agar–gelatin hybrid sponge-induced three-dimensional in vitro ‘liver-like’ hepg2 spheroids for...

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE R E S E A R C H A R T I C L EJ Tissue Eng Regen Med 2009; 3: 368–376.Published online 30 April 2009 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/term.172

Agar–gelatin hybrid sponge-inducedthree-dimensional in vitro ‘liver-like’ HepG2spheroids for the evaluation of drug cytotoxicity

Poonam Verma1, Vipin Verma1, Pratima Ray2 and Alok R. Ray1*1Centre for Biomedical Engineering, Indian Institute of Technology Delhi (IITD), New Delhi-110016, and All India Institute of MedicalScience (AIIMS), New Delhi-110029, India2Department of Paediatrics, All India Institute of Medical Science (AIIMS), New Delhi-110029, India

Abstract

Agar–gelatin hybrid sponges were used as scaffolds to induce the formation of three-dimensional(3D) spheroids of HepG2 cells. Agar and gelatin in 2 : 1 ratio were used to make films and sponges.The cell adhesive properties of the films were evaluated by the attachment kinetics. The growthkinetics of HepG2 cells was studied using MTT assay and morphology of the 3D spheroids wasobserved through inverted optical microscopy. The liver cell-specific functions of the 3D spheroidswere evaluated in terms of albumin secretion and urea synthesis. Paracetamol was used as a modeldrug to investigate the use of these 3D spheroids in the preliminary cytotoxicity evaluation ofdrugs. The results showed that the agar–gelatin hybrid sponges induced the formation of 3D HepG2spheroids with significant liver-specific functions. These spheroids exhibited higher amounts ofalbumin and urea synthesis than the control monolayer culture. These 3D spheroids were found tobe more sensitive to the drug (TCIC50 value of 4.6 mM) than the control monolayer (TCIC50 valueof 6.2 mM). The study shows that agar–gelatin-induced HepG2 3D spheroids can be used for thepreliminary evaluation of the toxicity of drugs and chemicals. Copyright 2009 John Wiley & Sons,Ltd.

Received 24 August 2008; Revised 17 January 2009; Accepted 9 March 2009

Keywords scaffolds; three-dimensional cell culture; HepG2 cells; drug cytotoxicity; paracetamol; tissueengineering

1. Introduction

Every year hundreds of chemicals are tested globallyfor their pharmacological properties to be used asdrugs. The liver is the organ which is involved in thecomplex metabolism of chemicals, toxins and drugsand remains one of the primary organs assessedfor safety and efficacy evaluation of new drugs andchemicals (Okey et al., 1986). Thus hepatocytes arethe preferred cells to be used for toxicity testing. Cellline methods measure concentration which representsbasal cytotoxicity distributed to the organs (Acosta et al.,

*Correspondence to: Alok R. Ray, Centre for BiomedicalEngineering, Indian Institute of Technology Delhi (IITD),Hauzkhas, New Delhi-110016, India.E-mail: [email protected]

1985; Balls and Fentem, 1992; Barile et al., 1993;Ekwall, 1980; Hopkinson, 1993). When extrapolated tothe in vivo situation, these values correspond to humanblood concentration and may be interpreted as doseswhich pose a risk to all human organs (Barile, 1994).HepG2 is one of the most extensively used cell linesthat has been used to evaluate the toxicity of chemicalsand drugs (Knasmuller et al., 2004). The HepG2 cellsfrom hepatocellular carcinoma retain many metabolicfunctions of human liver (Knoweles et al., 1980). Thecells have been used for toxicological evaluations intwo-dimensional (2D) cultures (Aden et al., 1979). Inthe human liver, hepatocytes are organized in a three-dimensional (3D) lobular structure, thus monolayer failsto accurately resemble the native microenvironment. 3Dcultures of HepG2 cells in the form of spheroids cantherefore be an in vitro alternative to native liver. Cells

Copyright 2009 John Wiley & Sons, Ltd.

HepG2 spheroids on agar–gelatin sponges for drug cytotoxicity testing 369

in spheroids maintain liver-specific functions better thana monolayer. The structural and functional similarity ofspheroids are the features which make them suitable foruse as in vitro model.

