cause of the difficulties inherent in attempting to use in vitromethods to reproduce the complexity of interactions inliving animals (1). But there are also distinct disadvantagesto animal models. They provide little information about thecontrolling mechanisms underlying the mode of action ofdrugs and, even more important, cannot guarantee reliableextrapolation ofeffects to humans (2).

In vitro methods have some evident advantages overanimal models (3,4). There are no ethical objections and,because of the exactly defined and reproducible experimental conditions, a mechanistic interpretation of the results isoften possible. Human cells can be used, which allowstransfer of the results to humans. In some cases, continuousreal-time measurements can be performed. Finally, in vitromethods are generally less expensive than in vivo methods.

On the other hand, there are disadvantages to in vitromodels. Most such systems are static and do not permit anappropriate simulation of the time course of drug concentrations in the body. Another problem of most in vitro systemsis that cells are often cultivated in an unphysiologic manner(e.g., in a 2-dimensional monolayer instead of in 3-dimensional tissues in the organism). This leads in many cases toalterations in cellular reactions and metabolism. One approach that offers the advantages of the in vitro method andovercomes some of the disadvantages is cell growth in a3-dimensional matrix, which can result in more naturalmetabolic and growth behavior and provide a potential basisfor mimicking important aspects of organ structure (5). Thishas been described for human gliomas, which show distinctdifferences in their extracellular matrix when grown astumor spheroids and as monolayers (6). The extracellularmatrix plays an important role in cell proliferation and celldifferentiation (7,8) and has been suggested to influencepenetration barriers to various cytostatic drugs (9,10).

The fluidized-bed cell-culture reactor is a well-establishedtool for the production of monoclonal antibodies or therapeutic proteins using animal cells (11, 12). This technique of3-dimensional cell cultivation in open porous microcarriersoffers several features that may overcome major drawbacksof other in vitro systems: precise, flexible, and adjustableexperimental conditions; physiologic in vivo-like cell culti

A fluidized-bed cell-culture reactor with on-line radioactivitydetectionwas developedfor the in vitro evaluationof radiopharmaceuticals.Thetechniquewasappliedtomeasurethedependency of the lumped constant (LC) of FDG on the glucoseconcentrationin the culture mediumin a humangliomacell line.Methods: Human glioblastoma cells (86HG39) immobilized inopen porous microcarriers were cultivated in a continuouslyoperating fluidized-bed bioreactor. Atdifferent glucose concentrations in the culture medium, step inputs (0.1 MBq/mL) of FDGwere performedand the cellular uptake of FDG was measuredon-line and compared with analyzed samples. From theseresults,the LC of FDG and its dependencyon the glucoseconcentrationwere calculated.Resufts: This fluidized-bedtechnique enabled precise and reproducible adjustment of all relevant experimental parameters, including radiotracer timeconcentration course, medium composition, pH, dissolved oxygenand temperature under steady-stateconditions, and an on-linedeterminationofthe intracellularradiotraceruptake.Theimmobilizedgliomacellsformed stable,3-dimensional,tumor-likespheroids and were continuously proliferating, as proven by anS-phase portionof 25%—40%.For furtherexaminationof thecells, an enzymatic method for detachment from the carrierswithout cellular destruction was introduced. In the FDG experiments, a significant dependency of the LC on the glucose levelwas found. For normoglycemic glucose concentrations, the LCwas determinedto be in the range of 0.7 ±0.1, whereas inhypoglycemiaLC increasedprogressivelyupto a valueof 1.22 ±0.01 at a glucose concentrationof 3 mmol/L. Conclusion: Thebioreactor representsan improved in vitro model for the on-lineevaluation of radiotracers and combines a wide range of experimentalsetups and 3-dimensional,tissue-likecell cultivationwitha techniquefor on-line radioactivitydetection.

