Optimal In-Vitro Expansion

of Chondroprogenitor Cells
In 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
2
Department of Chemical and Biochemical Engineering, and Centre for Synthesis and
Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland;
Tel: þ353 1 7161862; Fax: þ353 1 7161177; e-mail: m.al-rubeai@ucd.ie
3
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-
ered cartilage tissue products is required. Expansion of the
cell population in vitro has become an essential step in
the process of tissue engineering of articular cartilage and
theoptimizationof thecultureconditions is afundamental
problem that needs to be addressed. The analysis of both
seeding density and passage length was considered
crucial in the optimization of expansion processes, and
their correct selection should be taken as a requisite to
establish culture conditions for monolayer systems. The
determination of the optimal seeding density and the
corresponding passage length for cell expansion in a
serial passagingoperationwas foundtobe a compromise
between growth kinetics and process time. This optimal
determination was carried out using a mathematical
approach that led to values of 10
4
cell/cm
2
for seeding
density and 73 h for passage length. Additional consi-
derations concerning the running cost of the process
were introduced. Although the optimal passage length
gave the desired expansion factor in a minimum process
time, the selection of an alternative value of 120 h was
shown to reduce the cost of the expansion process in
more than 60%. The optimization approach presented
will contribute to the development of feasible large
scale expansion operations of chondroprogenitor cells
required by the cartilage tissue engineering industry.
ß2005 Wiley Periodicals, Inc.
Keywords: expansion; chondrocyte; cartilage; Gompertz;
chondroprogenitor; monolayer culture
INTRODUCTION
Damaged articular cartilage has a limited ability to repair
itself due to the absence of vascularization and nerve endings
in the tissue (Buckwalter and Mankin, 1997; Mankin, 1974;
Mankin, 1982; Ratcliffe and Mow, 1991). Traditional thera-
pies to repair damaged cartilage include alloplastic and
allogenic implants and more recently autologous chondro-
cyte transplantation. The former therapies require surgical
removal of healthy cartilage and is limited by the size of the
defect, while the latter therapies are limited by donor site
morbidity (Cancedda et al., 2003; Grande et al., 1989;
Hunziker, 2002). As an alternative to these current therapies,
efforts in tissue engineering of cartilage have led to the
development of biocompatible, biodegradable scaffolds onto
which chondrocytes are seeded. One of the challenges that
tissue engineers will have to address in the near future is the
development of feasible large scale cell expansion processes.
The estimated number of articular cartilage incidences
worldwide is around 30 million cases of knee osteoarthritis
and 1.2 million cases of focal defects every year (Medtech
Insight, 2002). If tissue engineering products are to be used
for the treatment of these incidences, it is crucial to estimate
the scale of cell production needed for all these repair
procedures, but such essential question is rarely approached
in reported tissue engineering studies. Table I gives an idea of
the dimensional aspect of articular cartilage repair and the
estimated cell necessities associated. Routine tissue culturing
methodologies can hardly cope with the scale of cell
production required for these procedures. Expansion of cell
population invitro has become an essential step in the process
of tissue engineering of articular cartilage, and optimization
of the culture conditions and expansion protocols are
fundamental issues that need to be addressed.
In the process of cartilage tissue engineering, it is
important to ensure that the expanded cell population retains
its phenotypic function. Chondrocytes derived fromarticular
cartilage biopsies have only a limited proliferative potential.
They dedifferentiate upon repeated passaging (Benya and
Shaffer, 1982) and the number of cell divisions chondrocytes
undergo in vitro decreases with age (Dozin et al., 2002). The
issue of phenotype expression and differentiation has led to
the investigation of the potential use of pluripotent stemcells
as a source for tissue engineering. Recently a newpopulation
ß2005 Wiley Periodicals, Inc.
Correspondence to: Mohamed Al-Rubeai
of chondroprogenitor cells isolated from the superficial zone
of the articular cartilage has been identified and partially
characterized (Dowthwaite et al., 2004). This population
of cells exhibits a significant degree of plasticity in its
differentiation pathways (Dowthwaite et al., 2004), a
requisite of any stem cell population (Morrison et al.,
1997). The engraftment of bovine surface zone-derived cells
in an embryonic chick tracking system resulted in the
formation of a variety of connective tissue types including
bone, tendon, and perimysium (Dowthwaite et al., 2004).