This article deals with the formation of 3D humanhepatocyte cell line HepG2 spheroids, using agar–gelatinhybrid sponges. We have been able to show thatagar–gelatin hybrid scaffolds fabricated in our laboratory(Verma et al., 2007) induce these HepG2 cells to form 3Dspheroids with better liver-specific functions than cells inmonolayer without applying any mechanical force. Theuse of these models in drug cytotoxicity testing has beenshown, using paracetamol as a model drug.

2. Materials and methods

2.1. Materials

Gelatin and agar were purchased from Difco Laboratories(MI, USA) and Himedia Laboratories Pvt. Ltd (Bombay,India), respectively. Glycine was purchased from SRL(Mumbai, India). Tissue culture media (MEM), sodiumbicarbonate and sodium carbonate were purchased fromSigma (St. Louis, MO, USA). Fetal bovine serum wasbought from Gibco BRL (USA). Urea and albuminestimation kits were purchased from Bayer (Germany).Other chemicals were of analytical grade and werepurchased locally. The drug paracetamol, under the brandname ‘Crocin’ (GlaxoSmithKline, India; Batch No. R 7158)was purchased from the local pharmacy.

2.2. Preparation of agar–gelatin matrices

The agar–gelatin hybrid matrices were prepared asdescribed in our previous article (Verma et al., 2007).Briefly agar and gelatin were taken in 2 : 1 ratioand were dissolved in phosphate-buffered saline (PBS),pH 7.4, making a 1% homogeneous solution. 0.15%glutaraldehyde was added to cross-link the gelatincomponent in the solution; 1 ml of solution was pouredinto each well of 24-well tissue culture plates (Nunc,Denmark). The solutions were allowed to gel at roomtemperature overnight. The gels were treated with 0.1M glycine for 1 h and washed repeatedly with double-distilled water and then with PBS, pH 7.4. The gelswere kept in an incubator for 12 h at 50 ◦C for makingfilms (11 ± 1 mm in diameter and <1 mm thick). Formaking sponges (11 ± 1 mm in diameter and 5 ± 1 mmin height), the gels were frozen at −80 ◦C for 24 h andfreeze-dried in a lyophilizer. The prepared matrices wereUV-sterilized overnight and kept in a sterile vacuumdesiccator until use.

2.3. Contact angle analysis

The wettability of the 2 : 1 agar–gelatin film wasdetermined by contact angle analysis of the surface at

25 ◦C using a NRL CA Goniometer (Rame-Hart Inc., NJ,USA; Model 100 00 230).

2.4. Maintenance of HepG2 cell line

The cell line was procured from the National Centre forCell Science (NCCS), Pune, India. The cells were routinelygrown in MEM medium (Sigma) supplemented with10% fetal bovine serum (Gibco), 1 mM sodium pyruvate(Sigma) and 1.5 g/l sodium bicarbonate (Sigma) in tissueculture flasks (Nunc) at 37 ◦C and 5% CO2 in an incubatorfor maintenance. The medium was changed every thirdday and subculture was carried out on a weekly basis,using a 1 : 4 splitting ratio. The viability of the cells wasdetermined by staining the cells with 0.4% Trypan blue(1 : 5 ratio); viable cells exclude the dye, while dead cellsretain it. The cells were counted using a haemocytometer.A cell suspension with >95% viability was used in eachexperiment.

2.5. Cell attachment studies

The cell attachment studies were done on agar–gelatinfilms and the control plate was without films. Briefly,the sterilized films were incubated with 2 ml MEMcomplete medium/well. After 3 h the medium wasreplaced with 1 ml cell suspension having a cell densityof 1 × 106 cells/ml. The films and control plates wereallowed to incubate at 37 ◦C in a 5% CO2 incubator. Atdifferent time intervals, the medium from each well fromboth plates was carefully collected in centrifuge tubes. Thetubes were spun at 800 rpm for 10 min, the supernatantfrom each tube was discarded and the cell pellet wasdispersed in 1 ml PBS. The unattached cells were countedusing a haemocytometer.

