Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/23569602

Stirredbioreactorsfortheexpansionofadultpancreaticstemcells

ArticleinAnnalsofanatomy=AnatomischerAnzeiger:officialorganoftheAnatomischeGesellschaft·December2008

DOI:10.1016/j.aanat.2008.09.005·Source:PubMed

CITATIONS

26

READS

52

7authors,including:

Someoftheauthorsofthispublicationarealsoworkingontheserelatedprojects:

Developingcomplex3Dhepaticspheroidstostudyliverdiseases.Viewproject

CatarinaBrito

iBET-InstitutodeBiologiaExperimentaleTe…

68PUBLICATIONS691CITATIONS

SEEPROFILE

ManuelJTCarrondo

iBET-InstitutodeBiologiaExperimentaleTe…

365PUBLICATIONS5,962CITATIONS

SEEPROFILE

PaulaMAlves

NewUniversityofLisbon

373PUBLICATIONS4,450CITATIONS

SEEPROFILE

AllcontentfollowingthispagewasuploadedbyManuelJTCarrondoon19September2014.

Theuserhasrequestedenhancementofthedownloadedfile.Allin-textreferencesunderlinedinbluearelinkedtopublicationsonResearchGate,lettingyouaccessandreadthemimmediately.

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Ann Anat 191 (2009) 104—115

RESEARCH ARTICLE

Stirred bioreactors for the expansion of adultpancreatic stem cells

Margarida Serraa, Catarina Britoa, Sofia B. Leitea, Erwin Gorjupb,Hagen von Briesenb, Manuel J.T. Carrondoa,c, Paula M. Alvesa,�

aInstituto de Tecnologia Quımica e Biologica–Universidade Nova de Lisboa/Instituto de Biologia Experimental eTecnologica (ITQB-UNL/IBET), Apartado 12, 2781-901 Oeiras, PortugalbFraunhofer Institute for Biomedical Engineering, FhG-IBMT, St Ingbert, GermanycFaculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Monte da Caparica, Portugal

Received 7 February 2008; received in revised form 31 July 2008; accepted 3 September 2008

KEYWORDSAdult stem cells;Stirred bioreactors;Expansion;Microcarriers;Pancreatic stem cells

SummaryAdult pluripotent stem cells are a cellular resource representing unprecedentedpotential for cell therapy and tissue engineering. Complementary to this promise,there is a need for efficient bioprocesses for their large scale expansion and/ordifferentiation.With this goal in mind, our work focused on the development of three-dimensional(3-D) culture systems for controlled expansion of adult pancreatic stem cells (PSCs).For this purpose, two different culturing strategies were evaluated, using spinnervessels: cell aggregated cultures versus microcarrier technology. The use ofmicrocarrier supports (Cytodex 1 and Cytodex 3) rendered expanded cell populationswhich retained their self-renewal ability, cell marker, and the potential todifferentiate into adipocytes. This strategy surmounted the drawbacks of aggregatesin culture which were demonstrably unfeasible as cells clumped together did notproliferate and lost PSC marker expression. Furthermore, the results obtainedshowed that although both microcarriers tested here were suitable for sustainingcell expansion, Cytodex 3 provided a better substrate for the promotion of celladherence and growth.For the latter approach, the potential of bioreactor technology was combined withthe efficient Cytodex 3 strategy under controlled environmental conditions (pH-7.2,pO2-30% and temperature-37 1C); cell growth was more efficient, as shown by fasterdoubling time, higher growth rate and higher fold increase in cell concentration,when compared to spinner cultures. This study describes a robust bioprocess for the

ARTICLE IN PRESS

www.elsevier.de/aanat

0940-9602/$ - see front matter & 2008 Elsevier GmbH. All rights reserved.doi:10.1016/j.aanat.2008.09.005

�Corresponding author. Tel.: +351 21 446 94 12; fax: +351 21 442 11 61.E-mail address: marques@itqb.unl.pt (P.M. Alves).URL: http://tca.itqb.unl.pt (P.M. Alves).

Author's personal copy

controlled expansion of adult PSC, representing an efficient starting point for thedevelopment of novel technologies for cell therapy.& 2008 Elsevier GmbH. All rights reserved.

Introduction

The potential of adult stem cells (ASCs) todifferentiate had been thought to be restricted tocell types related to the tissues in which theyreside, as their primary role is in tissue homeostasisand regeneration (Gokhale and Andrews, 2006).However, this concept has been challenged by theisolation of new lineage classes of uncommittedpluripotent stem cells, with remarkable versatilityin their differentiation potential, including pan-creatic stem cells (PSCs) (Kruse et al., 2004, 2006).PSCs were able to grow for more than 140 passagesmaintaining the expression of typical stem cellmarkers (alkaline phosphatase, SSEA-1, Oct-4 andNestin), as demonstrated by immunocytochemistryand RT-PCR (Kruse et al., 2006). PSCs have beenshown to possess great potential for self-renewaland multilineage differentiation. These cellsshowed spontaneous differentiation into lineagesof all three germ layers. When cultured in hangingdrops, PSCs formed organoid bodies, three-dimen-sional (3-D) aggregates containing cells of differentlineages (Kruse et al., 2004, 2006). This PSCplasticity makes them an appealing source for cellreplacement therapies and tissue engineering.