Key Words: cell culture;FDG; fluidizedbed; in vitro;lumpedconstant

J NucI Med2000;41:556—564

espite much discussion, substantial effort, and growingethical issues, animal models remain the standard forpharmacologic and toxicologic testing. This is mainly be

ReceivedJan. 4, 1999; revision accepted Jul. 21, 1999.Forcorrespondenceor reprintscontact:ManfredBiselli,PhD,Instituteof

Biotechnology,ForschungszentrumJülichGmbH,JQIichD.52425, Germany.

556 THEJoui@i OFNUCLEARMEDICINE•Vol. 41 •No. 3 •March 2000

A Cell-Culture Reactor for the On-Line Evaluationof Radiopharmaceuticals: Evaluation of theLumped Constant of FDG in Human Glioma CellsThomas Noll, Heinz Miihlensiepen, Ralf Engels, Kurt Hamacher, Manfred Papaspyrou, Karl-Josef Langen,and Manfred Biselli

Institutes ofBiotechnology, Medicine, and Nuclear Chemistry and Department ofElectronics, JülichResearch Center@Jülich,Germany

vation; feasibility of cell and medium sampling; on-linemeasurement, representative of in vivo-like exposure of thecells to the drug; and continuous operation under steadystate conditions. In this study we extended this system to theon-line evaluation of radiopharmaceuticals.

The high cell densities in the bioreactor allow an easy andaccurate determination of changes in the concentration ofunlabeled substrates, a process that is difficult in most invitro systems using low cell densities in monolayer cultivation, such as microtiter plates, tissue flasks, or Leightontubes. This high sensitivity of the bioreactor method allowsdetermination of metabolic rates similar to those in isolatedorgans or living animals.

FDG PET is being used in determination of the regionalmetabolic rate of glucose in the human body and has beenfound to be useful in many clinical applications, such as thediagnosis and grading of malignant gliomas (13—16).FDGandglucoseare transportedbetweenbloodandbrain tissuesby the same carrier. In the cytoplasm they compete forhexokinase, which phosphorylates to their respective hexose6-phosphates. Whereas glucose-6-phosphate is further metabolized, FDG-6-phosphate is trapped in the cell, becausethe phosphatase activity in the brain is negligible.

Because FDG and glucose differ in their rates of transportand phosphorylation,estimationof the metabolicrate ofglucose (MR@1@)from the net flux of FDG (MR@) requiresa proportionality constant, the lumped constant (LC), expressed as:

. Eq.l

An erroneous underestimation of LC will automaticallyyield a correspondingly erroneous overestimation for MR@1c(17). The LC in normal whole human brain has beenestimated to be in the range of 0.5 (18, 19) or 0.86 (17),whereas for gliomas significantly higher values, from 0.7 to3.1, were found (17). The LC also depends on the plasmaglucose concentration because of the different affinities ofhexokinase for transport and phosphorylation (20—23).

To test the potential of the fluidized-bed cell-culturereactor for the evaluation of radiopharmaceuticals we studied the kinetics of FDG in human glioma cells and analyzedthe influence of the glucose level on the LC of FDG.

MATERIALS AND METHODS

OrganismandCultivationConditionsThe glioblastoma cell line 86HG39, which was described by

means of immunocytochemical and morphologic criteria (24), wasobtained from Dr. Thomas Bilzer (Institut fürNeuropathologieHeinrich-Heine-Universitat, Düsseldorf,Germany). Cells weregrown in Iscove's modified Dulbecco's medium supplemented with10% fetal calf serum, 100 IU/mL penicillin, and 100 pg/mLstreptomycin (all from GibcofBRL, Eggenstein, Germany) anddivided twice a week in ratios between 1:4 and 1:10, depending on

the proliferation rate. Batch cultivations were performed in ahumidified atmosphere (37°C;5% C02).

Fluidized-Bed ReactorThe fluidized-bed cell-culture reactor consisted of a reactor tube

with a thermostat and a conical bottom inlet. The tube was insertedin a recycle loop containing the recirculation pump, dissolvedoxygen (DO) probe, external oxygenator for bubble-free aerationthrough gas-permeable silicone tubing, an application port forinjecting the radiotracer, and a sampling port for medium andcarriers (Fig. 1). Fresh medium was added continuously at rates inaccordance with cell consumption, which was measured periodically from medium samples, and supematant was simultaneouslyremoved. Temperature and pH were adjusted to 37°Cand 7.2,respectively, and the DO concentration was controlled at 30% of airsaturation by adding oxygen or nitrogen using mass flow valves.