2.6. Cell proliferation studies

The proliferation of cells on agar–gelatin sponges wasdetermined by MTT assay (Mosmann, 1983). For the cellculture experiments, agar–gelatin sponges and controlplates were UV-sterilized and washed with sterilizeddouble-distilled water. Before seeding the cells, thesponges were incubated with complete MEM mediumfor 3 h. After 3 h the medium was replaced with thecell suspension. Each plate had some wells left blank(without cells) for the purpose of making them controlsfor the MTT assay. 5 × 104 cells/well were seeded in 24-well plates containing only agar–gelatin sponges (notin blank wells) and control plates (leaving few wellsblank, as in agar–gelatin matrices). In blank wells ofboth the agar–gelatin sponges and the control plates, anequal volume of complete medium (without cells) waspoured. After inoculation, the plates were kept at 37 ◦Cin a 5% CO2 incubator. For MTT assay, the old completemedium from the wells was carefully replaced with 900 µlincomplete medium. 100 µl MTT (5 mg/ml in PBS, pH

Copyright 2009 John Wiley & Sons, Ltd. J Tissue Eng Regen Med 2009; 3: 368–376.DOI: 10.1002/term

370 P. Verma et al.

7.4) was added into two wells of the agar–gelatin matricesplate (one with agar–gelatin matrices film and the otherwith medium only) and two wells of the control plate (onewith cells and the other with medium only) after every24 h. The plates were again kept at 37 ◦C in a 5% CO2

incubator until purple crystals of formazan appeared. Themedium from the wells was carefully removed withoutdisturbing the cells. 1 ml acidic isopropanol (0.1 N HClin isopropanol) was added into each well to dissolve thecrystals by keeping them in the incubator at 37 ◦C for1 h. The solution from each well was collected in sterilecentrifuge tubes and spun at 13 000 rpm for 2 min toremove any cell debris. The ODs of the solutions wererecorded at 570 and 690 nm and background subtractionwas done.

2.7. Morphological study of the spheroids

The morphology of the 3D HepG2 cell spheroids wasobserved through an inverted optical microscope (Leica,Germany) at ×20 magnification. MTT assay indicated theviability of the spheroids.

2.8. Characterization of liver-specific functionsin HepG2 spheroids

The liver-specific functions of 3D HepG2 cell spheroidswere determined by measuring albumin secretion andurea synthesis.

2.8.1. Albumin secretion analysis

The agar–gelatin matrices plate and the control platewere seeded with 5 × 104 cells/well for albumin secretionstudies. These were kept at 37 ◦C in a 5% CO2

incubator. The media were collected after every 24 hinterval. The assay was performed using a commercialkit (Bayer, Germany; Code 702) according to theprotocol provided. Briefly, the samples were brought toroom temperature. Control pure albumin (5 mg/ml) andsamples were added with Bromo-cresol green reagent andincubated for 1 min. The change in colour was measuredspectrophotometrically at 628 nm. The concentration ofalbumin in the samples was determined by extrapolatingthe obtained values with the standard albumin curve.

2.8.2. Urea synthesis assay

The urea estimation was done using a urease-basedcommercial kit (Bayer, Germany; Code 792) accordingto the protocol provided. The samples collected forestimating albumin were used for the quantitativedetermination of urea. Briefly, 5 ml glass tubes weretaken for setting up the reactions and to each was added100 µl urease. For blank and standard, 10 µl completemedium without cells and standard pure urea were used.10 µl of each sample was added in a separate tube. The

reaction was allowed for 10 min at 37 ◦C. Then 1.5 mlreagents A (phenol) and B (hypochlorite) were added toeach tube and incubated for 15 min at 37 ◦C. The colourformed was measured spectrophotometrically at 546 nm.The concentration of urea in the sample was determinedby extrapolating the obtained values with the standardurea curve.

2.9. Paracetamol cytotoxicity study

Paracetamol (N-acetyl-p-aminophenol) was used as amodel drug for the evaluation of cytotoxicity.

