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Biomaterials 30 (2009) 4136–4142

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Biomaterials

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The use of vitrification to preserve primary rat hepatocyte monolayer on collagen-coated poly(ethylene-terephthalate) surfaces for a hybrid liver support system

Raquel Magalhaes a,b,1, Anil Kumar PR b,1, Feng Wen a, Xuying Zhao a, Hanry Yu b,c,d,e, Lilia L. Kuleshova a,*

a Low Temperature Preservation Unit, National University Medical Institutes, Yong Loo Lin School of Medicine, National University of Singapore, Block MD11 #03-01C,10 Medical Drive, Singapore 117597, Singaporeb Department of Physiology and RCE in Mechanobiology (for HY only), Yong Loo Lin School of Medicine, National University of Singapore, Block MD9,2 Medical Drive, Singapore 117597, Singaporec Singapore-MIT Alliance, Center for Singapore-MIT Alliance, E4-04-104 Engineering Drive 3, Singapore 117576d Institute of Bioengineering and Nanotechnology, Agency for Science, Technology and Research, 31 Biopolis Way, The Nanos, #04-01,Singapore 138669, Singaporee Department of Mechanical Engineering, MIT, Cambridge, MA 02139, USA

a r t i c l e i n f o

Article history:Received 1 April 2009Accepted 23 April 2009Available online 28 May 2009

Keywords:CryopreservationPolyethylene terephthalateCell cultureHepatocyteBioartificial liverVitrification

* Corresponding author. Tel.: þ65 6516 3359; fax:E-mail address: nmikl@nus.edu.sg (L.L. Kuleshova)

1 Both authors contributed equally to this work.

0142-9612/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.04.037

a b s t r a c t

We developed a scaled-up procedure for vitrifying hepatocytes for hybrid liver support system applica-tions. Hepatocyte monolayer cultured on collagen-coated polyethylene terephthalate (PET) discs consti-tuted the basic module for a hybrid liver support system. Freshly isolated rat hepatocytes were seeded oncollagen-coated PET discs with a diameter of 33 mm at a density of 5� 106 cells per disc, and were culturedfor 24 h before cryopreservation. The total duration of procedure starting from exposure to low concen-trations of cryoprotectants up to cryostorage is 10 min. Vitrification of the modules was achieved by usingtwo vitrification solutions sequentially with first vitrification solution containing two cryoprotectants,ethylene glycol (EG) and sucrose, while second vitrification solution contained additionally polymer, Ficoll.Direct exposure to liquid nitrogen vapours was followed by immersion into liquid nitrogen.Recovery procedure for vitrified modules included their warming in 1 M sucrose at temperature of 38–39 �C followed by subsequently washing in sucrose-based solutions of decreased concentration within15 min at room temperature. Viability, structural characteristics, and functions of cells were preserved byvitrification. Hepatocytes in the post-vitrified and warmed monolayer maintained differentiated hepa-tocyte characteristics both structurally and functionally. In average, protein synthesis measured asalbumin production was 181.00� 33.46 ng/million cells and 166.10� 28.11 ng/million cells, for controland vitrified modules respectively. Urea production was, in average, 1.52� 0.40 ng/million cells and1.36� 0.31 ng/million cells for a 7 day culture respectively, with no significant statistical differencebetween the control and vitrified modules.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The medical costs associated with caring for patients with endstage liver disease are high and continue to increase. Fulminanthepatic failure (FHF) is a disease condition that progress over a rela-tively short time course resulting in complete liver failure and relatedcomplications within 8 weeks. Chronic liver failure has a lengthycourse, compared to FHF and also progresses to the same end stagecondition. In both cases transplantation is the only available treat-ment. Shortage in donor liver increases the demand for findingalternatives to support liver by artificial means with the help of liver-

þ65 6773 5461..