The cells were immobilized on open, porous borosilicate glasscarriers (Siran; Schott, Mainz, Germany) modified with gelatin(25), with a diameter of 400—500pm and a porosity of 50%. Thereactor had a total volume of 98 mL, and the volume of settledcarriers was 30 mL. The bioreactor tube was equipped with 2detectors, consisting of a Nal scintillator, a photomultiplier, and anamplifier. They were connected to a standard readout electronicdisplay (main amplifier, discriminator, and counter), allowingsimultaneous data recording in time steps as short as 1 s. Onedetector faced the fluidized bed and the other faced the medium(Fig. 1). This arrangement enabled on-line determination of theintracellular tracer accumulation. The difference between the 2detectors, taking into account the attenuating effect of the glassportion of the fluidized bed, was equivalent to the tracer uptake ofthe cells.

Dosage of the radiopharmaceuticals can be varied in a widerange with this setup. A step function could be performed as well asa pulse input or an exactly defined profile using a perfusor. Toperform a step input, 200 mL supematant were collected during thesteady state ofthe fermentation, centrifuged (10 mm at 2000g), andfiltered. Radiotracer was added to the filtrate in an amount to givethe intended concentration (0.1 MBq/mL), and the solution wasused to replace the medium in the reactor within 25 s.

Samples of medium or carriers with immobilized cells wereremoved through the sampling port; alternatively, samples could betaken through the top in a laminar flow hood. The volumetric flowrate of the circulating medium could be adjusted from 10 to 116mL/min, corresponding to a medium velocity of 5—57.7cm/mm inthe reactor tube, depending on the target of the experiment.

AnalysisTo determine the concentration and viability of cells in tissue

flasks, the medium was withdrawn, and the cells were washed oncewith phosphate-buffered saline (PBS) without calcium and magnesium, pH 7.2 (GibcoIBRL), and detached by trypsinization (5 mmat 37°Cwith a solution of 0.5 g/L trypsin and 0.2 g/L ethylenediaminetetraacetic acid). The cell number was determined in ahemocytometer using the erythrosin B exclusion method (26).

For determination of the number of immobilized cells in thefluidized bed, a volume of approximately 0.5 mL open porousmicroc@rnerswas taken from the reactor. The medium was quicklywithdrawn and the carriers were washed once with 2 mL PBS.After addition of 5 mL solution of 0.1 mol/L citric acid, 0.1%(wtlvol) crystal violet, and 1% (wtlvol) t-octylphenoxypolyethoxy

LC - MR@- MR01@

CELL-CULTUREREACTORAS IN Vm@o MODEL •Noll et al. 557

flow directionlead brick collimator

detector 1 (medium)

detector 2 (fluidized-bed)

37°CHO

microcarriers withimmobilized cells

‘w@_..sampling port

aeration

FIGURE 1. Flow sheet of fluidized-bed bioreactor, equipped with 2 @yscintIllationdetectors.

ethanol (Triton X-lOO; Boebringer Ingelheim, Heidelberg, Ger using a Garnmaszint BF 5300 (Berthold, Bad Wildbad,Germany).many),the sample was shaken vigorously and incubated at 37°C.The sample was subsequently dried andweighed.After

a 6-h incubation, the violet-colored nuclei were counted inahemocytometer.The mic@o@ai@iierswere washed, dried for 48 h at

70°C,and weighed. Alternatively, the cells could be detached fromthe carriers by trypsinization as described in the Results section.

