• Journal of Plant Biotechnology
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• 제36권4호
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• pp.309-319
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• 2009

Transgenic plant cell cultures for the production of biopharmaceuticals including monoclonal antibodies, recombinant proteins have been regarded as an alternative platform in addition to traditional microbial fermentation and mammalian cell cultures. Plant-made pharmaceuticals (PMPs) have several advantages such as safety, cost-effectiveness, scalability and possibility of complex post-translational modifications. Increasing demand for the quantity and diversity of pharmaceutical proteins may accelerate the industrialization of PMP technology. Up to date, there is no plant-made recombinant protein approved by USFDA (Food and Drug Administration) for human therapeutic uses due to the technological bottlenecks of low expression level and slight differences in glycosylation. Regarding expression levels, it is possible to improve the productivity by using stronger promoter and optimizing culture processes. In terms of glycosylation, humanization has been attempted in many ways to reduce immune responses and to enhance the efficacy as well as stability. In this review article, all these respects of transgenic plant cell cultures were summarized. In addition, we also discuss the general characteristics of plant cell suspension cultures related with bioreactor design and operation to achieve high productivity in large scale which could be a key to successful commercialization of PMPs.

• KSBB Journal
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• 제15권6호
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• pp.609-614
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• 2000

최근 항암제로서 관심을 끌고있는 camptothecin올 생산하기 위한 희수(Camptotheca acuminata) 현탁세포 배양과 cam­p ptothecin의 생산을 촉진하는 연구를 수행하였다. 장기간 배양 으로 발생되는 이차대사산물 생산능력의 변이가 관찰되어 이 를 극복하고자 세포의 응집을 통한 camptothecin의 생산을 촉진하고자 하였다. 세포의 응집은 hybrid 배지와 sucrose 4 %일 때에 가장 많이 유도되었고, 이때 camptothecin의 생산량도 $18.04{\times}10^{-4} mg/L$로 가장 높은 생산성을 가졌다. 또한, 세포의 응집을 유도하기 위한 일환으로 교반속도를 변화시켜 이에 대한 효과를 알아보았는데, 100rpm에서 가장 많은 세포응집이 유도되었으며, camptothecin의 생산 역시 $19.4{\times}10^{-4} mg/L$로 가장 높은 결과를 나타내었다.

• 식물조직배양학회지
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• 제21권2호
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• pp.91-97
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• 1994

고구마 현탁배양세포로부터 POD 고생산세포주로 선발한 SP-47세포주를 사용하여 POD생산성을 향상시키기 위하여 식물생장조절제 및 탄소원의 종류와 농도, 세포접종량 등의 배양조건을 적정화하였다. 30 g/L Surcrose, 1 mg/L 2,4-D가 첨가된 LS배지 50mL을 함유한 30 mL Erlenmeyer flask에 세포생중량 1 g을 접종하여 $25^{\circ}C$ 암소에서 100 rpm으로 진탕배양하였을 때 세포생장은 배양 후 15일에 절정에 달하였으나 단위세포당 POD 활성(unit/g dry cell wt)은 배양 25일에 약 6,800으로 이는 실생 서양겨자무뿌리의 것보다 약 30배 높았다. 배양시기별 단위세포당 단백질 함량은 계대배양후 약간 높았다가 감소한 후, 배양 25일까지는 거의 일정한 값을 유지하다가 계속 배양함에 따라 감소하였으나 POD 비활성(unit/mg protein)은 배양후 12일부터 배양말기 (40일)까지 계속하여 증가하였다. 배양시기별 POD 동위효소의 패턴은 배양시기에 관계없이 거의 일정하였으나 배양 25일이후 산성의 주요 동위효소들의 활성이 약간 증가하였다. 이러한 결과로부터 고구마 세포배양의 POD는 세포생장 및 계대배양과 배지고갈로 인한 배양스트레스와 밀접한 관련 이 있을 것으로 간주된다. 본 연구에서 확립한 SP-47세포주는 하나의 동위효소가 강하게 발현되어 높은 활성을 보임으로써 새로운 POD 대량생산 시스템에 활용될 수 있을 것으로 기대된다.