Moreover, the chondrogenic ability of this cell population
has also been proved in vitro where cells synthesized hyaline
cartilage matrix when cultured in three-dimensional pellets
(Martin et al., 2005). Chondroprogenitor cells have also been
demonstrated to have a much higher expansion potential than
adult differentiated chondrocytes (Martin et al., 2005). The
cells retained the ability to synthesize a cartilage-like hyaline
matrix rich in glycosaminoglycans and collagen type II even
after 11 passages which equated to 25 population doublings.
In previous work, culture conditions and operation mode
were established for monolayer expansion of chondropro-
genitor cells (Martin et al., 2005). The proliferation enhance-
ment identified under these culture conditions led to cell
expansion factors that were considered more than sufficient
for autologous cell repair applications, where only few-fold
expansion would be eventually required (Brittberg et al.,
1994). Nevertheless, in addition to autologous applications,
the enhanced potential of chondroprogenitor cells to retain
the ability to form cartilage after extensive expansion in
culture (Dowthwaite et al., 2004; Martin et al., 2005) opens
up the possibility for more intense expansion processes that
may enable the generation of large cell banks for use in
allogeneic tissue engineering applications. The aim of this
paper was consequently to further the search for the culture
conditions and operation modes that will eventually optimize
cell expansion in a serial monolayer passaging process. For
this aim, the use of mathematical expressions to define the
growth curve of the cultures was found to be a valuable tool.
The optimization of chondroprogenitor cells expansion is
crucial in order to develop feasible large scale processes. To
our knowledge, this is the first reported work concerning a
mathematical approach to optimize the seeding density and
the passage length of a serial monolayer expansion process.
MATERIALS AND METHODS
Isolation of Chondroprogenitor Cells
Cartilage slices were harvested from the superficial zone of
articular cartilages of 2–3-week old bovine metatarsopha-
langeal joints by fine dissection. The slices were washed with
sterile 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) in serumfree DMEM(Sigma, UK) at
378C. After incubation the undigested cartilage fragments
were removed using a 70 mmFalcon filter (Becton Dickinson,
UK). The isolated cells were seeded at 4,000/mLonto 35 mm
plastic Petri-dishes (Iwaki, Japan) and incubated in serum
free DMEM at 378C for 20 min. These Petri-dishes were
previously coated with 10 mg/mL bovine fibronectin (Sigma,
UK) in PBS containing 1 mM MgCl
2
and 1 mM CaCl
2
overnight at 48C(Dowthwaite et al., 2004). After 20 min, the
media were removed from the Petri-dishes and discarded.
Tissue culture medium composed of DMEMþ (DMEM,
2 mM L-Glutamine, 1% Non-essential amino acids and
50 IU/mL penicillin/50 mg/mL streptomycin) supplemented
with 10%foetal calf serum(FCS; PAALaboratories, Austria)
was added to the remaining adherent cells which were
maintained in culture for up to 12 days. After 12 days in
cultures, the Petri-dishes were washed with PBS and
trypsinized (Trypsin–EDTA; Gibco, UK) until complete
cell detachment. Harvested chondroprogenitor cells were
then sub-cultured until different passage numbers by seeding
T-75 and T-150 tissue culture flasks (Iwaki, Japan) at a
density of 10
4
cells/cm
2
. Flasks were incubated at 378C in a
5% CO
2
atmosphere until sub-confluent and tissue culture
medium composed of DMEMþ supplemented with 10%
FCS changed every 2–3 days.
Cell Density and Viability
Chondroprogenitor cell cultures were washed with PBS and
trypsinized until complete cell detachment. Cells were then
Table I. Estimation of cell necessities for tissue engineering treatments.
