growth promotion of taxus brevifolia cell suspension culture

7/31/2019 Growth Promotion of Taxus Brevifolia Cell Suspension Culture

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B io te ch no l. B io pr oc es s E ng . 2000, 5: 350-354

Growth Promotion of Taxus brevifolia Cell Suspension CultureUsing Conditioned MediumMyung-Hwan Kim, Su Hwan Chun, and Dong-II Kim *Department of BiologicalEngineering and Institute of Industrial Biotechnology, Inha University, Inchon 402-751, Korea

Abstract The growth promotion of a T ax us b re vifo lia cell suspension culture was investi-gated using conditioning factors. The conditioning factors produced and secreted from cul-tured cells usually stimulate cell division and the production of secondary metabolites.Therefore, the effective incubation time for the optimal secretion of conditioning factors wasfirstly determined for the promotion of cellgrowth. Conditioned media obtained by cultivat-ing for 2 and 5 days showed the promotion of initial cell growth during the early cellgrowthperiod. However, the positive effect of the conditioning factors on the initial cell growth didnot continue because of the depletion of the medium nutrients. Accordingly, the addition ofa carbon source to the conditioned medium prolonged the positive effect on the cell growth.The addition of sucrose to the conditioned medium resulted in the maximum celldensity be-ing reached 4 days earlier compared to the control group and an increased substrate yield.Keywords: conditioning factor, conditioned medium, T a xu s b re vif ol ia

INTRODUCTIONAs the sources of various biochemical substances,

higher plants have been used to produce pharmaceuti-cals, flavors, fragrances, and insecticides. Traditionally,these substances have been obtained using direct ex-traction from the intact plant on a large scale; however,certain of these substances are actually produced throughchemical syntheses. Plant cells grow faster than theplants itself, are not limited to geographic or weatherconditions, and can promote the production of secon-dary metabolites by metabolic regulation under optimalconditions through artificially controlling the environ-ment [1]. However, the comercialization of plant cellcultures is not yet possible due to the difficulty of large-scale cultivation, low yields, the instability of cell lines,relatively slow growth characteristics compared to mi-crobial cells, and difficulties in purification and separa-tion [2].

Although secondary metabolites do not affect theimportant basic metabolism of cells, they help in thephysiological activation of cells and carry out the func-tion of a defense mechanism against various pathogens.The biosynthetic pathway of these secondary metabo-lites of plants is very complicated, implying a complexrelationship among various enzymes related to the pro-duction of the metabolites [3]. Plant cell biotechnologyis currently being considered as a method of producingsecondary metabolites and related research on increasedproductivity at a reduced cost needs to be continued [4].*Corresponding authorTel: +82-32-860-7515 Fax: +82-32-875-0827e-mail: [email protected]

In addition, to increase the productivity of secondarymetabolites, researches are progressing in bioreactordesign, optimization of culture conditions, elicitation,signal transduction, and redirecting the biosyntheticpathway in order to scale up and commercialize theplant cell culture process [5].

At the time of inoculation, a plant cell culture needsto maintain a minimum inoculation concentration. Ifthe inoculation level is under the minimum concentra-tion, the initial cell division becomes difficult since theconditioning factors that are either secreted from thecells or present in the culture do not accommodate theconditions required for growth. Normally, it is the cellthat produce the conditioning factors that change thesurrounding environment to accommodate cell growth.However, if a conditioned medium containing theseconditioning factors is provided, inoculation under theminimum concentration would be possible and cellgrowth could be promoted [6,7]. Yet, these condition-ing factors have not been clearly identified, and only afew potential substances have been reported. When theinitial cell concentration is relatively low and the phos-phate concentration in the medium is high, cell growthhas been observed to be reduced, indicating that an ex-cess amount of phosphate acts as a negative condition-ing factor for cell growth [8]. In contrast, Sun et a l. [9]reported that extracellular calmodulin promotes plantcell growth through a physiological interaction with anunknown extracellular component or various factors inthe cell wall. Conditioning factors not only promotecell growth but also increase productivity of the secon-dary metabolites. When anthocyanin is produced by astrawberry suspension culture, the productivity hasbeen reported to increase with a conditioned medium
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B iot echnol . B iop roce ss Eng . 2000, Vol.5, No.5and the ratio of the anthocyanin components changes[6,10]. The growth-stimulating effects of these condi-tioning factors and increased productivity of secondarymetabolites has been reported to be possible in varioustypes of plant cells [7]. Another study related with thissubject reported that anthocyanin production from arose suspension culture was enhanced when using aconditioned medium obtained from a strawberry sus-pension culture [11].

Accordingly, the present study attempted to improveproductivity by promoting the growth of a T ax us b re vi-folia cell culture, which produces the plant-originatedanti-tumor agent paclitaxel. Therefore, a conditionedmedium was introduced to obtain growth conditionswithout a lag phase during the initial growth periodand prolong the growth-stimulating effect for the pro-motion of cell growth.