Complementary to the promising potential ofpluripotent stem cells, there is a need for efficientculture systems for mass scale expansion and/ordifferentiation. The past years have witnessed anincreasing number of studies geared towards thisgoal. In particular, stirred suspension bioreactorshave gained special interest due to their advanta-geous characteristics: they are hydrodynamicallywell characterized, easy to increase, enableculture homogeneity and continuous monitoringand control of the culture parameters (Ulloa-Montoya et al., 2005; King and Miller, 2007). Sofar, these systems have been used for a wide rangeof applications: (i) to culture embryoid bodiesderived from mouse and human embryonic stemcells (ESCs) and differentiate them into hemato-poietic and cardiac lineages (Dang et al., 2002;Cameron et al., 2006); (ii) to expand undifferen-tiated murine ES cells as aggregates (Cormieret al., 2006; zur-Nieden et al., 2007); (iii) toenhance expansion and neuronal differentiation ofhuman teratocarcinoma stem cells (Serra et al.,2007); (iv) to culture several types of tissue-specific

ASCs as 3-D aggregates (Gilbertson et al., 2006;Youn et al., 2006; Chawla et al., 2006). Severalresearch groups have also employed stirred bior-eactors to culture ESCs using microcarriers assubstrate to support attachment and growth(Abranches et al., 2007; Fok and Zandstra, 2005).However, many challenges remain as no studiesfocused on large scale expansion of pluripotentASCs have been reported so far.

Herein, the feasibility of large scale adult PSCsexpansion was assessed by testing two differentapproaches: cell aggregated cultures versus micro-carrier technology.

The present work describes, for the first time, anefficient bioprocess for the controlled expansion ofrat PSCs using stirred bioreactors, overcoming oneof the drawbacks of stem cell technology.

Material and methods

Cell line

The PSC cell line RSAPank was derived from ratexocrine pancreas: acini from male Sprague Dawleyrats were isolated at the Fraunhofer Institute ofBiomedical Engineering-University of Lubeck andpurified as described previously (Kruse et al., 2004,2006).

Cell culture in static adherent conditions

Rat PSCs were routinely cultured in Dulbecco’smodified Eagle medium (DMEM, Invitrogen) supple-mented with 10% fetal bovine serum (FBS-Gold,PAA) and 100 U/mL penicillin–streptomycin (P/S,Invitrogen), at 37 1C, in a humidified atmosphere of5% CO2. Cells from passage 21 to passage 24 wereused for all stirred culture experiments.

Cell culture in stirred conditions

Suspension studies were performed in spinnervessels (Wheaton, USA) of 125mL volume (workingvolume-100mL) incubated at 37 1C in a humidifiedatmosphere of 5% CO2. Two different cultiva-tion strategies were carried out-cells cultured asaggregates (Strategy 1) and immobilized in micro-carrier supports (Strategy 2).

ARTICLE IN PRESS

Expansion of adult pancreatic stem cell 105

Author's personal copy

Strategy 1 – AggregatesSpinner vessels equipped with ball or paddle

impellers were tested. PSCs were inoculated at aconcentration of 4� 105cell/mL, in 70mL of DMEMsupplemented with 15% of FBS-Gold and 100U/mLP/S, and stirred at 50 rpm. After 6 h, culturemedium was added to yield a final volume of100mL and the percentage of serum adjusted to10%. During the cultivation period, the agitationrate was changed from 60 to 120 rpm to avoid celldamage or aggregate clumping.

Strategy 2 – MicrocarriersTwo types of microcarriers were tested, Cytodex

1 and Cytodex 3 (GE Healthcare), both at aconcentration of 3 g/L (dry weight). The micro-carriers were prepared and sterilized according tothe manufacturer’s recommendations. Cell inocu-lates (1� 105cell/mL) were obtained from adher-ent PSCs routinely cultured in static adherentconditions, harvested by trypsinization, collectedby centrifugation, resuspended in 5mL culturemedium (DMEM supplemented with 10% FBS-Goldand 100U/mL P/S), and immediately transferred tospinner vessels. Immobilization in the microcarrierswas carried out for 4–5 h: cells were allowed toattach to the beads with intermittent stirring(1min of stirring every 20min), in order to obtaina homogeneous cell distribution. The culturevolume was then adjusted to 75mL by additionof culture medium, and continuous agitationwas set to 45–50 rpm. Twenty four hours afterinoculation, culture medium was added to obtainthe final culture volume (100mL) and the agitationrate was gradually increased over time (up to80 rpm).

In all stirred experiments, culture medium waspartially replaced (50%) every 3 days. This was doneby stopping agitation, withdrawing spent mediumand refeeding fresh medium immediately aftersedimentation of microcarriers/aggregates.