Indication Incidence of surgical procedures Worldwide
Focal defects in articular cartilage 1.2 Â10
6
Osteoarthritis (OA of the knee) 3 Â10
7
Tissue engineering treatment Number of implants Cells required
a
Focal defects in articular cartilage
Typical human defect size (4 Â40 mm)
b
1 3.2 Â10
8
10% of focal defect incidences
c
1.2 Â10
5
3.9 Â10
13
Osteoarthritis
Typical human OA (2 Â4 Â40mm)
b
2 6.4 Â10
8
10% of OA defect incidences
c
3 Â10
6
1.9 Â10
15
a
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 a
haemocytometer. Viability was determined by the Trypan
blue exclusion method.
Growth Curve Fitted by Four-Coefficients
Gompertz Equation
Growth curves of chondroprogenitors in monolayer cultures
were characterized by a four coefficients generalization of
the Gompertz function, given by the following equation:
X ¼ X
0
þa Á exp Àexp
Àðt Àt
0
Þ
b
_ _ _ _
ð1Þ
Where t (h), corresponds to the culture time; X (cell/cm
2
),
corresponds to the value of viable cell density at any given
time t; X
0
(cell/cm
2
) corresponds to the initial value of viable
cell density; a (cell/cm
2
), corresponds to the difference
between the final value of viable cell density achieved at the
stationary phase of the culture (X
F
) and the initial value of
viable cell density (X
0
); t
0
(h), corresponds to the culture time
at which the increment of viable cell density is equal to 37%
of the final increment achieved at the stationary phase of the
culture; and b (h), corresponds to the culture time necessary
for the increment of viable cell density to pass from37 to 69%
of the final increment achieved at the stationary phase of the
culture. A schematic representation of (1) has been depicted
in Figure 1, where the qualitative meaning of each coefficient
in relation with the growth curve of a hypothetical culture has
been provided. For every growth curve analyzed, the
experimental data for viable cell density were acquired in
triplicates by the Trypan blue exclusion method. The
experimental data were fitted using a non-linear regression
algorithm provided with SigmaPlot (SigmaPlot 2002 for
Windows, Version 8.0). For each growth curve fitted, the
values of the equation coefficients with their respective
standard errors (SE), as well as the correlation coefficient (r
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 and
parameters related with the cost of the process (summarized
in Fig. 2) as follows.
Passage Parameters
Initial cell density, X
0
(cell/cm
2
), corresponds to the value of
viable cell density used to inoculate a single passage.
Final cell density, X
F
(cell/cm
2
), corresponds to the value
of final viable cell density observed at the time of harvesting
of a single passage.
Passage population doubling, n
p
(À), corresponds to the
number of doublings that cells undergo during a single
passage, from the inoculation to the time of harvesting.
Passage length, t
p
(h), corresponds to the duration of a
single passage, from inoculation to harvesting.
Passage expansion factor, E
p
(À), corresponds to the factor
by which the viable cell density is multiplied during a single
passage, from inoculation to harvesting.
Expansion Process Parameters
Initial cell number, I (cells), corresponds to the total viable
cell number available at the beginning of the expansion
process.
Final cell number, F (cells), corresponds to the total viable
cell number available at the end of the expansion process.
Final population doubling, n
f
(À), corresponds to the final
number of doublings that cells undergo during the expansion
process.
Total expansion time, T (h), corresponds to the duration of
the expansion process.
Total passage number, N
p
(À), corresponds to the total
number of passages that the cells undergo during the
expansion process.
Total expansion factor, E (À), correspond to the factor by
which the viable cell density is multiplied during the
expansion process.
I
Pi
(cell), corresponds to the total viable cell number
available at the beginning of the passage number i.
A
i
(cm
2
), corresponds to the surface area necessary to
inoculate the initial viable cell number (I
Pi
) at the beginning
of the passage number i.
Cost Parameters
C
MPA
($/cm
2
), correspond to the cost of culture medium per
unit of area necessary to carry out one single passage. For a
Figure 1. Chondroprogenitors growth curve fitted by four-parameters
Gompertz function. Schematic representation and qualitative meaning of a
four parameters generalization of the Gompertz function in relation with the
growth curve of a hypothetical culture of chondroprogenitor cells.