2.9.1. Preparation of the stock solutionof paracetamol (N-acetyl-p-aminophenol)

Paracetamol was dissolved into MEM complete mediumto form 50 mM stock solution. The pH of the medium wasadjusted to 7.4. The stock solution was filtered through a0.2 µm filter (Millipore, USA). Working solutions of thedrug were prepared by appropriately diluting the stocksolution with complete culture medium to give 1, 5, 10and 25 mM concentrations.

2.9.2. Treatment of cells with the differentconcentrations of the drug

For drug experiments, initial preparation of control platesand plates containing agar–gelatin sponges was the sameas mentioned in Section 2.6. Two wells in each platewere left empty as blanks. The medium in the wells waschanged every day until the beginning of the experiment.On day 5, the medium from the wells containingHepG2 spheroids and control (monolayer) was removed.After washing with PBS, working solutions of differentconcentrations were added in triplicate (1 ml/well). Theplates were kept at 37 ◦C in a 5% CO2 incubator for24 h. The viability of the cells was measured using MTTassay after 24 h (day 6) of incubation. In brief, themedium from each well was replaced with 1 ml freshmedium containing 100 µl MTT. The same fresh mediumwas placed in empty wells as blanks. The rest of theexperiment remained the same as described in Section 2.6.The absorbances of all the samples were recorded at 570and 690 nm and background subtraction was done. Thenumber of live cells was determined by extrapolating theobtained absorbance values with the standard formazanabsorbance MTT assay curve.

2.9.3. Calculation of TCIC50 value

The percentage cell death due to drug treatment (drugcytotoxicity) was calculated as follows:

L = number of live cells in drug-treated well/number

of live cells in untreated well × 100

% cell death = 100 − L



0 3

0

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ttach

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Figure 1. Attachment kinetics of Hep G2 cells on agar–gelatinfilms and control tissue culture plate. Each point representsthe mean ± SD of the results from three different experiments(n = 3)

The percentage cell death values were plotted againstdrug concentrations and the TCIC50 value (concentrationrequired to kill 50% cells) was calculated using regressionanalysis.

2.10. Liver-specific functions of HepG2 cellsafter drug treatment

The medium from the drug-treated wells was collectedbefore performing the MTT assay and used for albuminsecretion analysis and urea synthesis assay, as describedin Section 2.8.

2.11. Statistical analysis

The data obtained by studying cell proliferation, albuminsecretion, and urea synthesis were analysed by t-test,using Sigmastat 2.0 software.

3. Results

3.1. Contact angle analysis

The average contact angle of the water droplets on the2 : 1 agar–gelatin film was 24◦.

3.2. Cell attachment studies

The cell attachment study (Figure 1) indicated thatHepG2 cells adhered to the films much more rapidlythan the control plate. The trend continued throughoutthe experiment. During the first hour, approximately 42%cells adhered to the films, while control tissue cultureplate could adhere only ca. 20% cells. At the end of theexperiment, i.e. by the fifth hour, no unattached cellswere found on the films.

0 1 6 100.0

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maz

an a

bsor

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Figure 2. Growth kinetics of Hep G2 cells on agar–gelatinsponges and control plate. Each point represents the mean ± SDof the results from three different experiments (n = 3, p < 0.5)

3.3. Cell proliferation studies on agar–gelatinsponges

Cell proliferation on agar–gelatin film and in the controlwas similar up to day 4 (Figure 2). From day 5, the cellson the control plate began proliferating more than theagar–gelatin film and the trend continued up to day 9.The maximum proliferation was exhibited on day 8 byboth the control plate and the agar–gelatin film.

3.4. Morphological study of the spheroids

Figure 3 shows the formation and growth of HepG2spheroids on agar–gelatin sponges. Small clusters ofHepG2 cells were seen on day 3 (Figure 3a), whichbecame bigger on day 5 (Figure 3b). Well-organizedspheroid formation was seen on day 7 (Figure 3c) andday 9 (Figure 3d).