Cell culture in bioreactor

A 250mL bioreactor vessel designed in ourlaboratory was used to culture PSCs in a fullycontrolled environment. The internal geometry ofthe vessel is similar to the commercially availablespinner vessels but is equipped with pH and pO2

meters (from Mettler-Toledo, Urdorf, Switzerland),which allow on-line measurement and monitoringof these parameters. The pH was maintained at 7.2by injection of CO2 and addition of base (NaOH,0.2M). The dissolved oxygen concentration wasmaintained at 30% via surface aeration. The

temperature was kept at 37 1C by water recircula-tion in the vessel jacket controlled by a thermo-circulator module. This vessel was adapted to acommercially available bioreactor control unit(B-DCU, B-Braun Biotech International GmbH,Sartorius, Germany) that controls pH, pO2 andtemperature. Data acquisition and process controlwere performed using MFCS/Win SupervisoryControl and Data Acquisition (SCADA) software(B-Braun Biotech International GmbH, Sartorius,Germany).

PSCs were immobilized in Cytodex 3 microcar-riers (3 g/L) and directly inoculated in the bior-eactor (1� 105cell/mL) in a working volume of250mL. The agitation rate was kept at 50 rpmduring the first 24 h and then gradually increasedover the culture period up to 80 rpm. Culturemedium was partially replaced (50%) every 3 days,as described above for the spinner experiments.

Cell counts and viability

Cell culture samples collected daily were visua-lized using an inverted microscope with phasecontrast (DM IRB, Leica, Germany). The total cellnumber was determined by counting cell nucleiusing a Fuchs-Rosenthal hemacytometer, afterdigestion with 0.1M citric acid/1% Triton X-100(wt/wt)/0.1% crystal violet (wt/v) (Alves et al.,1996).

Cellular viability was assessed by measuringlactate dehydrogenase (LDH) activity from theculture supernatant. LDH activity was determinedspectrophotometrically (at 340 nm) following therate of oxidation of NADH to NAD+ coupled with thereduction of pyruvate to lactate. The specific rateof LDH release (qLDH) was calculated for each timeinterval using the following equation: qLDH ¼DLDH/(Dt�DXv), where DLDH is the change inLDH activity over the time period Dt, and DXv is theaverage of the total cell number during the sametime period.

Growth rate, doubling time and multipleincrease in cell expansion

Growth rates, doubling times and fold increaseparameters were also calculated for all cultures inmicrocarrier systems. Growth rates (m) werecalculated using a simple first-order kinetic modelfor cell expansion: dX/dt ¼ mX, where t (day) is theculture time and X (cell) is the value of viable cellsfor a specific t. The value of m was estimated usingthis model applied to the slope of the curves duringthe exponential phase. Doubling time (td) was

ARTICLE IN PRESS

M. Serra et al.106

Author's personal copy

calculated using the equation td ¼ [Ln(2)]/mU Thefold increase in cell expansion (FI) was evaluatedbased on the ratio XMAX/X0, where XMAX is the peakcell density (cell/mL) and X0 is the lowest celldensity (cell/mL).

Metabolite analysis

Glucose (GLC) and lactate (LAC) concentrations inthe culture were analyzed using a Bioprofile 100 plusAnalyzer (Nova Biomedical, USA). The specificmetabolic rates (qMet, mol.cell�1.day�1) were calcu-lated for the two periods between the first tworefeeds, using the equation: qMet ¼ DMet/(Dt DXv),where DMet is the variation in metabolite concentra-tion during the time period Dt and DXv the averageof adherent cells during the same time period. Theapparent lactate from glucose yield (YLAC/GLC) wascalculated as the ratio between qLAC/qGLC.

Telomerase activity

Telomerase is responsible for the maintenance ofchromosome length and significant telomeraseactivity in cells is fundamental for their infinitereplication capacity (Masters, 2000). Telomeraseactivity was determined using the Telo TAGGGTelomerase PCR ELISA PLUS kit (Roche, Basel,Switzerland) according to manufacturer’s instruc-tions. The assay uses the telomeric repeat ampli-fication protocol (TRAP), in which the telomerase-mediated elongation products are amplified by PCRand detected with an enzyme-linked immunosor-bent assay.

Immunocytochemistry

Cell cultured in microcarriersMicrocarrier cultures were transferred to 1.5mL

tubes and sedimented by gravity. The medium wasremoved and cells were rinsed with 0.5mM MgCl2solution in phosphate-buffered saline (PBS), fixedfor 20min in 4% (w/v) paraformaldehyde (PFA,Sigma) solution in PBS and permeabilized withTriton X-100 (TX-100, Sigma), 0.3% (w/v) diluted inPBS for 20min. After 1 h in blocking solution(bovine serum albumin, BSA, 1% (w/v), and TX-100 0.1% (w/v) in PBS), cells were incubated withprimary antibody for 2 h; washed 3 times with PBSand incubated with secondary antibody for 1 h.Microcarriers were transferred to glass slides andcovered with a drop of ProLong mounting mediumcontaining DAPI (Molecular Probes). For sampleobservation, slides were covered with glass cover-slips.