MELERO-MARTIN ET AL.: OPTIMAL EXPANSION OF CHONDROPROGENITOR CELLS 521
given culture condition(s), the concentration of medium
(expressed in mL/cm
2
) and the cost of the medium ($/mL)
can be considered as constants, independent of the passage
length. The value of C
MPA
($/cm
2
) depends directly on the
number of medium changes performed during the extent of
the passage. However, since all the cost analyses were done
for batch cultures the value of C
MPA
($/cm
2
) can be consi-
dered as a constant for each specific batch culture condition.
C
FPA
($/cm
2
), corresponds to the cost of tissue culture
flasks expressed per unit of area. For a given kind of tissue
culture flasks, this value can be considered as a constant,
independent of the passage length.
C
XPA
($/cm
2
), corresponds to the rest of the running cost of
any single passage expressed per unit of area (excluding the
cost considered in C
MPA
and C
FPA
). This cost includes the
cost of PBS buffer and trypsin-EDTA solution required for
cell harvesting, labor cost, and all the other operating costs.
By its own definition, it is clear that the value of this
parameter depends on the length of the passage. Never-
theless, to ease the analysis, this value was considered as a
constant. The reasons for such hypothesis are based on the
fact that some of the costs included in this parameter are
independent of the passage length (e.g., the cost of PBS
buffer and trypsin–EDTA solution required for cell harvest-
ing, and the labor cost attributed to the inoculation and cell
harvesting). Taking also into consideration that C
XPA
is
added to C
MPA
and C
FPA
on the overall passage running cost,
the hypothesis of considering C
XPA
as a constant is reason-
able.
C
PA
($/cm
2
), corresponds to the total running cost of any
single passage expressed per unit of area. Since all C
MPA
,
C
FPA
, and C
XPA
were considered as constants (for a given
culture conditions and under the hypotheses stated before),
the value of C
PA
was also considered as a constant.
C
Pi
($), corresponds to the total running cost of passage
number i.
C ($), corresponds to the total running cost of the
expansion process. This parameter can be calculated by
summing the cost of every single passage constituting the
expansion process.
x (À), corresponds to a dimensionless expression of the
total running cost of the expansion process, C.
Figure 2. Definitions and equations of passage and expansion process parameters. Passage definitions refer to those parameters in relation with the growth of
the cell population during a single monolayer passage. Expansion and cost definitions refer to those parameters in relation with the whole expansion process as a
sum of every monolayer passage.
522 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 93, NO. 3, FEBRUARY 20, 2006
Determination of Passage Length for
Optimal Cell Expansion
For a given initial cell number (I), the optimal passage length
(tp) for cell expansion was determined by maximizing the
final cell number (F) for a given total expansion time (T) as
follows:
MaxðFÞ ¼ Max I Á E
T=t
p
_ _
¼ Max
LnðE
p
Þ
t
p
_ _
ð2Þ
The term Max(z) refers to the value of t
p
that maximizes the
function z.
Determination of Passage Length for Optimal Cost
For a given initial cell number (I), the optimal passage length
(t
p
) for minimal process cost was determined by minimizing
the dimensionless cost (x) normalized to the total expansion
factor (E) for a given final cell number (F) as follows:
Min
x
E
_ _
¼ Min

n
f
=n
p
i¼1
E
iÀ1
p
E
n
f
=n
p
p
_
¸
¸
¸
_
_
¸
¸
¸
_
ð3Þ
The term Min(z) refers to the value of t
p
that minimizes the
function z.
Experimental Outline
A brief overview of the experiments performed is given
below.
Experiment 1: Effect of Seeding Density
6-well tissue culture plates (Iwaki, Japan) were seeded at five
different initial densities corresponding to 10
5
cells/cm
2
,
5 Â10
4
cells/cm
2
, 10
4
cells/cm
2
, 5 Â10
3
cells/cm
2
, and 10
3
cells/cm
2
(passage 2 chondroprogenitor cells). All the
cultures were supplied with 0.3 mL/cm
2
of DMEMþ
supplemented with 40% FCS and 1 ng/mL of transforming
growth factor beta 1 (TGF-b1; R&D Systems) (Martin et al.,
2005). Plates were kept at 378C in a 5% CO
2
atmosphere for
10 days. Viable cell densities were evaluated in triplicates at
intervals for each of the different seeding densities tested.