3.5. Characterization of liver-specific functionsin HepG2 spheroids


The study showed that albumin secretion increased up today 3 and then gradually kept decreasing up to day 9 inHepG2 spheroids (Figure 4). In monolayer control cells,a decrease in the secreted albumin was observed betweendays 2 and 9. The concentration of the albumin was muchgreater in spheroids than the control cells.


The HepG2 spheroids on agar–gelatin sponges exhibitedsignificant increase in urea synthesis on day 2, then asteady decrease up to day 8, and on the day 9 the amountof urea decreased very sharply (Figure 5). On the otherhand, in control cells, increase in urea concentration was


372 P. Verma et al.

Figure 3. Optical photographs showing the growth of Hep G2 cells on agar–gelatin sponges: (a) day 3; (b) day 5; (c) day 7; and(d) day 9. All photographs are at ×20 magnification

0 1 4 7 105

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Alb

umin

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Figure 4. Albumin secretion by Hep G2 cells in spheroids andcontrol monolayer. Each point represents the mean ± SD of theresults from three different experiments (n = 3, p < 0.5)

observed on day 2, but from day 3 it was sharply decreasedup to the day 5 and then maintained a very low amount.

3.6. Paracetamol cytotoxicity study

The mean percentage cell deaths, observed after treatingthe spheroids with 1, 5, 10 and 25 mM paracetamolwere 31%, 52%, 77% and 98%, respectively (Figure 6a).The TCIC50 value, calculated using linear regression plot,was 4.6 mM. The mean percentage cell deaths, observedafter treating the monolayer with 1, 5, 10 and 25 mM

paracetamol, were 25%, 45%, 70% and 94%, respectively

0 2 4 6 8 10

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Ure

a C

once

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06 cel

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Figure 5. Urea synthesis by Hep G2 cells in spheroids and controlmonolayer. Each point represents the mean ± SD of the resultsfrom three different experiments (n = 3, p < 0.5)

(Figure 6b). The TCIC50 value calculated using linearregression plot was 6.2 mM.

3.7. Liver-specific functions of HepG2 cells afterdrug treatment


The concentration of the albumin secreted by thecells after drug treatment is shown in Figure 7. Thedecrease in albumin is evident with the increase in drugconcentration. Drug treated HepG2 spheroids secretedless albumin than the control monolayer.



0 5 10 15 20 2520

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(a)

(b)

Figure 6. (a) Percentage cell death in paracetamol treated3D Hep G2 spheroids. Each point represents the mean ±SD of the results from three different experiments (n = 3,p < 0.5). (b) Percentage cell death in paracetamol-treated HepG2 monolayer. Each point represents the mean ± SD of theresults from three different experiments (n = 3, p < 0.5)


Figure 8 shows urea synthesis by the HepG2 cells afterdrug treatment. Like albumin, urea synthesis also declinedwith increasing drug concentration. Similarly, drug-treated HepG2 spheroids synthesized less urea than thecontrol monolayer.

3.8. Statistical analysis

The proliferation data of HepG2 cells over agar–gelatinmatrix and the control plate were analysed by t test.The data passed the normality test (p = 0.287) andthe equal variance test (p = 0.362). The difference inthe mean values of the two groups was not greatenough to reject the possibility that the difference wasdue to random sampling variability. There was not astatistically significant difference between the control andthe agar–gelatin matrix (p = 0.530).

When the albumin secretion data were compared usingthe t-test, the data passed the normality test (p = 0.733)

Concentration of Paracetamol (mM)

1 10 25

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ay)

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50HepG2 SpheroidsControl monolayer

5

Figure 7. Albumin secretion by the drug-treated Hep G2 cellsin spheroids and control monolayer on sixth day. Each pointrepresents the mean ± SD of the results from three differentexperiments (n = 3)

Concentration of Paracetamol (mM)

1 5 10 25

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HepG2 SpheroidsControl monolayer

Figure 8. Urea synthesis by the drug-treated Hep G2 cells inspheroids and control monolayer on day 6. Each point representsthe mean (± SD) of the results from three different experiments(n = 3)

and the equal variance test (p = 0.996). The differencein the mean values between the data was found to begreater than what would be expected if occurring bychance. There was a statistically significant difference(p = <0.001).