Cell aggregatesAfter sampling, PBS-washed aggregates were

transferred to a tissue protecting compound (TissueTeck, OCTTM Compound, Sakura) and frozen. Sam-ples were kept at �80 1C until cryosectioning in aLeica Cryostat. For the immunofluorescence assay,10mm sections were rehydrated with PBS and fixedin methanol at �20 1C for 10min. After 1 h in asolution of 2% (w/v) BSA and 0.1% (v/v) TX-100 inPBS, the slides were incubated with the primaryantibody for 1 h. After 2 washing steps with PBS, theslides were incubated for an additional 45min withthe secondary antibody. Finally, a drop of ProLongmounting medium (Molecular Probes) was added tothe slide that was then covered with a coverslip.

Incubations were done at room temperature.Primary and secondary antibodies used were:monoclonal mouse anti-Nestin (Chemicon) andanti-mouse AlexaFluor 488 (Molecular Probes),respectively, diluted in blocking solution.

All samples were visualized under a fluorescencemicroscope (DMRB, Leica, Germany).

Adipocyte differentiation

Cell samples were transferred to static cultures(Tissue culture flasks 75 cm2) and cultured untilreaching 100% confluency. At this time, cultureswere induced to differentiate into adipocytes byreplacing the growth medium with differentiationmedium (DMEM containing 10% heat inactivated FBSGold, 100U/mL P/S, 10 nM dexamethasone, 0.5mMisobutylmethylxantine, 60 mM indomethacin and100 ng/mL insulin, all from Sigma). Control cultureswere run in parallel in medium without additives.The medium was replaced 3 times per week andadipogenesis was observed over a period of 14 days.Differentiation was assessed on the basis ofmorphological characteristics and lipid staining byOil Red O. Briefly, adipocyte differentiated cultureswere washed with PBS and fixed with formalin 10%(Biorad) at room temperature for 30min. Cellswere then washed twice with sterile water andincubated with 60% isopropanol (Sigma) during2min. After that, cultures were stained with freshsolution of Oil red O (1:3 v/v) and counterstainedwith hematoxylin.

Results

Culture of PSCs as aggregates

The hypothesis of culturing PSCs as cell aggre-gates (Strategy 1) in stirred suspension systems was

ARTICLE IN PRESS

Expansion of adult pancreatic stem cell 107

Author's personal copy

evaluated using 125mL spinner vessels. With thisstrategy, the effect of impeller geometry on cellaggregation and growth was assessed; two impellerconfigurations were tested: ball (SP-AB) and paddle(SP-AP). Initially, the agitation was kept at low rate(50 rpm) in medium supplemented with 15% of FBSin order to promote cell aggregation (Masters,2000). One day after inoculation, small cellaggregates (70–90 mm) were observed in bothcultures by phase contrast microscopy (Figure 1Aand C). Although cells were able to assemble,single cells were also detected in the culturesupernatant. This was particularly observed in SP-AP (Figure 1A), confirming previous reports showingthat the ball impeller geometry favours cellaggregation (Moreira et al., 1995; Santos et al.,2007). From day 1 onwards, SP-AP aggregates losttheir integrity (Figure 1B) and culture viability hada pronounced decrease (data not shown).

In SP-AB, cell aggregates increased in size, withdiameters ranging from 90 to 120 mm upon 3 days ofcell cultivation (Figure 1D). However, no cellproliferation occurred suggesting that the increasein size was a result of cell assembling and aggregateclumping. On day 13 (Figure 1E), SP-AB aggregatesamples were collected and analyzed for thepresence of nestin positive cells, a marker of adultPSCs (Kruse et al., 2006), which was evaluated byimmunofluorescence microscopy in cryosections.Figure 1F shows that only a small percentage ofcells stained positive for nestin.

Cell growth and viability of PSCs cultured inmicrocarriers

For the second strategy, Cytodex 1 microcarriers,based on a positively charged cross-linked dextran

ARTICLE IN PRESS

Figure 1. Phase contrast photomicrographs of PSCs cultured as small aggregates in spinner vessels equipped withpaddle (A,B) and ball impeller (C–E). Cells were visualized by day 1 (A,C), day 3 (B,D) and day 13 (E).Immunofluorescence photomicrograph of cell aggregates cryosections on day 13 of ball spinner culture (F). PSCswere identified by nestin (green) labelling and nuclei were stained with DAPI (blue) Scale bars: (A–E) 100 mm, (F) 50 mm.

M. Serra et al.108

Author's personal copy

matrix, and Cytodex 3, in which the dextran matrixis covered with a layer of collagen, were tested fortheir ability to support the growth of PSCs inspinner vessels (SP-Cyt1 and SP-Cyt3 experiments).In both studies, cells were inoculated at 1� 105

cell/mL, corresponding to 5 and 8 cells permicrocarrier in SP-Cyt1 and SP-Cyt3, respectively.For all experiments, the microcarrier concentrationwas 3 g/L, the agitation rate was increased duringthe culture period from 50 to 80 rpm, and mediumreplacement was performed every 3 days.

Four hours after inoculation, the majority of cells(87.5% of the cell inoculum) were already attachedto the microcarriers in SP-Cyt3 (Figure 2A), whichwere able to support cell growth until day 4 (Figure2B). A similar behavior was observed in SP-Cyt1culture (data not shown), except that a lowernumber of cells adhered to the supports (76.3% ofthe cell inoculum).