The glucose concentration in the medium was determinedenzymatically, using a commercial automatic analyzer (Ebio Cornpact; Eppendorf, Hamburg, Germany) according to the manufacturer's instructions.Determination

of LCThe LC, the conversion factor between the utilization of FDO

and glucose in cells, was determined as the ratio of the metabolicrates of FDG and glucose (Eq. 1). FDG utilization was calculated

@ the intracellular uptake measured in the carrier samplesduring the first 15 mm of theexperiment:For

cell cycle analysis, cells either from tissue flasks or frommic@@riern were trypsinized, centrifuged (10 miii at 200g),resuspended, fixed in 70% ethanol, and stored at —20°Cuntilq

r mmol ],

MR@ = ç [@@@ cellsmeasurement.

Before measurement the cells were centrifuged andresuspended in PBS. After staining and incubation with propidiumiodide (18 pig/mL/l0@cells) and ribonuclease (40 pg/mIJl0@cells)for 1 h, analysis was performed with an Epics XL flow cytometer(excitation at 488 am, emission at 630 run; Coulter, Krefeld,where

q = cell-specific radiotracer uptake rate (cpm/l0@cells), and@, specific activity (cpm/mmol glucose). The cell-specific

glucose utilization was determined at the same time from analyzedmeiium samples under steady-stateconditions:Germany).

DNA histograms were analyzed by a computer model(MultiCycle; Phoenix Flow Systems, San Diego, CA). The S-phasecompartment of the cell CultUreSwas used as a parameter for theMFR

X (C0 —Cr) I mmol

@@1c N, [mm x io@cells1'proliferative

activity of the cells.where MFR = medium feed rate (mL/min), C0 = glucose conFor each determination of the cell-specific radiotracer uptake, 3centration fresh medium (mmol/L), C@= glucoseconcentrationcarrier

samples (0.25 mL) were taken from the fluidized bed. Eachreactor (mmoIfL), and N@ total number of cells in thereactor.samplewas washed 3 times with PBS. The radioactivity (cpm;FDG was synthesized as described (27). The averagespecificdecay-corrected

with half-life [t1@J= 109.7 mm) was measuredactivity was <7.4 X 1013Bq/mmol.

Eq.2

Eq.3

558 THEJoui@i OFNucLEAi@MEDICINE•Vol. 41 •No. 3 •March 2000

supernatant

medium supply

application portfor radiotracer recirculation pump

Cu0a)

0

>@Cl)CCuV

Cu

C0

00.CuCl)Cu.C9.(0

oFIGURE 2. Cell densityduringcontinuous cultivation of 86HG39 glioma cells influidized-bed bioreactor. 0 = Number ofimmobilized cells/g carrier; 0 = S-phaseportionas parameterforproliferativeactivityof culture.

RESULTS

Cultivation in Tissue Flask and Fluidized-Bed BioreactorFigure 2 shows the results of a continuous cultivation of

86HG39 glioma cells in the fluidized-bed reactor. Thebioreactor was inoculated with 3 X l0@ viable cells/mL.Within 30 mm all cells were immobilized on the carriers inthe fluidized bed (data not shown). Carrier samples wereused to determine cell density (cells/g carrier) and S-phaseportion as parameters for the proliferative activity of theimmobilized cells as described above.

After a short lag phase, the number of immobilized cellsincreased exponentially until the carriers were completelycovered with cells. The number of immobilized cells reacheda steady-state value of 1.15 X 10@cells/g carrier (1 g carrieris approximately equivalent to 2 mL settled carriers). Thecells formed a 3-dimensional tumor-like culture (Fig. 3) butwere still proliferating, as shown by the S-phase portion,which varied from 40% during the exponential phase to 25%in the steady state. Because of limited space on the carriers,the newly built cells left the carriers and were continuouslywashed out with the supematant. This steady state could bemaintained for up to 2 mo (data not shown). The MFRremained constant, as did the composition of the supematant. An S-phase portion of 25%—40%was equivalent to thecorresponding value during the exponential growth phase ofa batch cultivation of 86HG39 glioma cells in tissue flasks,as shown in Figure 4.