All rights reserved.

assisted devices. Many patients may benefit from supplementaryliver support, to prevent the disease condition to worsen. The liversupport is essential to patients with FHF until the patient’s own liverregenerates or until the patient with chronic liver failure obtainsa compatible donor liver for transplantation. This requirement hasdeveloped over decades to the concept of liver support systems.Initially such devices were solely based on mechanical systems orprocesses. However, liver support requires more than mechanicalremoval of toxic substance. It should also provide detoxification andprotein synthesis functions of the liver. Hence it is necessary toinclude biological components to develop hybrid liver supportsystem. Usually the biological component is hepatocytes grown onsynthetic substrate. However, maintaining viability of cells for pro-longed culture periods is difficult. Cryopreservation permits thepooling of cells to reach a critical cell number, their storage and

R. Magalhaes et al. / Biomaterials 30 (2009) 4136–4142 4137

transportation to different locations. Cryopreservation also permitsquality control testing of the cell-based products.

The main objective in tissue engineering approach is to restore,maintain or improve tissue functions. The potential clinical appli-cations of linking tissue engineered constructs and functionalprimary cells with the ability to effectively cryopreserve them arevery promising. An optimized procedure, yielding good cell surviv-ability and maintaining cell metabolic levels similar to those ofnon-cryopreserved cells would result in the development of liver cell-based devices that could be further used in treatment. The devicemay also be employed for other applications such as drug testing.Polyethylene terephthalate (PET) is a polymer that can be fabricatedinto thin film allowing it to be cut into desired shape and size to beused in cell culture. It was demonstrated that PET substrate anchorshepatocytes stably as a monolayer with tight cell–cell interactionsand high levels of liver-specific functions typically seen in 3Dhepatocyte culture [1]. In this study, we aimed to develop vitrifica-tion (ice-free cryopreservation) to preserve larger cell-containingconstructs where hepatocytes were pre-seeded/cultured on PETfilms which were coated with collagen. Each construct constitutesa module of the hybrid liver support system, which will be referredto as ‘PETh’ or ‘module’ further throughout the text.

The specific vitrification solution compositions were deter-mined based on previous reports of our own and other researchgroups where a mixture of ethylene glycol (EG) and sucrose wasdeveloped [2–6]. In the past, vitrification solutions used onlypenetrating cryoprotectants (PCs) [7] but since the early 1990s, thebenefit of replacing PC with sugars for vitrification of biologicalmaterial has been identified [8–10]. We previously reported thatratio of 1:2 for penetrating cryoprotectants (EG or EGþ 1,2-pro-panediol) to sucrose is particularly effective for vitrification oftissue-like self assembled aggregates. Although analysis of differ-ential scanning calorimetry experimental data showed that thesolution containing EGþ 1,2-propanediol (which had lower totalsolution concentration, therefore with lower toxicity impact onhepatocytes) was capable of supporting vitrification at the coolingand warming rates employed in our study, the efficacy of vitrifi-cation procedure in preserving the functional ability of cells was notimproved by introduction of 1,2-propanediol [3,8,11].

We have studied polymer-supplemented vitrification solutions[12]. Generally, supplementation with polymer (dextran, Ficoll andpolyvinylpyrrolidone) as a concept of cryopreservation of sensitivecells containing nucleus found to be effective for small objects such asembryos of different species including human [8,13–15]. In contrast,vitrification of large objects such as liver slices [16,17] or vasculargrafts used only mixture of penetrating cryoprotectants [18–20].

We have taken into consideration surface property and geometryof PET seeded with hepatocytes – a flat polymer film cut into a disc of33 mm diameter, coated with collagen and seeded with a monolayerof cells (PETh) – therefore we introduced Ficoll to prevent excessivedehydration prior to cooling. In contrast to polyvinylpyrrolidone, Ficollis devoid of impurities and it has been proven effective as an additivein cryopreservation of biological material as supplied by manufacturer[8,11,14]. Its high molecular weight (70 kDa) will not allow it topermeate the cell membrane and therefore form a protective layer onthe surface of the PETh. The addition of the polymer to vitrificationsolutions hypothesized and here tested was aimed at minimizing theadverse effect of the unfavourable geometry of the monolayer tosustain stable amorphous state during cryopreservation, since suchconstruct has a high surface area/volume ratio.