Figure 3A shows the total cell concentrationprofile of adherent PSC in SP-Cyt1 and SP-Cyt3experiments. In both cultures, the exponentialgrowth phase lasted until day 4, where themaximum cell concentrations were reached:1.7� 105 and 1.9� 105 cell/mL for SP-Cyt1 andSP-Cyt3, respectively (Figure 3A). These valuescorrespond to a 2.2-fold increase in cell concentra-tion for both experiments (Table 1), considering thepercentage of inoculated cells that attached to thesupports. Growth rates (m) and doubling times (td)were calculated and compared (Table 1). A highergrowth rate was achieved for SP-Cyt3 (0.26 day�1)than for SP-Cyt1 (0.21 day�1), since the exponen-tial phase period was shorter (3 days) in the firstculture. For the same reason, td was lower in SP-Cyt3 (2.6 days). Culture viability was analyzed bymeasuring the cumulative values of specific LDHrelease rates (qLDH) in the culture supernatant.Figure 3B shows that, from day 4 onwards, asignificant increase in cumulative qLDH was ob-served for both experiments (more pronounced inSP-Cyt1). Simultaneously, a decline in total cell

number was detected, indicating that cultures hadreached the death phase (Figure 3A).

Metabolic characterization of PSCs culturedin microcarriers

Partial medium exchange was performed every 3days in order to: (i) assure supply of nutrients,(ii) partially remove metabolic waste products and,more importantly (iii) avoid accumulation ofgrowth factors or other secreted molecules capableof inducing PSC differentiation. Cell metabolismwas compared in Cytodex 1 and Cytodex 3 micro-carrier cultures to ensure that nutrient consump-tion and waste production were not limiting PSCgrowth. Measurement of metabolite concentrationsin the medium (namely glucose and lactate) wereperformed daily and are presented in Figure 4. Theresults showed that for both cultures and up to 9days, there was no depletion in glucose levels(Figure 4A). Concerning metabolic products, thelevels of lactate never reached 20mM, except inSP-Cyt1 at the last 2 days of cultivation (Figure 4B).

In order to better characterize and compare thecell metabolism pattern of the two microcarriercultures, the specific rates of nutrient consumption(qGLC) and metabolite production (qLAC) werecalculated. Two phases were considered: one untilthe first refeed (Phase 1, from day 0 to 3) and theother between the first and second refeeds (Phase2, from day 3 to 6) (Table 2). The values obtainedfor both cultures were similar. Overall, the specificconsumption and production rates were higherwithin Phase 1 which is consistent with the highactivity of cells at the beginning of the exponentialphase in cell growth. In Phase 2, a decrease inall specific rates (qGLC and qLAC) was detected;at this time, cells were at the end of theexponential phase and entering the death period,usually associated with a slowdown of theirmetabolic activity. The yields of lactate productionfrom glucose consumption (YLAC/GLC) were also

ARTICLE IN PRESS

Figure 2. Phase contrast photomicrographs of PSCs cultured in Cytodex 3 microcarriers under stirred suspensionconditions. Cells were visualized 4 h after inoculation (A) and by day 4 (B). Scale bar: 100 mm.

Expansion of adult pancreatic stem cell 109

Author's personal copy

calculated in order to evaluate the efficiency ofglucose metabolism (Table 2). The results showedthat, overall, YLAC/GLC were higher than 2 suggest-ing that glucose was totally converted to lactate viaglycolysis and that other carbon sources areeventually being canalized to generate lactate.

These findings indicate the occurrence of oxygenlimitation in both spinner experiments.

PSCs expansion in a fully controlledbioreactor

Since Cytodex 3 microcarriers have been shownto preferentially support PSCs growth on stirredsuspension systems (best cell adherence and fastercell growth), the next challenge was to increasethe scale and control of PSCs growth by culturingcells in a 250mL controlled bioreactor (BR-Cyt3).In this vessel, cells were cultured under thesame conditions used for SP-Cyt3 (agitation rate– 50–80 rpm, inoculum concentration – 1� 105 cell/mL and bead concentration – 3 g/L), exceptthat the cells previously attached to Cytodex 3

ARTICLE IN PRESS

Figure 3. PSCs cultured in microcarriers using stirred suspension systems. (A) Growth curves in terms of total adherentcells per millilitre and (B) viability expressed by the cumulative values of specific LDH release rates.

Table 1. Growth rate (m), doubling time (td) and foldincrease (FI) values of PSC cultured in Cytodex 1 andCytodex 3 microcarriers, using spinner vessels and a250mL bioreactor.

SpinnerCytodex 1

SpinnerCytodex 3

BioreactorCytodex 3

m (day�1; h�1) 0.21; 0.0089 0.26; 0.011 0.35; 0.015td (day; h) 3.2; 78 2.6; 63 2.0; 47FI 2.2 2.2 4.7

M. Serra et al.110

Author's personal copy

microcarriers were directly inoculated into thebioreactor. Furthermore, cultivation was per-formed in a fully controlled environment (tempera-ture-37 1C, pH-7.2, and pO2-30%), in order toovercome the oxygen limitation observed in thespinner culture (described above).