The values of viable cell density were fitted by a four
coefficients Gompertz equation and the values of passage
expansion factor (E
p
) and passage population doubling (n
p
)
calculated as functions of the passage length (t
p
). Addition-
ally, the values of the specific growth rates were calculated
for each of the different seeding densities tested by the
following equation:
m ¼
1
X
Á
dX
dt
%
2
ðX
2
þX
1
Þ
Á
ðX
2
ÀX
1
Þ
ðt
2
Àt
1
Þ
ð4Þ
Where m (/h), corresponds to the value of specific growth
rate at any given culture time; t (h), corresponds to the
culture time; and X (cell/cm
2
), corresponds to the value of
viable cell density at any given culture time.
Experiment 2: Reproducibility of Cell Density with
Passage Number
Chondroprogenitor cells were serially cultured in batch for 6
passages (from passage 2 to passage 7). For each passage, T-
25 tissue culture flasks (Iwaki, Japan) were seeded at 10
4
cells/cm
2
using 0.3 mL/cm
2
of DMEMþsupplemented with
40% FCS and 1 ng/mL of TGF-b1. Flasks were kept at 378C
in a 5%CO
2
atmosphere for 3 days. Viable cell densities were
evaluated in triplicates at day 3.
Experiment 3: Effect of Different Feeding Strategies
Three different culture conditions were evaluated in 6-well
tissue culture plates seeded at 10
4
cells/cm
2
(passage 2
chondroprogenitor cells). These culture conditions corre-
sponded to: (I) 0.3 mL/cm
2
of DMEMþ supplemented with
10% FCS and medium replaced every 48 h. These culture
conditions were referred to as ‘‘Standard Culture Condi-
tions,’’ (II) 0.3 mL/cm
2
of DMEMþsupplemented with 40%
FCS and medium replaced every 12 h. These culture
conditions were referred to as ‘‘High FCS Feeding Culture
Conditions,’’ and (III) 0.3 mL/cm
2
of DMEMþ supplemen-
ted with 40% FCS and 1 ng/mL of TGF-b1 in batch. These
culture conditions were referred to as ‘‘Optimized Batch
Culture Conditions’’ (Martin et al., 2005). Viable cell
densities were evaluated in triplicates at intervals for each
of these culture conditions. The values of viable cell density
were fitted by a four coefficients Gompertz equation and the
values of passage expansion factor (E
p
) and passage
population doubling (n
p
) calculated as functions of the
passage length (t
p
). Additionally, the values of the specific
growth rates were calculated. Finally, the optimal passage
length (t
p
) for cell expansion was determined for each of the
culture conditions investigated.
RESULTS
Effect of Initial Cell Density on Cell Proliferation
Fromthe results depicted in Figure 3 it was observed howthe
initial cell density played a central role in the proliferation
performance of the cultures, affecting both the kinetics of cell
growth and the final cell density achieved. These results were
expected since the proliferation of chondroprogenitor cells is
controlled by cell –cell contact inhibition (Martin et al.,
2005), mechanism that is essentially density-dependent
(Dietrich et al., 1997). Therefore, it could be expected that
higher seeding densities will necessarily lead to a more rapid
generation of inhibitory contacts and consequently to a more
rapid appearance of the stationary phase. While cultures with
seeding densities of 10
5
cell/cm
2
reached the stationary phase
at day 5, the cultures with seeding density of 10
4
cell/cm
2
required 8 days.
MELERO-MARTIN ET AL.: OPTIMAL EXPANSION OF CHONDROPROGENITOR CELLS 523
The experimental data obtained for the growth curve at
each of the seeding densities were fitted to a mathematical
function. This mathematical expression of the growth curve
provided a detailed analysis of the effects that the seeding
density has on both cell growth kinetics and expansion
process performance. A four coefficients Gompertz equation
(Fig. 1) was selected to fit the experimental data for cell
growth. The results are represented in Figure 3 and the values
of each parameter are summarized in Table II. The
experimentally obtained growth curves of chondroprogenitor
cells (data points in Fig. 3) were fitted with high accuracy by
the selected Gompertz function for each of the seeding
densities evaluated. The least-squares correlation coeffi-
cients were found to be above 0.99 for all cases (Table II).