2. Materials and methods

Chemicals and reagents used in the experiments were purchased from SigmaChemicals (St. Louis, MO) unless otherwise stated.

2.1. Primary culture of rat hepatocytes

Hepatocytes were isolated from adult male Wistar rats weighing 250–300 g bytwo-step collagenase perfusion method, with slight modification [21]. Animals werehandled according to the guidelines and protocol by the Institutional Animal Careand Use Committee of National University of Singapore. Viability of cells wasdetermined to be >90% by Trypan Blue exclusion assay. Freshly isolated hepatocytes(5�106 cells) were seeded on collagen-coated PET films (33 mm diameter) andcultured in Hepatozyme medium containing 10�8 M dexamethazone, 100 units/mlpenicillin, and 100 mg/ml streptomycin. Cells were allowed to adhere for 2 h in 5%CO2 at 37 �C and 95% humidity before first medium change was performed and thenmaintained for specific duration.

2.2. Cryopreservation

2.2.1. Preparation of cryopreservation solutionsThe solutions were prepared as reported previously [3–5]. The solutions for

cryopreservation were prepared with DMEM (Gibco, Invitrogen) (1) pre-equilibrationsolutions: 10% (v/v) and 25% (v/v) EG; (2) vitrification solutions (VSs): VS1: EG andsucrose [40% (v/v) EG, 0.6 M sucrose], VS2: EG, sucrose and Ficoll [40% (v/v) EG, 0.6 M

sucrose 9% (w/v) Ficoll]; (3) three basic dilution solutions: 1 M, 0.5 M sucrose, 0.25 M

sucrose in DMEM respectively. The intermediate step with sucrose concentration0.70 M was achieved by adding 0.5 M sucrose in drop-wise manner to the modulewhich was in a dish with 1 M sucrose. DMEM solution was then used for washing.

2.2.2. Cryopreservation proceduresFreshly isolated hepatocytes were seeded on PET films and cultured for 24 h

before cryopreservation. PEThs were divided into two experimental groups. The PETfilms were prepared with the diameter of 33 mm in order to suit inside 35 mmculture dish. Control group remained in continuous culture throughout. In vitrifi-cation group, PEThs undergone through equilibration, cooling and warmingprocedures, followed by removal of cryoprotectants (complete vitrification–warm-ing cycle).

The modules were transferred to 35 mm culture dish and pre-equilibrated with10% EG, followed by 25% EG (each step for 3 min) and finally to vitrification solu-tions. Exposure to vitrification solutions was done in two consecutive steps: 1st step,2.5 min exposure to VS1, followed by 0.5 min exposure to VS2. Equilibration in pre-equilibration and vitrification solutions prior to cryopreservation was performed atroom temperature (23� 2 �C), 1 day after seeding. PEThs were handled using a pairof fine forceps. After exposure, one at a time, modules were exposed to vapour phaseof liquid nitrogen for 10 s, cell surface down, before plunging into liquid nitrogen.Metabolic activities at temperatures below temperature of solidification (glasstransition temperature of cryoprotectants) are arrested. Therefore the time in liquidnitrogen can be considered as suspended, not affecting outcomes of vitrification. Forwarming process, PEThs, cell surface down, were placed, into 50 ml of 1 M sucrosesolution, warmed to 38 �C in a 60 mm culture plate over a temperature controlledheated stage. After 1 min, the warmed sample was relocated to 35 mm culture plateand incubated in 4 ml 1 M sucrose at room temperature for 4 min. Further dilutionwas carried out at room temperature at the intervals of 2.5 min in 4 steps from0.70 M sucrose to DMEM solutions described in 2.2.1. (point 3) of this section. Thisprocedure has been designed to remove cryoprotectant from cells gently.