MATERIALS AND METHODSSuspension Cell Culture and Media

The Ta xu s b re vi fo li a (Pacific yew) suspension cell cul-ture used in the present study was induced from anexplant of a Pacific yew tree provided by Dr. HenrikPedersen (Rutgers University, USA) and the cells weregrown in Schenk and Hiderbrandt (SH) medium [12]with the addition of 20 giL of sucrose as the carbonsource. Five mg/L of a-naphtaleneacetic acid (NAA) and0.2 mg/L of 6-benzylaminopurine (BAP) were added asgrowth regulators along with a vitamin concentratedsolution supplement. The pH was set at 5.8 with theaddition of 1 N NaOH before autoclaving and a subcul-ture was done every 12 days. The cell culture was per-formed in a rotary shaker under dark conditions at 120rpm while maintaining a temperature of 25C.Preparation of Conditioned Medium

The conditioned medium was prepared from the T.brevifolia suspension cell culture. Eighty mL of the cul-ture broth including cells was inoculated into 170 mL ofthe new culture medium and subculture was takenevery 12 days. Based on the time lapse after each sub-culture, different conditioned media were made byseparating the cells and the culture in a vacuum using aWhatman No.1 filter in a Buchner funnel under sterileconditions.Analytical Methods

After the removal of the culture medium in a vacuumusing a Whatman No. 1 filter paper, the cells werewashed with the same amount of distilled water twiceto eliminate any remaining sugar on the cell surface andthen dehydrated under vacuum. The filtered cells weretransferred to an aluminum weighing-tray and the freshcell weight (FCW) was measured using a chemical bal-ance. After measuring the FCW, the cells were dehy-

351

14

10

. . .. .. . .. con t ro l_ conditioned m ed ium

12

12 15Time( days)

Fig. 1. Effect of conditioned medium on initial cell growth insuspension culture of T. brevifolia.

drated in a drying oven at 60C until a constant weightwas achieved then the dry cell weight (DCW) wasmeasured.

To determine the amount of sugar used relative tothe cell growth, the concentrations of sucrose, glucose,and fructose were analyzed using an HPLC system con-sisting of a pump (Model 910, Young-In Scientific Co.,Korea), refractive index (RI) detector (Waters Co., USA),and a carbohydrate column (4.6 x 250 mm) as a sta-tionary phase. As for a mobile phase, a mixture of ace-tonitrile and water at the ratio of 80:20 was used underisocratic conditions with a flow rate of 1.0 mUmin.

RESULTS AND DISCUSSIONEffect of Conditioned Medium in Taxus brevifoliaSuspension Culture

To observe the initial cell growth of T. brevifolia dueto a conditioned medium, experiments were conductedusing the conditioned medium obtained 5 days aftersubculture. As shown in Fig. 1, when the conditionedmedium obtained on the 5th day after subculture wasused, the results showed no lag phase, which is nor-mally experienced during the initial cell growth period,plus the initial cell growth increased compared to thatof the control group. The maximum cell concentrationwas seen on the 6th day after applying the conditionedmedium, after which there was no more cell growthand the stationary phase finished. This result may bedue to intracellular sugar depletion, as shown in Fig. 2.When the specific growth rate was calculated on the3rd day, the rate was 0.08 and 0.11 day 1 for the controlgroup and the group using the conditioned medium,respectively, thereby indicating that the specific growthrate increased by 38% when the conditioned mediumwas used.

A conditioned medium usually promotes cell growth.Immobilized C atharathus roseus cells cultured in the


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. .. . .. . .. con t ro l___ conditioned medium

12 15Time (days)

Fig. 2. Time course changes of sugar concentration in freshmedium and conditioned medium.

. .. .. ., _ c o n tr o l_ conditioned 2

4

4 10 12Time ( days)

Fig. 3. Appropriate conditioning time for initial cell growthpromotion in T. brevifolia suspension culture. The conditionednumbers (2,5,8, 12) indicate the conditioning period (days).

presence of a conditioned medium enhanced both thefresh-weight accumulation and the growth rate [13].However, in the previously published reports [6,10], noclear effects of conditioned media on strawberry sus-pension cell growth were obtained during the early pe-riod of cultivation. According to Mori and Sakurai [7],the plant type is a determining factor as to whether ornot a plant is capable of stimulating cell growth.Determination of Optimal Conditioning Time

Fig. 3 shows the results of the experiment conductedto determine the most effective conditioning time forthe initial cell growth of T. brevifolia. As the condition-ing periods, 2, 5, 8 and 12 days were applied for theconditioned medium, representing the lag phase, initialexponential growth phase, late exponential growthphase, and stationary phase, respectively. In the case ofthe conditioned medium obtained on the 2nd and 5th

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B io te ch no l. B io pr oc es s E ng . 2000, Vol.5, No.518.--------------------------------------.