Higher seeding densities corresponded to both shorter lag
phase (characterized by lower values of t
o
) and to sharper
growth curve (lower values of b). These differences in growth
kinetics were also observed by the evaluation of the specific
growth rate (Fig. 4A). Again, those cultures inoculated with
higher cell densities showed both shorter lag phase
(characterized in this case by the time at which the specific
growth rate abruptly raises) and lower maximum specific
growth rate (peaks in Fig. 4A). Although higher initial cell
densities produced faster growth kinetics attention should be
Figure 3. Effect of initial cell density on cell proliferation. 6-well tissue
culture plates were seeded at five different initial densities corresponding to
10
5
cells/cm
2
, 5 Â10
4
cells/cm
2
, 10
4
cells/cm
2
, 5 Â10
3
cells/cm
2
, and 10
3
cells/cm
2
(passage 2). All the cultures were supplied with 0.3 mL/cm
2
of
DMEMþsupplemented with 40%FCS and 1 ng/mL of TGF-b1. Viable cell
densities were evaluated at intervals for each of the different seeding
densities tested. Each data point represents the mean of three separate
cultures ÆSD. Data points fitted by Gompertz functions (lines).
Table II. Coefficients estimation for the Gompertz equation at different seeding density.
Seeding density
(cell/cm
2
)
Gompertz, four coefficients (value ÆSE)
X ¼ Xo þa Á exp Àexp
ÀðtÀtoÞ
b
_ _ _ _
X
0
(cell/cm
2
)
t
0
(h)
b
(h)
a
(cell/cm
2
) r
2
100,000 95,000 Æ7,000 46 Æ2 23 Æ2 358,000 Æ8,000 0.9979
50,000 48,000 Æ5,059 57 Æ1 27 Æ1 404,000 Æ6,000 0.9995
10,000 10,500 Æ2,600 83 Æ1 39 Æ1 304,000 Æ4,000 0.9994
5,000 5,000 Æ1,000 109 Æ1 39 Æ1 181,000 Æ2,000 0.9997
1,000 1,300 Æ1,000 155 Æ2 47 Æ3 135,000 Æ3,000 0.9994
Figure 4. Optimization of passage length at different initial cell densities.
6-well tissue culture plates were seeded at five different initial densities
corresponding to 10
5
cells/cm
2
, 5 Â10
4
cells/cm
2
, 10
4
cells/cm
2
, 5 Â10
3
cells/cm
2
, and 10
3
cells/cm
2
(passage 2). All the cultures were supplied with
0.3 mL/cm
2
of DMEMþsupplemented with 40%FCS and 1 ng/mLof TGF-
b1. Viable cell densities were evaluated in triplicates at intervals and the data
points fitted by Gompertz functions (Fig. 3). A: Specific growth rate
expressed as a function of the passage length. B: Optimal passage length was
determined by the maximum of (Ln (E
p
)/t
p
).
524 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 93, NO. 3, FEBRUARY 20, 2006
also plaid to the expansion factor achieved for each seeding
density. The expansion factors (calculated as 1þa/X
0
; see
Fig. 1) achieved for each of the cultures investigated cor-
responded to 5, 9, 30, 36, and 103 as the initial cell density
was increased (10
5
, 5 Â10
4
, 10
4
, 5 Â10
3
, and 10
3
cell/cm
2
respectively). Consequently; while higher initial cell den-
sities produce faster growth kinetics, it also imposed a lower
value of the expansion factor achievable in a single passage.
Determination of Seeding Density and Passage
Length for Optimal Cell Expansion
Two questions were considered essential before proceeding
with the establishment of an optimal culture condition for
monolayer cell expansion. Firstly, it was necessary to
determine the seeding density for optimal cell expansion.