The urea synthesis data, when compared using thet-test, also passed the normality test (p = 0.511) andthe equal variance test (p = 0.446). The difference inthe mean values between the data was found to begreater than what would be expected if occurring bychance. There was a statistically significant difference(p = <0.001).


374 P. Verma et al.

The drug cytotoxicity data were compared using thet-test. It also passed the normality test (p = 0.708) andthe equal variance test (p = 0.893). The difference inthe mean values of the two groups was not greatenough to reject the possibility that the difference wasdue to random sampling variability. There was not astatistically significant difference between the control andagar–gelatin matrix (p = 0.781).

4. Discussion

We report here the formation of 3D HepG2 spheroidson an agar–gelatin matrix, studies of their liver-specificfunctions and the evaluation of their role in preliminarydrug toxicity testing, using paracetamol as a modeldrug. The agar–gelatin matrices were fabricated in ourlaboratory using the mutual entrapment method. Inthis paper their suitability for 3D cell growth has beenreported.

Hydrophilic surfaces favour cell growth. A contactangle of <45◦ indicates a hydrophilic surface with goodwetting properties. The contact angle of 2 : 1 agar–gelatinfilms indicates hydrophilic surface characteristics for cellproliferation.

A cell attachment study was done to assess the adhesivenature of agar–gelatin matrices for HepG2 cells. Thisstudy was also important as it gave a primary clueabout the adhesion and proliferation of HepG2 cellson agar–gelatin matrices, as cell–matrix interaction isa very complicated phenomenon and is dependent onboth surface properties (Lee et al., 1994) and cells. Thestudy indicates that the HepG2 cells exhibited better celladhesion on an agar–gelatin surface than on the controltissue culture plate.

The agar–gelatin matrices induced the formation of 3DHepG2 spheroids, while the control tissue culture plateshowed 2D monolayer growth. This is because the in vitro3D assembly of the cells depends not only upon cell–cellinteractions but also upon cell–matrix interactions. Agaris a mixture of sulphated heteropolysaccharides made upof D-galactose and an L-galactose derivative ether linkedbetween C-3 and C-6. Studies have shown that galactosecarrying synthetic ECMs bind hepatocytes through areceptor-mediated mechanism, resulting in enhancedhepatocyte functions. ECM geometry, as well as thetype, density and orientation of galactose, affect theattachment and functions of hepatocytes (Cho et al.,2006). Hepatocytes are anchorage-dependent cells andthey rapidly lose liver-specific functions and viabilityin vitro. It is vital to direct galactose-mediated hepatocyterecognition to increase interactions between galactose-carrying polymers and hepatocytes for liver-specificfunctions (Cho et al., 2007). Gelatin is a denatured formof the natural extracellular matrix collagen, the proteinwhich maintains the integrity of the tissues. Possibly,gelatin provided the cue to HepG2 cells to orientateand organize themselves into a definite pattern to form

a 3D architecture. In the case of the polypropylenetissue culture control plate, the 2D cell–cell contactswere present but cell–matrix interactions were lacking.Thus, the HepG2 cells cultured on the agar–gelatin matrixorganized themselves into 3D spheroids.

MTT assay was done to study the growth kinetics ofHepG2 cells over agar–gelatin matrices. MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]is a yellow compound which is converted into purpleformazan in the reaction catalysed by mitochondrialreductase enzyme in living cells. Thus, purple formazan isan indicator of live cells. The HepG2 cells exhibited similarproliferation up to day 4 on both the control plate andagar–gelatin matrices. The difference in the proliferationbecame evident from day 5 onwards, when the growthof the cells over the agar–gelatin matrices was less thanthe control. This was due to the fact that the cells startedreorganizing in the form of 3D spheroids on agar–gelatinmatrices, and this reorganization induced differentiationrather than proliferation. The cells in the spheroids keptproliferating and increasing in number up to day 8.