Total cell concentrations of adherent cells arerepresented in Figure 3A, as well as the cumulativeqLDH values achieved throughout culture period inFigure 3B. The first 4 h were characterized by apronounced decrease in cell number; 50% of thetotal starting cells were detached from the beads.

ARTICLE IN PRESS

Table 2. Metabolic characterization of PSC cultured in Cytodex 1 and Cytodex 3 microcarriers, using spinner vesselsand a 250mL bioreactor.

Spinner Cytodex 1 Spinner Cytodex 3 Bioreactor Cytodex 3

Phase 1 Phase 2 Phase 1 Phase 2 Phase 1 Phase 2

qGLC mmol.10�6cell.day�1 15.1 10.6 13.0 8.0 12.7 9.5qLAC mmol.106�6cell.day�1 32.5 23.5 29.0 17.9 20.3 11.4YLAC/GLC 2.1 2.2 2.2 2.2 1.6 1.2

The specific rates of glucose consumption (qGLC) and lactate production (qLAC) were calculated from day 0 to 3 (Phase 1) and from day 3to 6 (Phase 2). The apparent lactate from glucose (YLAC/GLC) yields was calculated for the two phases.

Figure 4. Glucose uptake and lactate release of PSC cultured in Cytodex 1 and Cytodex 3 microcarriers, using spinnervessels and a 250mL bioreactor. Metabolite concentrations (glucose and lactate) are shown over 9 days of culture.

Expansion of adult pancreatic stem cell 111

Author's personal copyARTICLE IN PRESS

Figure 5. Phase contrast photomicrographs of PSCs cultured in Cytodex 3 microcarriers in a 250mL bioreactor. Cellswere visualized after inoculation (A), by day 4 (B) and at the end of cell cultivation, i.e., by day 9 (C). Scale bar:100 mm.

Figure 6. Characterization of PSCs cultured in Cytodex 3 microcarriers. By day 7, PSCs growing adherent to the carrierswere identified by nuclear DAPI staining (blue) and by nestin labelling (green), using immunofluorescence microscopy(A). Adipogenic differentiation was evaluated by lipid staining with Oil Red O (B). Scale bars: (A) 50 mm and (B) 100 mm.Telomerase activity of PSCs was analyzed by day 7 of culture using a Telo TAGGG Telomerase PCR ELISA PLUS kit (C). Thewhite bars show the results obtained for the PSCs expanded in static (Static) and stirred (SP-Cyt1, SP-Cyt3 and BR-Cyt3)conditions. Negative controls (black bars) represent telomerase activity following inactivation by heat.

M. Serra et al.112

Author's personal copy

Simultaneously, qLDH was significantly higher at thistime point than in spinner culture, confirming thatincreased cell lysis was taking place. Phase contrastphotomicrographs showed that a smaller amount ofcells remained attached to microcarriers in BR-Cyt3than in SP-Cyt3 (Figures 5A and 2A, respectively),confirming that the inoculation process wassomehow aggressive for the cells. After 24 h, cellsstarted to divide with a growth pattern similarto that obtained for SP-Cyt3, except that theexponential phase was extended until day 5(Figure 3A). At this time, a high percentage ofsupports were totally covered by PSC (Figure 5B);cell concentrations reached the maximum value(2.3� 105 cells/mL) yielding a two times higherfold increase in cell concentration than in SP-Cyt3(4.6 versus 2.2) (Table 1). From day 5 onwards, cellsreached the death phase and detached from thebeads (Figure 5C), resulting in increasing qLDHlevels (Figure 3B).

Growth characteristics were also compared forboth culture systems (Table 1). The results ob-tained showed the apparent growth rate was higher(0.35 day�1) and the doubling time was lower (47 h)in BR-Cyt3 than in SP-Cyt3, confirming that cellgrowth was faster in the bioreactor culture.

Overall these results can be justified by themetabolic performance of cells when cultured in afully controlled environment. Neither the depletionof nutrients nor the accumulation of inhibitorywaste concentrations was detected over theculture period (Figure 4). The specific rates ofmetabolite consumption and production wereestimated (Table 2). Furthermore no changes weredetected in glucose consumption, while the levelsof lactate production were significantly lower,resulting in yields of YLAC/GLC lower than 2 (1.6and 1.2 for Phase 1 and Phase 2, respectively); dueto the higher oxygen availability to the cells therewas a more efficient use of glucose thus resulting ina higher production of biomass.

Characterization of PSCs expanded inmicrocarriers

The distribution of PSCs on the microcarriersurface was visualized by nestin staining usingimmunofluorescence microscopy. In the experi-ments using the Cytodex supports, adherent cells,identified by nuclear DAPI staining, stained positivefor nestin (Figure 6A). To evaluate the efficiency ofeach microcarrier culture system in expandingPSCs, all populations were characterized in termsof self-renewal capacity and differentiation poten-tial by evaluation of telomerase activity and

induction of adipocyte differentiation, respec-tively. Telomerase activity was analyzed for ratPSCs derived from both static and microcarriercultures (Figure 6C). In all instances, the absor-bance readings of samples (A450 nm–A690 nm) werehigher than twice the value of the respectivenegative control (heat-treated samples), confirm-ing that all samples were telomerase positive. Inmicrocarrier cultures, the levels of telomeraseactivity were similar to the control, showing thatexpanded PSCs maintained the capacity to prolif-erate extensively in vitro. To examine the differ-entiation potential, PSCs were cultured inadipocyte differentiation medium for 2 weeks.The presence of lipid deposits stained with OilRed O (Figure 6B) demonstrated that these cellswere able to differentiate into adipocytes.