2.3. Cell viability

PEThs were vitrified for 1 h and warmed to physiological condition (described inpart 2.2.2 of this section). The modules were then warmed and cultured in Hep-atozyme medium overnight. Trypan Blue exclusion assay was used to assess theimpact of vitrification on the viability of cells in the PETh. Monolayers were stained onday 1 and day 5 post-vitrification and compared to control in time-matching points.

2.4. Structural study

2.4.1. Phase contrast microscopyStructural integrity of the vitrified hepatocyte monolayers was evaluated as

short- and long-term assessments. Continuous culture without vitrification wasused as control. Morphological examination of monolayer in the vitrification groupwas performed one day prior to vitrification (day 1), immediately after vitrification(day 0) and to a period of up to 5 days after vitrification. Results of vitrification werecompared to those of control in two time-matching points.

2.4.2. Immunofluorescence stainingThe biological characteristics of bile canalicular structures of hepatocyte

monolayer were determined by immunofluorescence staining of apical membraneprotein, the Multidrug Resistant Protein 2 (MRP2). Vitrified modules were warmedto physiological conditions and cultured in Hepatozyme medium. After 24 h,monolayers were fixed in 3.7% paraformaldehyde for 1 h at room temperature. Thefixed samples were rinsed with PBS and non-specific binding sites were blockedwith freshly prepared 1% BSA in PBS for 30 min. Cells were incubated sequentiallywith primary antibody (anti-MRP2, 1:50) and secondary antibody (anti-rabbit IgG

Fig. 1. Effect of vitrification on viability and structure integrity of the hepatocyte monolayer imaged by phase contrast microscopy is shown in (A)–(D). For vitrification group PEThswere assessed (A) one day before vitrification (day 1); (B) immediately after vitrification (day 0) and (C) a period of up to 5 days culture post-vitrification (C1: day 1; C2 day 5). Forcontrol group (D1: day 1; D2: day 5) at time-matching points to vitrified PEThs. Experiment time-matching layout is shown in (E). For vitrification group PEThs were assessed (A)one day before vitrification (day 1); (B) immediately after vitrification (day 0) and (C) a period of up to 5 days post-vitrification (C1: day 1; C2: day 5). For control group (D1: day 1;D2: day 5) at time-matching points to vitrified PEThs. Experiment time-matching layout is shown in (E). Cell viability on day 5 post-vitrification assessed by Trypan Blue exclusion isshown in (F), viable cells remained unstained while dead cells retained blue colour (F1: vitrified PETh; F2: time-matching control).

R. Magalhaes et al. / Biomaterials 30 (2009) 4136–4142 4139

tagged with TRITC, 1:50) for 1 h each with intermitted rinse using PBS (3 times, eachlasting 5 min). Labelled samples were mounted with FluorSave (Calbiochem, SanDiego, CA) and examined under a confocal microscope (LSM 510 Meta, Zeiss).

2.5. Metabolic activities

2.5.1. Quantification of albumin secretionProtein synthesis ability of vitrified hepatocyte monolayer was evaluated by

estimating the secreted albumin over a period of 7 days in culture post-vitrificationand time-matching controls. The culture medium of all samples was completelyreplenished after each collection and results represented on day basis, on the day ofcollection (average from the day of medium change to the day of media collection ifthe medium change is more than one day). The secreted albumin was measured byperforming an enzyme-linked immunosorbent assay (ELISA) using a commercial kit(Bethyl Laboratories, TX, USA).

2.5.2. Determination of urea productionThe ability of hepatocytes to transform ammonium into urea was estimated as

previously described [5]. Briefly, culture medium was replaced with 1.5 ml of 1 mM

ammonium chloride (NH4Cl) and kept for 90 min inside growth chamber. Ureaproduction was determined calorimetrically using a commercial urea nitrogen kit(StanBio Laboratory, TX, USA) to a period of 7 days post-vitrification for vitrifiedPEThs and time-matching controls. Actual amount was calculated by plottingstandard curve with urea solutions of known concentrations.