A number of reports show that the functions, via-bility and proliferation of the cells are related to theirmorphology and organization of the cells (Robertsand Soames, 1993; Hamamoto et al., 1998; Younget al., 2000; Baldwin and Saltzman, 2001). The liver-specific functions of the spheroids were measured interms of albumin secretion and urea synthesis activ-ity by the HepG2 cells. The significantly more albuminsecretion shown by the spheroids on the agar–gelatinmatrix may be due to matrix-induced 3D growth ofthe HepG2 cells, mimicking native tissue structure. Anadult human produces around 12 g albumin/day (Tavilland McCullough, 1992). The average number of livercells is assumed to be 2 × 1011, which is compara-ble to approximately 60 µg/106 cells/day (Khalil et al.,2001). Our observations match with this value, i.e. range40–64 µg/106 cells/day.

Urea is the main end-product of protein synthesis andis another key marker of hepatocytes. The greater ureaproduction exhibited by the spheroids on the matrix isthe result of the more differentiated and functionallyactive state of the cells comparable to the native tissue.Urea (24.96 g/l) is synthesized by an adult human being(Rudman et al., 1973), which is equivalent to about200 µg/106 cells/day.

The drug paracetamol (acetaminophen; CAS No. 103-90-2) was chosen to test the feasibility of agar–gelatininduced 3D HepG2 model for the determination of itsTCIC50. The TCIC50 values correlate with in vivo LD50 data(Clemedson et al., 2000; Ekwall et al., 1998) and also canbe used to determine the starting dose for in vivo testing(Spielmann et al., 1999). We tested four concentrationsof the drug on HepG2 spheroids as well as in themonolayer control and calculated the percentage celldeath using MTT assay. The TCIC50 value obtained for theparacetamol in the monolayer control was 6.2 mM, whichwas close to the reported value of 6.6 mM using humanhepatocytes (Jover et al., 1992). We also found many



variations in the TCIC50 values after comparing with thepublished data. Huveneers-Oorsprong et al. (1997) havereported the TCIC50 values of 10 and 2.8 mM in rat andpig hepatocytes, respectively. Singh and Reen (1999) havereported CC50 (TCIC50) of the drug about 0.7 mM usingH4IIEC3/Gy hepatoma cells in 48 h. Patierno et al. (1989)have shown a decrease in the survival of C3H/10T1/2cells from 49% to 9% with a paracetamol concentrationof 3.3–13.2 mM. The variations observed may be due tothe use of cell lines from different sources in monolayerculture and exposure of the drug for different periods.The lower TCIC50 value of paracetamol in 3D spheroidsthan in the control monolayer shows that HepG2 cellsgrowing in three dimensions are more sensitive to thesame concentration of drug than the control cells. Thisis supported by the lower concentration of albumin andurea in 3D spheroids than in the control monolayer. Thehigher sensitivity towards the drug shown by cells inspheroids than in monolayer can be attributed to the 3Dorganization of the cells.

The statistical analysis shows that the growth ofHepG2 cells on agar–gelatin matrix was not significantlydifferent, but it induced cells to secrete more albumin andsynthesize more urea than the cells over control plate,comparable to liver tissue.

It is evident from the above discussion that agar–gelatinmatrices have the capability to induce 3D growthof HepG2 cells. These spheroids may prove to be abetter model system than monolayers for preliminarycytotoxicity evaluations, due to the higher expressionof liver-specific functions and more sensitivity to testcompounds.

5. Conclusion

Agar–gelatin sponges induced the organization of HepG2cells into 3D spheroids mimicking native liver cyto-architecture and also upregulated liver-specific functionsin them. The cytotoxicity evaluation of the differentconcentrations of paracetamol on these spheroids showsmore sensitivity than the monolayer control. Thesespheroids can be proved a superior model to monolayerculture for the preliminary drug and chemical toxicityapplications.

Acknowledgements

Poonam Verma is grateful to the Council for Scientific andIndustrial Research (CSIR), New Delhi, India, and Vipin Vermais grateful to the Indian Council of Medical Research (ICMR),New Delhi, India, for providing Senior Research Fellowshipsenabling them to carry out this research.

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agar–gelatin hybrid sponge-induced three-dimensional in vitro ‘liver-like’ hepg2 spheroids for...

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