Cell Detachment from CarriersAn important requirement for examination ofthe immobi

lized cells and for determination of cell-specific parametersis a technique that enables complete detachment of theimmobilized cells without cellular destruction. This can beperformed enzymatically using trypsin. Approximately 0.5mL carriers with immobilized cells were washed once withPBS and incubated with 2 mL trypsin solution (2.5 g/L;GibcofBRL) at 37°C.The detached cells were quantifiedusing the erythrosin B exclusion method (26). The total

number of immobilized cells was determined using thecrystal violet method. Figure 5 shows the portion ofdetached cells and their viability in dependence on the timeof incubation. After 10 mm, all cells were detached withinthe scope of the accuracy of measurement. A longerincubation resulted in a slight decrease in the number ofcounted cells, caused by enzymatic lysis of cells. Theviability was nearly constant at 85%.

FDG Uptake of Immobilized Glioma Cellsin Fluidized-Bed Bioreactor

Figure 6 shows a transport experiment using FDG in thefluidized-bed bioreactor and performed at the steady state ofthe fermentation. The glucose concentration in the mediumwas 3.2 mmollL, and the MFR was 13 mLih. At the markedtime, a step change in FDG concentration was performedwith a final concentration of 0.1 MBq/mL. Fresh mediumwas also supplemented with 0.1 MBq/mL FDG to avoiddilution effects. Figure 6A shows the on-line signals of the 2detectors. The lower activity in the fluidized bed is caused bythe glass portion of the carriers, which replace activitycontaining medium. But because borosilicate glass carriersdo not enrich FDG (e.g., by adhesion phenomena), this is aconstant effect. Therefore, it can be mathematically corrected using the activity values of the 2 detectors measuredimmediately after finishing the step input. At this time, theintracellular uptake is negligible, and the quotient of the 2signals represents the glass portion in the fluidized bed:

glass portion 100 — @—[%], Eq. 4

where Affi = radioactivity in the fluidized bed (Bq), andAm radioactivity in the medium (Bq).

Figure 6B shows the on-line signals after decay correction(t1,@of 18F = 109.7 mm) and mathematical consideration ofthe glass portion in the fluidized bed. The FDG concentration in the circulating medium was nearly constant, whereasthe value in the fluidized bed increased as a result of the

CELL-CULTUREREACFORAS IN Vm@o MODEL •Noll et al. 559

Time [hJ

FIGURE 3. Scattered-electronmicroscopy picturesfrom open porousglasscarriers, diameter 400—500pm, modified withgelatin. (A) Cell-freecarrier.(B) Carrierwithimmobilized cells (1.15 x 10@cells/g carrier).

intracellular enrichment. At different times, carrier andmedium samples were taken from the reactor and analyzedfor determination of intracellular tracer accumulation, glucose consumption rate, and tracer concentration in themedium. A good correlation between on-line differencesignal (difference between both detector signals in Figure6B) and off-line analyzed samples was obtained (Fig. 6C).The glucose consumption rate was determined to be 200 ±5.5 ng/l0@ cells/mn, and the LC was 1.14 ±0.05.

To investigate the dependence of the LC on the glucoseconcentration in vitro for FDG, 7 experiments with differentsteady-state levels corresponding to different glucose concentrations in the medium (2.7—12.33mmolIL) were performed.Figure 7 shows the results for the LC compared with thevalues obtained from Sokoloff et al. (20) and Suda et al. (23)for [‘@CJ-2-deoxyglucosein rats. For glucose concentrationsabove 5 mmol/L the LC was found to be in the range of 0.7

±0.1, whereaswith glucose concentrations below 5 mmol/L,the LC increased progressively up to a value of 1.22 ±0.01at a glucose concentration of 2.7 mmol/L.

DISCUSSION

The specific goal of this study was to develop and validatea fluidized-bed cell-culture reactor as an in vitro model forthe on-line evaluation of radiopharmaceuticals. A humanglioma cell line was chosen as the cellular model for thisapproach. The cells, immobilized in open porous microcarriers, formed stable 3-dimensional spheroids and were continuously proliferating, comparable to cells in the exponentialgrowth phase during batch cultivation.