In conclusion, all these findings demonstratethat, after expansion in microcarriers, cells main-tained the expression of PSC marker, their self-renewal ability, and differentiation potential,confirming that they retained their stem cell behavior.

Discussion

The first purpose of stem cell cultivation is toexpand cells while maintaining their pluripotentpotential, in order to produce large numbers ofhighly pure populations, adequate for controlleddifferentiation. In the present study, we success-fully developed an efficient strategy for theexpansion of adult PSC cultures in Cytodex 3microcarriers using fully controlled bioreactors.

A vast range of microcarriers is currently avail-able, employing different materials and surfacetypes, in order to allow the culture of differentanchorage-dependent cell types in stirred condi-tions. This strategy has been widely used by ourgroup in the development of scalable bioprocessesto grow mammalian cells in suspension bioreactors(Alves et al., 1996; Santos et al., 2005). In thiswork, the two microcarriers tested (Cytodex 1 andCytodex 3), provided a suitable matrix for theexpansion of PSCs under stirred conditions. A moreefficient and robust cell adherence and prolifera-tion was obtained using Cytodex 3 beads (Figure 3),which may be explained by the better adhesion ofPSCs to the collagen layer that covers the surfaceof the carriers. Cytodex 3 has been speciallydesigned for culturing sensitive cells and has beenreported to be suitable for culture of primary braincells (Santos et al., 2005), as well as for theexpansion of mouse ESCs in suspension stirredsystems (Fok and Zandstra, 2005; Abrancheset al., 2007).

ARTICLE IN PRESS

Expansion of adult pancreatic stem cell 113

Author's personal copy

Expanding PSCs adherent to Cytodex 3 micro-carriers resulted in a stem cell population thatretained its pluripotent characteristics: PSC mar-ker, self-renewal ability and differentiation poten-tial (Figure 6), proving to be of great advantageover culturing the cells as aggregates, where cellsclumped together, failed to proliferate and losttheir nestin labeling (Figure 1). Although cellaggregates have been used in previous reports toexpand undifferentiated murine ESCs (Cormieret al., 2006; zur Nieden et al., 2007), neural stemcells (Gilbertson et al., 2006) and carcinoma stemcells (Youn et al., 2006), this approach revealed tobe unfeasible for culturing PSCs (Figure 1). Thesedifferences in cell behavior may reflect the distincttissue origins, as PSCs are pluripotent ASCs derivedfrom pancreas (Kruse et al., 2004, 2006). Ourresults suggest that PSCs are anchorage-dependentand that adherence plays a role in the control ofcell fate decisions.

The potential of bioreactor technology wascombined with the efficient Cytodex 3 strategy,resulting in a robust bioprocess for the expansion ofPSCs. Under controlled environmental conditions(pH, pO2 and temperature), cell growth was moreefficient, as shown by faster doubling time, highergrowth rate and higher fold increase in cellconentration, when compared to spinner cultures(Table 1). This improvement may be explained bythe higher availability of oxygen (supplied by thebioreactor controller) to the cells. Comparison ofapparent lactate from glucose yields obtained instirred bioreactor and spinner vessels (YLAC/GLC ¼1.6–1.2 and 2.2, respectively, Table 2) indicatesthat cells metabolized glucose more efficiently inthe bioreactor, with lower production of lactate.Thus, the oxygen limitation observed in thespinners was successfully overcome in the bioreac-tor experimental setup, leading to an increasedlevel of cell proliferation.

The fold increase in cell concentration obtained inthe bioreactor was not as high as those achieved formESC cultured in Cytodex-3 microcarriers in spinnervessels (Fernandes et al., 2007) and neonatal porcinepancreatic cells cultured as spherical-islets (Chawlaet al., 2006). PSCs are fusiform cells, with sizes up to50–100mm, which limited the cell expansion ratio inmicrocarriers to an approximately 5-fold increaseobtained in this study. Thus, in order to obtain higherexpansion ratios, serial passaging with addition offresh microcarriers can be incorporated. Moreover,alternative strategies, such as perfusion or fed-batchculture mode allowing the microcarrier feeding maybe considered.

In summary, this controlled and robust culturesystem for expansion of pluripotent adult PSCs,

using stirred bioreactors, has proven to be a strongstarting point for the development of noveltechnologies for cell therapy.

Acknowledgements

The authors acknowledge Antonio Roldao andMarcos Sousa for helpful discussions on this manu-script. The work was supported by the EuropeanCommission (Cell Programming by NanoscaledDevices- NMP4-CT-2004-500039).

