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Optimal In-Vitro Expansionof Chondroprogenitor CellsIn Monolayer Culture
Juan M. Melero-Martin,
1
Mary-Ann Dowling,
3
Mark Smith,
3
Mohamed Al-Rubeai
1,2
1
Department of Chemical Engineering, School of Engineering,University of Birmingham, Birmingham, B15 2TT, United Kingdom
Department of Chemical and Biochemical Engineering, and Centre for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland; Tel: 
þ
353 1 7161862; Fax: 
þ
353 1 7161177; e-mail: m.al-rubeai@ucd.ie 
Smith & Nephew Research Centre, York Science Park, Heslington, York, YO10 5DF,United Kingdom
Received 1 June 2005; accepted 1 September 2005 
Published online 28 October 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20735 
Abstract:
A continuous production of large quantities of chondroprogenitor cells for the manufacture of engine-eredcartilagetissueproductsisrequired.Expansionofthecell population in vitro has become an essential step intheprocessoftissueengineeringofarticularcartilageandtheoptimizationofthecultureconditionsisafundamentalproblem that needs to beaddressed. The analysisof bothseeding density and passage length was consideredcrucial in the optimization of expansion processes, andtheir correct selection should be taken as a requisite toestablish culture conditions for monolayer systems. Thedetermination of the optimal seeding density and thecorresponding passage length for cell expansion in aserialpassagingoperationwasfoundtobeacompromisebetween growth kinetics and process time. This optimaldetermination was carried out using a mathematicalapproach that led to values of 10
4
cell/cm
2
for seedingdensity and 73 h for passage length. Additional consi-derations concerning the running cost of the processwere introduced. Although the optimal passage lengthgave the desired expansion factor in a minimum processtime, the selection of an alternative value of 120 h wasshown to reduce the cost of the expansion process inmore than 60%. The optimization approach presentedwill contribute to the development of feasible largescale expansion operations of chondroprogenitor cellsrequired by the cartilage tissue engineering industry.
ß
2005 Wiley Periodicals, Inc.
Keywords:
expansion; chondrocyte; cartilage; Gompertz;chondroprogenitor; monolayer culture
INTRODUCTION
Damaged articular cartilage has a limited ability to repairitselfduetotheabsenceofvascularizationandnerveendingsin the tissue (Buckwalter and Mankin, 1997; Mankin, 1974;Mankin, 1982; Ratcliffe and Mow, 1991). Traditional thera-pies to repair damaged cartilage include alloplastic andallogenic implants and more recently autologous chondro-cyte transplantation. The former therapies require surgicalremoval of healthy cartilage and is limited by the size of thedefect, while the latter therapies are limited by donor sitemorbidity (Cancedda et al., 2003; Grande et al., 1989;Hunziker, 2002). As an alternativeto these current therapies,efforts in tissue engineering of cartilage have led to thedevelopmentofbiocompatible,biodegradablescaffoldsontowhich chondrocytes are seeded. One of the challenges thattissue engineers will have to address in the near future is thedevelopmentoffeasiblelargescalecellexpansionprocesses.The estimated number of articular cartilage incidencesworldwide is around 30 million cases of knee osteoarthritisand 1.2 million cases of focal defects every year (MedtechInsight, 2002). If tissue engineering products are to be usedfor the treatment of these incidences, it is crucial to estimatethe scale of cell production needed for all these repairprocedures, but such essential question is rarely approachedinreportedtissueengineeringstudies.TableIgivesanideaof the dimensional aspect of articular cartilage repair and theestimatedcellnecessitiesassociated.Routinetissueculturingmethodologies can hardly cope with the scale of cellproduction required for these procedures. Expansion of cellpopulationinvitrohasbecomeanessentialstepintheprocessof tissue engineering of articular cartilage, and optimizationof the culture conditions and expansion protocols arefundamental issues that need to be addressed.In the process of cartilage tissue engineering, it isimportant to ensure that the expanded cell population retainsitsphenotypicfunction.Chondrocytesderivedfromarticularcartilage biopsies have only a limited proliferative potential.They dedifferentiate upon repeated passaging (Benya andShaffer,1982)andthenumberofcelldivisions chondrocytesundergo invitro decreases with age (Dozin et al., 2002). Theissue of phenotype expression and differentiation has led totheinvestigationofthepotentialuseofpluripotentstemcellsasasourcefortissueengineering.Recentlyanewpopulation