2.6. Statistical analysis

Values obtained for vitrified samples were compared to those of the controls byStudent’s t-test (SPSS version 12, SPSS Inc., Chicago, IL, USA). p< 0.05 was consideredstatistically significant. Results were presented as mean� standard deviation (SD).

3. Results

3.1. Cell viability and tissue structure integrity

The effects of vitrification on the structure and viability ofhepatocytes constituting the modules for the hybrid liver support

Fig. 2. MRP2 immunostaining of control and vitrified hepatocyte monolayer. Cells showedimmediately after vitrification (day 0) and 24 h after vitrification (day 1). Red colour indica

system were assessed by phase contrast microscopy (Fig. 1).Hepatocytes developed bile canalicular like structures on collagen-coated PET films within 24 h (day 1). Vitrified and control PEThswere maintained in culture and morphologically examined toa period of up to 5 days post-vitrification.

Qualitative verification on day 0 showed no detrimental effect tothe structural organization of cells in the films and there was novisible difference between the vitrified PEThs and the time-matching controls (time-matching scheme shown in Fig. 1E). Theresults are presented in Fig. 1. After vitrification, PEThs cultured upto 5 days preserved bile canalicular structures indicating the successof the experiment.

With any cryopreservation method it is important to maintainthe structural integrity of the sample during the course of cryo-preservation. In this study, an attempt was made to vitrify mono-layer of rat hepatocytes method on collagen-coated PET substrate.Hepatocytes are highly susceptible to injury during the freeze–thaw process. Therefore, the cryopreservation method itself isa focus area yet to be developed [22]. The morphology of hepato-cytes in culture is one of the parameters which can be assessedusing light microscopy. Hepatocytes form bile canalicular likestructures in culture as an indication of the appropriate and optimalculture condition. Hepatocyte monolayers were vitrified, stored,warmed and assessed under phase contrast microscope. Cells inmonolayer maintained similar morphology compared to the timematched control monolayer cultures. Continuous culture revealedthat the vitrified PEThs showed no detriment to the structuralorganization of cells in the film during a period of up to 5 days.Throughout this period there was no visible difference between thevitrified PEThs and the time matched controls. The results pre-sented in Fig. 1(A–D2) show preserved bile canalicular structureswhich indicates the success of vitrification in preserving these

characteristic apical polarity staining. Vitrified hepatocyte monolayers were examinedtes positive MRP2 staining.

Fig. 3. Albumin secretion of PEThs. (A) Albumin secretion on day 1, day 5 and day 7post-vitrification matched with control at each corresponding time point (n¼ 4–5).Albumin secretion was determined on a per day basis. (B) Average albumin secretionduring culture period, i.e. 7 days post-vitrification for vitrification group or for thecorresponding length for the control group (n¼ 14). No statistical difference wasobserved between control and vitrified PEThs within one day of observation (A) as wellas between average values (B) (p> 0.05); except for * which indicates statisticallysignificant difference (p¼ 0.002).

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structures. Trypan Blue staining of PEThs on 5 day post-vitrificationrevealed comparable viability of cell monolayer with respect totime matched control (Fig. 1F1 and F2). This confirmed that therewas no impact of vitrification on viability of hepatocytes on PETs inlong term.

3.2. Polarity of vitrified PEThs

Polarization of hepatocytes in tissue structure has beendemonstrated to be of great importance for the maintenance ofliver tissue-like function of hepatocyte cultures [23]. Isolatedhepatocytes restore their polarized aspects when appropriateconditions are provided. Morphological polarization of PEThmonolayers was determined by immunostaining of MRP2 proteinpresent at the canalicular membrane. MRP2 expression of vitrifiedsamples on day 0 and day 1 showed polarized nature of hepato-cytes which is an indicator to the preservation of structural proteinsin monolayer (Fig. 2). The functional polarization confirmed by thepositive MRP2 stainings on post-vitrified samples was shown to becomparable to that of control monolayers.