• 원예과학기술지
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• 제33권2호
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• pp.251-258
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• 2015

Eurycoma longifolia is an important rare medicinal plant that contains valuable bioactive compounds. In the present study, cell suspension culture of E. longifolia was established for the production of biomass and phenolic compounds. Various medium parameters, such as concentration of auxin, salt strength of the medium, and sucrose and nitrogen concentrations, were optimized for the production of biomass at the flask-scale level. Full strength Murashige and Skoog (MS) medium supplemented with $3.0mg{\cdot}L^{-1}$ naphthaleneacetic acid (NAA), 3% (w/v) sucrose, 0:60 $NH{_4}^+:NO{_3}^-$ was found suitable for biomass accumulation. Based on the optimized flask-scale parameters, cell suspension cultures were established in balloon-type bubble bioreactors, and bioprocess parameters such as inoculum density and aeration rate were optimized. Inoculum density of $50g{\cdot}L^{-1}$ and increasing aeration rate from 0.05 to 0.3 vvm, with increases every 7 days, were suitable for the accumulation of both biomass and phenolic compounds. With the optimized conditions, $14.70g{\cdot}L^{-1}$ dry biomass, $10.33mg{\cdot}g^{-1}$ DW of phenolics and $3.89mg{\cdot}g^{-1}$ DW of flavonoids could be achieved. Phenolics isolated from the cell biomass showed optimal free radical scavenging activity.

• 한국식생활문화학회지
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• 제27권6호
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• pp.743-750
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• 2012

A tissue cultured wild ginseng (TCWG) suspension was inoculated with lactic acid bacteria and fermented to improve the functionality of TCWG. The utilization of TCWG was increased directly using the freeze-dried powder. The optimal ratio of TCWG powder and water for fermentation was 1:19 (5%), which was selected by measuring the fluidity and viable cell count according to concentration. The effects on ADH activation and immune cell activation by each ferments with 10 kinds of Lactobacillus sp. strains were examined. The ferments with the Lactobacillus casei KFRI 692 strain showed 5.4 times higher ADH activity and 1.3 times higher ALDH activity than the non-fermented TCWG powder (control). The level of NO production and cytotoxicity was also measured by Raw 264.7 cells. The ferment with the Lac. casei KFRI 692 strain showed the highest level of NO production and lower cytotoxicity than the others. Therefore, the Lac. casei KFRI 692 strain was selected as a strain for fermentation of a TCWG suspension to maximize its functionality. To identify the optimal fermentation time of the selected Lac. casei KFRI 692 strain on the 5% TCWG suspension, the viable cell count of lactic acid bacterial and the changes in pH were observed for 72 hours. 24-hrs was found to be the optimal fermentation time. In this way, fermented TCWG with lactic acid bacteria showed higher ADH activation efficacy and immune cell activation than non-fermented TCWG.

• Plant Biotechnology Reports
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• 제4권2호
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• pp.125-128
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• 2010

Culture conditions were established for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Nymphoides coreana. Zygotic embryos formed pale-yellow globular structures and calluses at a frequency of 85.6% when cultured on half-strength Murashige and Skoog (MS) medium supplemented with 0.3 $mg\;l^{-1}$ of 2,4-D. However, the frequency of pale-yellow globular structures and white callus formation decreased slightly with an increasing concentration of 2,4-D up to 10 $mg\;l^{-1}$ with the frequency rate falling to 16.7%. Cell suspension cultures were established from zygotic embryo-derived calluses using half-strength MS medium supplemented with 0.3 $mg\;l^{-1}$ of 2,4-D. Upon plating onto half-strength MS basal medium, over 92.3% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted into potting soil and achieved full growth to an adult plant in a growth chamber. The high frequency plant regeneration system for Nymphoides coreana established in this study will be useful for genetic manipulation and cryopreservation of this species.

