions. Rotifer was fed on condensed freshwater Chlorella. The productivity of rotifer in the high density culture system was compared to that of rotifer in the batch culture system, in which rotifer was fed on baker's yeast. The growth rate of rotifer increased as temperature increased in the culture system supplied with air or oxygen gas. The maximum density of rotifer in the culture systems supplied with air was in range of 16,300$\~$17,000 ind./ml at $24^{\circ}C$. In the culture systems supplied with oxygen gas, it ranged 26,300$\~$30,500 ind/ml at $28^{\circ}C$. When the concentration of dissolved oxygen in the culture system supplied with air reached to below 1 ppm or when the concentration of undissolved ammonia in the culture system supplied with oxygen gas reached 16.6$\~$22.6 ppm, the growth of rotifer decreased. When oxygen gas was supplied and pH was adjusted to 7, the maximum density of rotifer reached to 43,000 ind/ml at $32^{\circ}C$. The production costs for 10 billion rotifer in the high density culture and batch culture were 693,000 and 961,000 won, respectively. Therefore, it was concluded that the productivity of rotifer in the high density culture was higher than that in a batch culture.

• Horticultural Science & Technology
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• v.28 no.5
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• pp.845-849
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• 2010

This study was conducted to compare various culture methods and evaluate economic feasibility of each method for mass propagation of new ever-bearing strawberry 'Goha'. Four different methods such as semi-solid culture, solid culture, liquid suspension culture and bioreactor culture were compared. The solid culture and bioreactor culture showed the shortest and longest root length, such as 3.6 cm and 8.3 cm, respectively. Fresh weights of plants cultured in bioreactor were 2,261 mg, which were heavier than those of cultures. Dry weights of plants cultured in bioreactor were the heavier compared to those in other cultures. The number of axillary bud developed in bioreactor was seven, but axillary bud was not developed in other cultures. Production cost through bioreactor culture was calculated to be 303 won per plant which was 542 won less than that of solid culture. As a result, we found that the bioreactor culture was the most cost effective culture method for in vitro mass propagation in new ever-bearing strawberry 'Goha'.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.150-150
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• 1993

목적 :크롬친화세포는 카테콜아민 (CA)을 분비하는 새포로서 무신 수질내에 주로 존재한다. 따라서 부신피질의 영향을 배제하여 여러가지 자율신경계 작용 약물의 약리작용을 연구하는데 중요한 조직이다. 그러므로, 부신으로부터 크롬친화세포를 분리하여 배양하는 방법을 습득하여 자율신경계 작용약물연구에 이용코자 히스타민을 이용하여 CA 분비작용을 연구하였다. 방법: 도살장에서 소를 즉사시킨 후 좌우양측 부신을 분리하여 collagenase digestion으로 부신수질로부터 분리하고 Percoll gradient에 의해서 chromaffine cell을 정제하였다. 이렇게 정제한 크롬친화세포는 Dulbecco's modified Eagle medium에 10% fetal calf serum과 함께 culture dish에다 넣어 5% $CO_2$, incubator에서 유지시켜 주면서 실험을 시행하였다. 배양세포는 분리후 일주일 이내에 사용하였다. Catecholamine측정은 electrochemical detector를 연결하여 HPLC로 측정하였다.

• KSBB Journal
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• v.13 no.1
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• pp.108-113
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• 1998

The cell growth and amino acid metabolism of a hybridoma cell line in T-flasks, spinner flasks, and a 2L bioreactor were compared. Similar growth and metabolic behaviour were observed for spinner flask and bioreactor cultivations, while those in T-flasks differed significantly. Through a detailed analysis of nutrients and wastes, 7 amino acids were found to be consumed to a much higher extent than the rest of the amino acids. Supplementing the based medium with selected amino acids, glucose, and vitamines increased the cell density by 70%. The addition of vitamines was found to increase the metabolic rates of glucose and lactate.