ß
2005 Wiley Periodicals, Inc.
Correspondence to:
Mohamed Al-Rubeai
 
of chondroprogenitor cells isolated from the superficial zoneof the articular cartilage has been identified and partiallycharacterized (Dowthwaite et al., 2004). This populationof cells exhibits a significant degree of plasticity in itsdifferentiation pathways (Dowthwaite et al., 2004), arequisite of any stem cell population (Morrison et al.,1997). The engraftment of bovine surface zone-derived cellsin an embryonic chick tracking system resulted in theformation of a variety of connective tissue types includingbone, tendon, and perimysium (Dowthwaite et al., 2004).Moreover, the chondrogenic ability of this cell populationhas also been proved invitrowhere cells synthesized hyalinecartilage matrix when cultured in three-dimensional pellets(Martinetal.,2005).Chondroprogenitorcellshavealsobeendemonstratedtohaveamuchhigherexpansionpotentialthanadult differentiated chondrocytes (Martin et al., 2005). Thecellsretainedtheabilitytosynthesizeacartilage-likehyalinematrix rich in glycosaminoglycans and collagen type II evenafter 11 passages which equated to 25 population doublings.In previous work, culture conditions and operation modewere established for monolayer expansion of chondropro-genitorcells(Martinetal.,2005).Theproliferationenhance-ment identified under these culture conditions led to cellexpansion factors that were considered more than sufficientfor autologous cell repair applications, where only few-foldexpansion would be eventually required (Brittberg et al.,1994). Nevertheless, in addition to autologous applications,the enhanced potential of chondroprogenitor cells to retainthe ability to form cartilage after extensive expansion inculture (Dowthwaite et al., 2004; Martin et al., 2005) opensup the possibility for more intense expansion processes thatmay enable the generation of large cell banks for use inallogeneic tissue engineering applications. The aim of thispaper was consequently to further the search for the cultureconditionsandoperationmodesthatwilleventuallyoptimizecell expansion in a serial monolayer passaging process. Forthis aim, the use of mathematical expressions to define thegrowth curve of the cultures was found to be a valuable tool.The optimization of chondroprogenitor cells expansion iscrucial in order to develop feasible large scale processes. Toour knowledge, this is the first reported work concerning amathematical approach to optimize the seeding density andthe passage length of a serial monolayer expansion process.
MATERIALS AND METHODSIsolation of Chondroprogenitor Cells
Cartilage slices were harvested from the superficial zone of articular cartilages of 2–3-week old bovine metatarsopha-langealjointsbyfinedissection.Thesliceswerewashedwithsterile phosphate buffered saline (PBS, pH 7.4) and sequ-entially digested in pronase (0.1% w/v; 3h) and collagenase(0.04%w/v;overnight)inserumfreeDMEM(Sigma,UK)at37
8
C. After incubation the undigested cartilage fragmentswereremovedusinga70
m
mFalconfilter(BectonDickinson,UK).Theisolatedcellswereseededat4,000/mLonto35mmplastic Petri-dishes (Iwaki, Japan) and incubated in serumfree DMEM at 37
8
C for 20 min. These Petri-dishes werepreviously coated with 10
m
g/mL bovinefibronectin (Sigma,UK) in PBS containing 1 mM MgCl
2
and 1 mM CaCl
2
overnight at4
8
C(Dowthwaiteetal., 2004).After 20min, themedia were removed from the Petri-dishes and discarded.Tissue culture medium composed of DMEM
þ
(DMEM,2 mM
L
-Glutamine, 1% Non-essential amino acids and50 IU/mL penicillin/50
m
g/mL streptomycin) supplementedwith10%foetalcalfserum(FCS;PAALaboratories,Austria)was added to the remaining adherent cells which weremaintained in culture for up to 12 days. After 12 days incultures, the Petri-dishes were washed with PBS andtrypsinized (Trypsin–EDTA; Gibco, UK) until completecell detachment. Harvested chondroprogenitor cells werethensub-cultureduntildifferentpassagenumbersbyseedingT-75 and T-150 tissue culture flasks (Iwaki, Japan) at adensity of 10
4
cells/cm
2
. Flasks were incubated at 37
8
C in a5% CO
2
atmosphere until sub-confluent and tissue culturemedium composed of DMEM
þ
supplemented with 10%FCS changed every 2–3 days.
Cell Density and Viability
Chondroprogenitor cell cultures were washed with PBS andtrypsinized until complete cell detachment. Cells were then
Table I.