3.3. Albumin and urea production of hepatocytes seeded on PETs

Vitrified modules were maintained in continuous culture periodof up to 7 days after vitrification for the assessment of their metabolicfunctions. Time-matching controls were maintained for the sameperiod of time (i.e. 8 days after seeding). The ability of vitrifiedhepatocytes to secrete albumin in levels that were not statisticallydifferent from control was maintained during culture period, exceptfor day 7 where significant difference was recorded (p¼ 0.002). Thedecline in the amount of albumin secreted from day 1 to day 7 wasobserved in both samples (n¼ 4–5), which is consistent withpreviously reported results of other groups [3,4,24] (Fig. 3A). Overall,the average amount of albumin secreted during continuous culturewas of 181.00� 33.46 ng/million cells and 166.10� 28.11 ng/millioncells (n¼ 14), for control and vitrified samples, respectively, withoutsignificant statistical difference (Fig. 3B). The ability to changeammonium to urea was determined during 90 minutes on the day today basis (n¼ 3–6). Urea production rates did not show any signifi-cant differences between vitrified and control PEThs, neither ona day by day comparison nor for an overall comparison (Fig. 4A andB). In average the detoxification rates for control and vitrified PEThswere 1.52� 0.40 ng/million cells and 1.36� 0.31 ng/million cells,respectively (n¼ 12). Results obtained demonstrated that for theculture period of 7 days, post-vitrified modules were capable ofmaintaining their detoxification function.

These results together demonstrate that vitrification was effec-tive in preserving the metabolic capacity of hepatocytes asa monolayer on PETs, during a culture period that exceeds the usageperiod of the hybrid liver support system for which these filmswere designed.

4. Discussion

In this study, we have demonstrated that cryopreservationemploying two vitrification solutions in sequence can lead to highviability of hepatocytes cultured in monolayer.

One of the challenges of cell-based therapy, despite of its vastadvancement, is the ready availability of functional cells in limitedtime. An optimized procedure, yielding good cell survivability andmaintaining cell metabolic levels similar to non-cryopreservedcells would serve as a base for the development of human livercell–matrix system, that could be further used in the course oftreatment for the benefit of the same patient, as life expectancyof such group of patients is increasing significantly. The objective

of this study was to evaluate vitrification developed for rathepatocyte monolayer on collagen-coated polymer film asmodules in hybrid liver support system applications and otherssuch as drug testing.

Clinical experiences with bioartificial liver (BAL) supportsystems are available with devices developed by Demetriou, Suss-man [Extracorporeal Liver Assist Devices (ELAD)] and Gerlach [25–27]. Such devices with different configurations were successful inmaintaining the patients for a mean duration of 3–75 h (approxi-mately up to 3 days) [28]. Our results showed that there is nosignificant statistical difference between vitrified and time-matching control modules at the end of 7 day post-vitrificationculture period, which clearly indicated that our modules hadexceedingly sufficient period of exploitation. Meanwhile, duringthe whole period of continuous culture, the cryopreservedmodules, retained the ability to secrete albumin and urea at levelsthat were consistently similar to those of control modules.Although it is rapid to assemble these modules into one wholecomplete system, the process of fabricating modules for suchsupport systems is time consuming. Therefore, our vitrifiedmodules can be used to replace newly produced modules to savetime required. This method is clearly better than conventionalfreezing as it is readily available in scientific literature that somekey characteristics of sensitive cells are unable to recover up to 3days post-freezing, which suggests freezing approach is inappli-cable for this substitution [29].

Fig. 4. Urea production of PEThs after 90 min incubation of ammonia chloride inculture media at 37 �C. (A) Urea production on day 1, day 3 and day 7 post-vitrification,matched with control at each corresponding time point (n¼ 3–6). (B) Average of ureaproduction during culture period, i.e. 7 days post-vitrification for vitrification group orfor the corresponding length for the control group (n¼ 12). No statistical differencewas observed between control and vitrified PEThs within one day of observation (A) aswell as between average values (B) (p> 0.05).