For examinationof the immobilized cells and determination of cell-specific parameters, a trypsinization method thatenabled a complete detachment of the immobilized cellswithout cellular destruction was introduced.

560 THE Joui@w OF NUCLEARMEDIcir@ •Vol. 41 •No. 3 •March 2000

I LV

@90.@ @:

@ ........ 50

(@@

: ...‘%% C,,. . ,-

@@. , r .@ :@ 33:Time [h]

FIGURE 4. Batchcultivationof 86HG39gliomacellsin tissueflasks(25 cm2).0 =Number of viable cells; •= S-phase portion;n = 5.

The fluidized-bed reactor enables consideration of a widerange of experimental setups and offers the advantages ofsteady-state conditions in a continuous cultivation and withon-line measurement. The long duration of the stable steadystate permits a large number of experiments using the sameculture over a long time period, provided that the cells arenot affected by the experiments. This can be assumedespecially for radiotracers. The DO concentration, pH, andmedium composition, in addition to temperature, can beadjusted according to the experimental requirements—forexample, to examine the effects of heat treatment on cellulartransport and metabolism. The flow rate of the circulatingmedium can be adapted to the flow rate of the blood in thecorresponding organism, (e.g., mouse, rat, or human). Theinput function of the radiotracer is freely adjustable andenables the simulation of in vivo conditions. In contrast toother 3-dimensional systems, such as hollow-fiber or fixed

bed bioreactors, sampling and examination of the immobilized cells and their compounds are possible at any time.Because of the high cell densities in the bioreactor (totalnumber of cells, —1.5 X l0@ viable cells), an easier andmore accurate determination of metabolic rates is possible,in comparison with in vitro systems using low cell densitiesin monolayer cultivation, such as microtiter plates, tissueflasks, or Leighton tubes.

The fluidized-bed bioreactor is suitable for cultivation of awide spectrum of cells. We performed fermentations withcell lines including hybridoma, Chinese hamster ovary(CHO), baby hamster kidney (BHK), murine mast cells,amd different human gliomas, in addition to primary material such as hepatocytes from liver or stromal and hematopoietic cells from bone marrow (12,28,29).

To prove the applicability of this in vitro system, FDGuptake in human glioma cells was examined. The noise of

FIGURE 5. Enzymatic detachment of86HG39 glioma cells from carriers withtrypsin (2.5 g/L). •= Portion of detachedcells; 0 = viability;n = 3.

@;‘ioo@ ......., .. -@•@ -F.@ —r@

0@@@@

Cl)@ ---@ 8O@:@ 80 @4@q'@

.@@ 60@

.C .,i@. Cu0 60 :Cu:@@@ 40

‘@ @°@:

C S.2 200 20

@ , S@ 1@. 0@ ‘ i . ISO@ 1@5 20 25:@ 5

: Time of incubation [minj

CEi.L-Cuuruiu@ REACTORAS IN VITRo MODEL •Noll et al. 561

@60000E

‘@ 50000

@40000

@ 30000

@ 20000

@l0000C

0

B120000

L@ 100000•

@, 80000

C.)

@ 60000.@

@ 40000@

j 2000:

C40000

@30000

@20000

20@l0000

0

0 5 10 15 20 25 30 35 40 45

radioactivity of fluidized-bed

radioactivity of medium

@ ddition of FDG

0 5 10 15 20 25

Time[mm]30 35 40 45

1',gw@ @l)L@UUV

0C.)

9000@

6000';@

FIGURE6. FOGuptakeof86HG39gliomacells influidized-bed bioreactor. Before starting experiment, cells were cultivated untilsteady state of number of immobilized cellswasreachedat 1.15 x 10@cells/gcarrier.(A)Timecourseof radioactivitysignalsmeasured in medium and fluidized bed afterstep inputof FOGyielding radioactivityconcentration of 0.1 MBq/mL. (B) Oecaycorrected time—activitysignals measured inmedium and fluidized bed, allowing calculation of intracellular FDG accumulation.(C)Comparisonof intracellularFDGaccumulation,obtainedbyon-lineradioactivitydetection, with off-lineanalyzed samples (0,n = 3).