• Journal of Microbiology and Biotechnology
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• 제8권3호
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• pp.237-244
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• 1998

A study was carried out to elucidate the relation between the production of flavonol glycosides and the change of phenylalanine ammonia-lyase activity in cell suspension cultures of Ginkgo biloba by the unassisted and synergistic effects of various factors. The quercetin production showed a mixed-growth-associated pattern in cell suspension cultures. Fluorescent light and UV radiation increased phenylalanine ammonia-lyase (PAL) activity, and resulted in the increase of the production of quercetin and kaempferol ten- and four-fold, respectively, as compared to that obtained in the normal culture condition. The cell growth of Ginkgo biloba was enhanced .at higher temperatures whereas the quercetin production was at its maximum at low temperatures. Moreover, the quercetin production was increased by temperature change during the culture period. In particular, the quercetin production was at the highest level when the culture temperature was elevated from $10^{\circ}C\;to\;30^{\circ}C$. The addition of phenylalanine as a precursor in the culture medium stimulated an 8-fold increase in the production of quercetin; the addition of naringenin caused a l0-fold increase. The quercetin production was also greatly increased by feeding enzyme cofactors such as 2-ketoglutarate and ascorbic acid in the culture medium, but specific PAL activity was not increased except with phenylalanine feeding. The synergistic effect of UV radiation and naringenin feeding was observed, resulting in the increase of flavonol glycoside production at a rate higher than in any other case investigated.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권5호
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• pp.396-401
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• 2006

The objective of this study was to investigate the antitumor activity of suspension cultures of tea callus cells grown in the presence of different concentrations of the growth regulator 2,4-dichlorophenoxy acetic acid (2,4-D) with or without light irradiation. The methanol and ethanol extracts of precipitated cells (MEP, EEP) exhibited stronger inhibitory effects on the growth of tumor cell lines than the water extract of precipitated cells (WEP) or the supernatant Compared to culture under dark conditions, exposure to light irradiation led to significantly higher antitumor activity. The MEP from light irradiated cells at $250{\mu}g/mL$ with 2.0mg/L 2,4-D displayed more than 64% growth inhibition of HEP-2 cells, whereas normal cells showed less than 25% growth inhibition. The some fractions of MEP obtained from Diaion HP-20 column chromatography displayed the majority of inhibitory activity against the HEP-2 cell line. These results show that 2,4-D, and light stimulated the synthesis of antitumor compounds.

• 한국약용작물학회지
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• 제3권2호
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• pp.96-99
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• 1995

본 실험은 쪽 세포의 현탁배양에서 indirubin 생산을 위한 indole의 처리 시기와 처리 기간 및 첨가량을 찾기 위하여 수행하였으며 얻어진 결과는 다음과 같다. 1. Indole 첨가시 세포 생장량은 억제되었다. 2. Tryptophan 첨가시 세포 생장량의 증가는 크지 않았다. 3. Indole과 L-tryptophan을 고체레지에 첨가한 경우 indole을 첨가한 배지에서 만 indirubin이 검출되었다. 4. Indirubin 생산에 적절한 indole 첨가 농도는 고체배지에서 200mg으로 나타났다. 5. 현탁배양 중 indole 첨가시기를 달리한 결과 배양 20일 후 indole을 첨가한 배지에서 세포와 배지내 indirubin 농도가 높았다. 6. 고체배양보다 현탁배양에서 세포내에 축적된indirubin이 많았으며 배지 내 로 상당량의 indirubin이 유출되었다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.329-333
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• 2003

본 연구에서는 형질전환된 N. tabacum 배양에 있어서 배양 중반 저온으로의 변환이 세포에 미치는 영향과 hGM-CSF의 생산성 변화를 관찰하였다. 배양 중반 저온으로의 변환은 DCW의 증가와 세포크기의 감소를 보였다. Ascorbic acid의 첨가는 배양초기 세포 생존율의 감소를 완화시켰으며, 배양 중반 저온으로의 변환은 약간의 세포생존율 감소를 보였다. 저온으로의 변환, 저온 배양에서의 betaine 첨가, ascorbic acid 첨가 모두 배양 후반 세포 lysis 억제에 효과가 있었다. 배양 중반 저온으로의 변환시 배지내 단백질 분해 효소의 활성을 측정한 결과, 대조구 세포에 비해 낮은 단백질 분해 효소 활성을 나타내었다. 그로인해 배양 중반 이후 단백질 분해 효소에 의한 급격한 hGM-CSF 분해를 감소시킴으로써 상대적으로 대조구 세포에 비해 높은 hGM-CSF 생산성을 유지시켰다. Ascorbic acid를 첨가한 후 배양 도중 betaine(1 mM)을 첨가하여 저온으로 온도를 변환시, hGM-CSF의 생산성 대조구 세포에 비하여 최대 2.1배 까지 높게 유지시켰다.