References

Abranches, E., Bekman, E., Henrique, D., Cabral, J.S.,2007. Expansion of mouse embryonic stem cells onmicrocarriers. Biotechnol. Bioeng. 96, 1211–1221.

Alves, P.M., Moreira, J.L., Rodrigues, J.M., Aunins, J.G.,Carrondo, M.J.T., 1996. Two-dimensional versus three-dimensional culture systems: Effects on growth andproductivity of BHK cells. Biotechnol. Bioeng. 52,429–432.

Cameron, C.M., Hu, W.S., Kaufman, D.S., 2006. Improveddevelopment of human embryonic stem cell-derivedembryoid bodies by stirred vessel cultivation.Biotechnol. Bioeng. 94, 938–948.

Chawla, M., Bodnar, C.A., Sen, A., Kallos, M.S., Behie,L.A., 2006. Production of Islet-like structures fromneonatal porcine pancreatic tissue in suspensionbioreactors. Biotechnol. Prog. 22 (2), 561–567.

Cormier, J.T., Nieden, N.I.Z., Rancourt, D.E.,Kallos, M.S., 2006. Expansion of undifferentiatedmurine embryonic stem cells as aggregates in suspen-sion culture bioreactors. Tissue Eng. 12 (11),3233–3245.

Dang, S.M., Kyba, M., Perlingeiro, R., Daley, G.Q.,Zandstra, P.W., 2002. Efficiency of embryoid bodyformation and hematopoietic development fromembryonic stem cells in different culture systems.Biotechnol. Bioeng. 78 (4), 442–453.

Fernandes, M.A., Fernandes, T.G., Diogo, M.M., Lobatoda Silva, C., Cabral, J.S., 2007. Mouse embryonic stemcell expansion in a microcarrier-based stirred culturesystem. J. Biotechnol. 132 (2), 227–236, (31).

Fok, E.Y.L., Zandstra, P.W., 2005. Shear-controlledsingle-step mouse embryonic stem cells and embryoidbody-based differentiation. Stem Cells 23, 1333–1342.

Gilbertson, J.A., Sen, A., Behie, L.A., Kallos, M.S., 2006.Scaled-up production of mammalian neural precursorcell aggregates in computer-controlled suspensionbioreactors. Biotechnol. Bioeng. 94, 783–792.

Gokhale, P.J., Andrews, P.W., 2006. A prospective onstem cell research. Semin. Reprod. Med. 24 (5),289–297.

King, J.A., Miller, W.M., 2007. Bioreactor developmentfor stem cell expansion and controlled differentiation.Curr. Opin. Chem. Biol. 11, 394–398.

ARTICLE IN PRESS

M. Serra et al.114

Author's personal copy

Kruse, C., Birth, M., Rohwedel, J., Goepel, A., Wedel, T.,2004. Pluripotency of adult stem cells derivedfrom human and rat pancreas. Appl. Phys. A 79,1617–1624.

Kruse, C., Kajahn, J., Petschnik, A.E., MaaB, A., Klink,E., Rapoport, D.H., Wedel, T., 2006. Adult pancreaticstem/progenitor cells spontaneously differentiate invitro into multiple cell lineages and form teratoma-like structures. Ann Anat. 188, 503–517.

Masters, J.R.H., 2000. Anima Cell Culture: A PracticalApproach, third ed. Oxford University Press,Oxford.

Moreira, J.L., Alves, P.M., Aunins, J.G., Carrondo, M.J.T.,1995. Hydrodynamic effects on BHK cells grown assuspended natural aggregates. Biotechnol. Bioeng. 46,351–360.

Santos, S.S., Fonseca, L.L., Monteiro, M.A.R., Carrondo,M.J.T., Alves, P.M., 2005. Culturing primary brainastrocytes under a fully controlled environment in anovel bioreactor. J. Neurosc. Res. 79, 26–32.

Santos, S.S., Leite, S.B., Ursula Sonnewald, U., Carron-do, M.J.T., Alves, P.M., 2007. Stirred vessel cultures ofrat brain cells aggregates: Characterization of majormetabolic pathways and cell population dynamics.J. Neurosc. Res. 85, 3386–3397.

Serra, M., Leite, S.B., Brito, C., Costa, J., Carrondo,M.J.T., Alves, P.M., 2007. Novel culture strategy forhuman stem cell expansion and neuronal differentia-tion. J. Neurosc. Res. 85 (16), 3557–3566.

Ulloa-Montoya, F., Verfaillie, C.M., Hu, W.S., 2005.Culture systems for pluripotent stem cells. J. Biosci.Bioeng. 100, 12–27.

Youn, B.S., Sen, A., Behie, L.A., Girgis-Gabardo, A.,Hassell, J.A., 2006. Scale-up of breast cancer stemcell aggregate cultures to suspension bioreactors.Biotechnol. Prog. 22, 801–810.

zur Nieden, N.I., Cormier, J.T., Rancourt, D.E., Kallos, M.S.,2007. Embryonic stem cells remain highly pluripotentfollowing long term expansion as aggregates in suspen-sion bioreactors. J. Biotechnol. 129 (3), 421–432.

ARTICLE IN PRESS

Expansion of adult pancreatic stem cell 115