0 5 10 15 20 25 30 35 40 45

Time [mm]

the on-line signal is caused by the fluidization of the carriers.Detector 2 measures a limited volume of the fluidized bedwith fluctuation oflocal carrier density (Fig. 1). The numberof cells in this volume depends on the size and porosity ofthe carriers and on the cell density per g carrier as well as on

the expansion of the fluidized bed (dependent on the flowrate of the circulating medium). Therefore, detector 2 cannotmeasure the cell-specific uptake directly, and 1 carriersample per experiment is necessary for calibration. Thissample can also be used for further analysis. After calibra

562 THE Jouiu@i OF Nuci@E@ MEDICINE •Vol. 41 •No. 3 •March 2000

Time [mm]

A A

I ,4

I ,2@

C

C,)C0C.)0,8•0U)0.E 0,6

@@1

0,4•

2 4 6 8 10 12 14 16

Glucoseconcentration[mM]

FIGURE7. Valuesof LC ±SD (n = 3) atdifferentglucose concentrations:those determined in this study (•),normoglycemiain rats using [1@C]-2-deoxyglucose[A] (Sokoloff et al. (20)), and hypoglycemiausing[1@'C]-2-deoxyglucose[0] (Sudaet al. (23)).Becausethere was no obvious mathematical function to fit the observed relationshipbetween LC and glucose concentration,continuouslinesweredrawnbyinspectionby Suda et al. (23) and by ourselves,respectively.

A

0

0

tion it was shown by off-line analyzed carrier samples thatthe intracellular radiotracer uptake is measurable on-linewith this system.

To test the potential of the fluidized-bed cell-culturereactor we have analyzed the influence of the glucoseconcentration on the LC of FDG. Sokoloff et al. (20) andSuda et al. (23) described the influence of the plasmaglucose level on the LC of [‘4C]-2-deoxyglucose in vivo inrat brain. LC values were described in the literature to behigher in hypoglycemia and to decline from a value of 1.2for the lowest arterial plasma glucose concentration (1.9mmol/L) to a constant value of about 0.48 in normoglycemia. Thus, our evidence suggests that the LC of FDG isdependent on the plasma glucose concentration in humanglioma cells. For glucose concentrations above 5 mmol/L,the LC was found to be in the range of 0.7, whereas withglucose concentrations below 5 mmol/L, the LC increasedup to a value of 1.2 at a glucose concentration of 3 mmol/L.The significantly higher value for the LC in normoglycemiain gliomas compared with normal brain has also beendescribed in other studies of rat (30,31) and human gliomas(21). Earlier studies assumed the LC for gliomas to be the

same as that for normal brain (16,32), estimated to be 0.42(18) or 0.52 (19). But the LC also varies widely from tumorto tumor, as shown by Spence et al. (17), who performed astudy including 40 patients with supratentorial malignantgliomas and found the LC to be in a range of 0.72—3.1.

Llmftations of the BioreactorThe fluidized-bed bioreactor described is a simplified in

vivo model for an organ or a tumor that has no blood vesselsor a blood—brain barrier. Furthermore, this system cannotprovide information regarding interaction of different celltypes or the influence of metabolism of radiopharmaceuticals by other organs, such as the liver. Nevertheless, this

method may improve and enlarge the possibilities of in vitromethods and therefore contribute to a further reduction ofthe number of animals used for pharmacologic and toxicologic testing.

CONCLUSION

The fluidized-bed cell-culture reactor for on-line evaluation of radiotracers in vitro combines precise and flexiblecontrol ofexperimental conditions and physiologic, 3-dimensional cell cultivation with an on-line radioactivity detectionsystem. Using this system we studied the kinetics of FDG inhuman glioma cells and showed that the dependence of theLC of FDG on the glucose level is similarto thatobtainedinrat brain studies.

ACKNOWLEDGMENTS

The authors thank Prof. Dr. Christian Wandrey for hisexcellent support of this work and Marcus Cremer fortechnical assistance.

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