R. Magalhaes et al. / Biomaterials 30 (2009) 4136–4142 4141

Majority of the available BAL configurations are based on hollowfiber membrane, but have a limitation in efficient mass transfer [30].The availability of hepatocytes and the time required to establish theculture for BAL bioreactors are practical hindrances to all adoptedmethods. Preserving monolayer of hepatocytes for future use inbioreactors can address this limitation. Hence this study attempts toprove that hepatocytes can be preserved by vitrification in mono-layer form, to find application as immediate cell stock for hybrid liversupport system.

Usual practice of cryopreservation by slow cooling requiresanimal or human proteins to improve the results. The method ofcryopreservation by vitrification proposed here does not requirethe use of any animal and human protein/serum, making it ideallysuitable for clinical application.

Conventionally hepatocytes are cryopreserved as cell suspensionor in different 2- and 3D culture configurations, using slow cooling[31–38]. Freezing has induced a decrease in qualitative results evenfor hepatocytes in suspension [39,40]. We have reported thatvitrification as an excellent method for cryopreservation of micro-encapsulated hepatocytes and hepatocyte spheroids with smalldimensions (<150 mm) [8,11,12]. The success of this study lays on theoriginal strategy of introducing a second polymer-supplemented VSin addition to the first VS in which the hepatocytes in the PEThs hadbeen fully exposed to penetrating cryoprotectants. The applicationof this original strategy, has yielded very promising results in bothviability of post-warmed cells and the maintenance of their meta-bolic functions during continuous culture, with no detriment totheir survival or seeding orientation.

Even though the exposure to the first VS leads to sufficient EGpenetration into the hepatocytes, the addition of a second VS con-taining polymer will coat the majority of the outer surface of the

modules. The same could apply to other objects with large surfacearea/volume ratio not only to protect the cells but also to prevent EGfrom further penetration into the cells, and thus to prevent over-toxicity. Initially, during our preliminary studies we employ only onevitrification solution, namely VS1. While only VS1 is used, we founda difference in the level of functionality compared to that of control,even though this level of functionality was still considered highenough during optimization protocol (data not shown). Therefore,we believe that the addition of second vitrification solution, i.e. VS2,can protect the surface of cells from dehydration as a viscous outerlayer is formed. Additionally, since VS2 has much higher total soluteconcentration (74% (w/v)) than VS1 (65% (w/v)), ice crystal forma-tion is highly unlikely in VS2 even for lower cooling and warmingrates below 100 �C/min, expected for the immersion of largeconstructs into liquid nitrogen. This makes VS2, the polymerenriched solution an attractive option for larger constructs.

5. Conclusions

This report describes a scale-up method for vitrification ofconstructs involving hepatocytes, intended as a module designed tobe applied in a hybrid liver support system. Monolayer expressedsufficient viability and cellular characteristics and was well main-tained in post-vitrification culture. It is conceivable that preservinghepatocytes in ready to use condition with required functionalcapabilities for future use will improve the performance of theintended applications. With the complete avoidance of products ofhuman or animal origin in the cryopreservation process, we lookforward to apply this protocol in future clinical or pharmacologicalapplication.

Acknowledgments

This research was supported by a grant from the BiomedicalResearch Council of Singapore (Grant No.: 04/1/21/19/317) to LLK;and in part by National Medical Research Council Grant (R-185-000-099-213) and Singapore-MIT Alliance Computational andSystems Biology Flagship Project funding to HY. Ms R. Magalhaes isa graduate research scholar of the Yong Loo Lin School of Medicine,National University of Singapore, Singapore. We gratefullyacknowledge Dr Y. Kuleshov and Dr S.S. Gouk for critically readingthe manuscript.

Appendix

Figures with essential color discrimination. Fig. 2 in this articlemay be difficult to interpret in black and white. The full colorimages can be found in the on-line version, at doi:10.1016/j.biomaterials.2009.04.037.

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