Estimation of cell necessities for tissue engineering treatments.Indication Incidence of surgical procedures WorldwideFocal defects in articular cartilage 1.2
Â
10
6
Osteoarthritis (OA of the knee) 3
Â
10
7
Tissue engineering treatment Number of implants Cells required
a
Focal defects in articular cartilageTypical human defect size (4
Â
40 mm)
b
1 3.2
Â
10
8
10% of focal defect incidences
c
1.2
Â
10
5
3.9
Â
10
13
OsteoarthritisTypical human OA (2
Â
4
Â
40mm)
b
2 6.4
Â
10
8
10% of OA defect incidences
c
3
Â
10
6
1.9
Â
10
15a
64 million cells/cm
3
as seeding density (Puelacher et al., 1994).
b
Typical sizes provided by Smith & Nephew Research Centre, York, UK.
c
Assuming 10% of incidences are appropriate for treatment (Aroen et al., 2004).
520 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 93, NO. 3, FEBRUARY 20, 2006
 
resuspended in tissue culture medium and counted using ahaemocytometer. Viability was determined by the Trypanblue exclusion method.
Growth Curve Fitted by Four-CoefficientsGompertz Equation
Growth curves of chondroprogenitors in monolayer cultureswere characterized by a four coefficients generalization of the Gompertz function, given by the following equation:
 X 
¼
 X 
0
þ
a
Á
exp
À
exp
Àð
À
0
Þ
b
ð
1
Þ
Where
(
h
), corresponds to the culture time;
(cell/cm
2
),corresponds to the value of viable cell density at any giventime
;
 X 
0
(cell/cm
2
) correspondstothe initial value ofviablecell density;
a
(cell/cm
2
), corresponds to the differencebetween the final value of viable cell density achieved at thestationary phase of the culture (
 X 
F
) and the initial value of viablecelldensity(
 X 
0
);
0
(
h
),correspondstotheculturetimeat which the increment of viable cell density is equal to 37%of the final increment achieved at the stationary phase of theculture; and
b
(
h
), corresponds to the culture time necessaryfortheincrementofviablecelldensitytopassfrom37to69%of the final increment achieved at the stationary phase of theculture. A schematic representation of (1) has been depictedinFigure1,wherethequalitativemeaningofeachcoefficientinrelationwiththegrowthcurveofahypotheticalculturehasbeen provided. For every growth curve analyzed, theexperimental data for viable cell density were acquired intriplicates by the Trypan blue exclusion method. Theexperimental data were fitted using a non-linear regressionalgorithm provided with SigmaPlot (SigmaPlot 2002 forWindows, Version 8.0). For each growth curve fitted, thevalues of the equation coefficients with their respectivestandarderrors(SE),aswellasthecorrelationcoefficient(
2
)were reported.
Definitions of Parameters
Several parameters were defined according to three cate-gories: parameters concerning a single monolayer passage,parameters related to the whole expansion process andparameters related with the cost of the process (summarizedin Fig. 2) as follows.
Passage Parameters 
Initial cell density,
0
(cell/cm
2
), corresponds to the value of viable cell density used to inoculate a single passage.Final cell density,
F
(cell/cm
2
), corresponds to the valueof final viable cell density observed at the time of harvestingof a single passage.Passage population doubling,
n
p
(
À
), corresponds to thenumber of doublings that cells undergo during a singlepassage, from the inoculation to the time of harvesting.Passage length,
p
(
h
), corresponds to the duration of asingle passage, from inoculation to harvesting.Passageexpansionfactor,
p
(
À
),correspondstothefactorby which the viable cell density is multiplied during a singlepassage, from inoculation to harvesting.
Expansion Process Parameters 
Initial cell number,
(cells), corresponds to the total viablecell number available at the beginning of the expansionprocess.Finalcellnumber,
(cells),correspondstothetotalviablecell number available at the end of the expansion process.Final population doubling,
n
(
À
), corresponds to the finalnumber of doublings that cells undergoduring the expansionprocess.Total expansion time,
(
h
), corresponds to the duration of the expansion process.Total passage number,
p
(
À
), corresponds to the totalnumber of passages that the cells undergo during theexpansion process.Total expansion factor,
(
À
), correspond to the factor bywhich the viable cell density is multiplied during theexpansion process.
 I 
Pi
(cell), corresponds to the total viable cell numberavailable at the beginning of the passage number
i
.
 A
i
(cm
2
), corresponds to the surface area necessary toinoculate the initial viable cell number (
 I 
Pi
) at the beginningof the passage number
i
.
Cost Parameters 
MPA
($/cm
2
), correspond to the cost of culture medium perunit of area necessary to carry out one single passage. For a
Figure 1.
Chondroprogenitors growth curve fitted by four-parametersGompertz function. Schematic representation and qualitative meaning of afour parameters generalizationofthe Gompertzfunctionin relationwith thegrowth curve of a hypothetical culture of chondroprogenitor cells.
MELERO-MARTIN ET AL.: OPTIMAL EXPANSION OF CHONDROPROGENITOR CELLS 521

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