____ conditioned med um__._ conditioned med um + glucose--.- conditioned med um + sucrose --!~=:::S..

4~-----.----r_---.----._--_,r_--_r----~4 10 12 14

Time (days)Fig. 5. Time course change of dry cell weight on effect ofsugar addition into conditioned medium during lag phase.Addition of Sugar for Extension of Conditioning Effect

It was confirmed that the initial growth-stimulatingeffect due to the conditioning factors did not last be-cause of the lack of nutrients. Thus, an experiment toprolong the production promoting effect was conductedby adding the conditioned medium before the mini-mum inoculum density was achieved. Compared to theinitial sugar concentration of 20 giL in the control cul-ture, glucose and sucrose were added separately in theconditioned medium so that the predicted sugar con-centration would be 15 giL. The effects of the sugaraddition in the conditioned medium on the cell growthare shown in Fig. 5. Although an increase in the initialcell growth was observed compared to that of the con-trol when the conditioned medium was used as agrowth medium, the cells rapidly entered into the sta-tionary phase on the 6th day when no additional sugarwas used. In contrast, when glucose or sucrose was ad-ditionally provided, a steady cell growth was observedand the same maximum cell concentration as that ob-tained in the control culture was achieved 4 days earlier.Fig. 6 shows that the initial sugar concentration afteradding sugar into the conditioned medium was 15 giLand Fig. 7 shows that the osmotic difference relative tothe sugar concentration difference was maintained untilthe latter stage of the culture, indicating that the cellgrowth occurred more actively and effectively despite arelative low concentration of sugar during the initialstage. When only the conditioned medium was used,the sugar ran out on the 6th day of cultivation, recon-firming that the cell growth ceased due to the depletionof sugar. To confirm that the effective cell growth oc-curred due to the conditioning factors, the cell yieldagainst the substrate was also analyzed. The result, asshown in Fig. 8, shows that the yield increased by 0.34compared to that of the control when additional sugarwas provided to the conditioned medium.

35320 ,-------------------------------------,__ control

__ conditioned medium.....,_ conditioned medium + glucose~ conditioned medium + sucrose

15

....IC,.. . 10" ''""n

5

oo 2 4 6 8 10 12 14

Time (days)Fig. 6. Time coursechangeof sugar concentration on effectofsugar addition into conditioned medium during lag phase.

_____ control180

160- 1400N:J:'" 120< ' ! :EIn 1000E.? BO. .0 60EIn0

40

20

00 2

____ oonditioned medium__'_oonditioned medium + glucose----T- conditio ned med iu m + sucrose

128 10Time (days)

Fig. 7. Time course change of osmolarity on effect of sugaraddition into conditioned medium during lagphase.

4 14

Effect of Sugar Addition TimeBased on the results observed for prolonging the

growth-stimulating effect due to the conditioning fac-tors through adding sugar during the initial culturestage, the possibility as to whether a similar effectwould be seen with the addition of sugar after the sta-tionary phase was also investigated. Therefore, the ef-fect on the growth with the addition of glucose andsucrose was examined when the conditioned mediumwas used 4 days after the beginning of the stationaryphase. For the control culture, glucose and sucrose wereadded into the same volume of SH basal medium. Asshown in Fig. 9, no similar growth-stimulating effectwas observed when the sugar was added during the sta-


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Control CM CM .. glucose CM" sucroseFig. 8. Comparison of biomass yield on effect of sugar addi-tion into conditioned medium during lag phase.tionary phase as when the sugar was added during theinitial stage, and only the maintenance of the station-ary phase was observed.

CONCLUSIONInitial cell growth stimulation with the application of

a conditioned medium in suspension cultures of Taxusbrevifolia was investigated. The initial cell growth-stimulating effect observed the using conditioned me-dium was not a supplemental effect of low osmoticpressure but rather the result of the conditioning fac-tors. For suspension cultures of T. brevifoila, the optimalconditioning time for the preparation of the condition-ing factors applicable for cell growth promotion was 5days. An important factor for maintaining the initialcell growth-stimulating effect due to the conditioningfactors was an additional supply of sugar. This addi-tional sugar supplementation promoted cell growth,decreased the time taken to reach the maximum cellconcentration, and increased the substrate yield. Thetime when the sugar was added to the conditioned me-dium was important and was found to be more effec-tive when it was during the initial culture stage.

Acknowledgements This work was supported by spe-cial research funding for biotechnology program fromInha University (2000).

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sugar addition_______ontrol___ conditloned medium... .._ condit ioned medium" g lucose_,,_ conditioned medium" sucrose

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Fig. 9. Effect of sugar addition into conditioned medium dur-ing stationary phase.

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[10] Sakurai, M., T Mori, M. Seki, and S. Furusaki (1996)Changes of anthocyanin composition by conditionedmedium and cell inoculum size using strawberry suspen-sion culture. B io te c h no l . L e tt . 18: 1149-1154.

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[13] Lee, C. W. T and M. L. Shuler (2000) The effect of inocu-lum density and conditioned medium on the productionof ajmalicine and catharanthine from immobilized C a-t ha ra n th u s r os eu s cells. B io te c h no l . B i o en g . 67: 61-71.

[Received August 9, 2000; accepted October 3,2000]

growth promotion of taxus brevifolia cell suspension culture

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