Secondly, for any selected seeding density, it was necessary
to establish the value of the passage length (t
p
) that makes the
expansion process optimal in a serial operation.
This optimization implied the maximization of the final
cell number (F) for a given initial cell number (I) and a total
expansion time (T). The optimal passage length (t
p
) was
found by plotting Ln(E
p
)/t
p
against t
p
and determining the
maximum of the resulting curve. The results of this analysis
for each of the seeding densities investigated are depicted in
Figure 4B. It was observed that the optimal seeding density
corresponded with 10
4
cell/cm
2
as reflected by the maximum
of the curves. In addition, it was found that the optimal
passage length for this seeding density corresponded to 73 h
(time at which the maximum of the curve was reached). The
selection of the adequate passage length was considered
crucial. While longer passage lengths will lead to higher
expansion factors in a single passage (see growth curves in
Fig. 3), it would be detrimental in a serial operation process as
it would impose longer total expansion time (or lower final
expansion factor if the process time is constant). Conse-
quently, although each seeding density was associated with
an optimal value of passage length, 10
4
cell/cm
2
for seeding
density with 73 h of passage length were found to be the
optimal conditions overall for cell expansion.
Determination of Passage Length for Optimal Cost
Independent of the seeding density, the selection of the
passage length will have a direct influence on the final cost of
the expansion process. Although the optimal passage length
will always give the desired expansion factor in a minimal
process time, longer values of the passage length could
eventually create a situation where the same desired
expansion factor is achieved with a lower total process cost
(in detriment of the process time) by reducing for example the
number of passage required. Therefore, it would be
interesting to analyze the effect that the passage length has
on the expansion cost. The approach followed and the
hypotheses considered are described in Materials and
Methods where a dimensionless expression of the total
expansion cost (x) is defined to ease the analysis.
The optimal passage length (t
p
) for a given initial and final
cell numbers (I and F) was found by plotting x/E against t
p
and determining the minimum of the resulting curves
(Fig. 5D). The results observed agreed with our previous
expectations. The longer the passage length selected for the
expansion process, the lower costing required for the
achievement of a desired final cell number. Interestingly,
the results depicted in Figure 5D were found to be
independent of the final population doubling considered
(n
f
), making this chart useful for estimating running cost for
any final expansion factor desired. According to this
estimation, longer passage length produced a progressive
reduction of the process cost (see the perceptual reductions
illustrated in Fig. 5D). Consequently, the final decision
should necessarily be a compromise between proliferation
performance and costing. From the costing point of view a
value around 120 h seems more reasonable (an increase in the
length of each passage from 73 to 120 h will produce a
62.81% reduction in process cost), but this will be in the
expense of longer process time and will not nevertheless
change the optimal seeding density of the cultures (Fig. 4B).
Effect of Passage Length on Expansion
Process Performance
To further illustrate the consequences of the passage length, its
effects on several process parameters (such as final population
doubling, n
f
, total expansion time, T, total passage number, N
p
,
and total expansion factor, E) were quantified. The results of
these quantifications are depicted in Figure 5. These charts can
be used as a tool for the estimation of the expansion process
characteristics of chondroprogenitor monolayer cultures.
There are several ways to estimate these process
parameters from a practical point of view. One way of
analysis could be the consideration of a desired final
expansion factor (E) as the starting point. Assuming that
the value of the passage length (t
p
) has been already selected
by costing considerations (for instance 120 h), then the total
number of passages required (N
p
) could be estimated from
Figure 5B. In addition, the total expansion time (T) and the
final population doubling (n
f
) corresponding to the desired
total expansion factor (E) could be determined by using
Figures 5A and C, respectively. Another situation could be
the consideration of a certain total expansion time (T) as the
starting point. In this occasion, the final population doubling
(n
f
) could also be estimated fromFigure 5A. Additionally, the
total passage number (N
p
) and the final expansion factor (E)
imposed by the selected expansion time (T) could be
determined by using Figure 5C and B, respectively.
Independent of the analysis performed, the value of the
dimensionless process cost (x) corresponding to any given
value of the total expansion factor (E) can be obtained from
Figure 5D. Finally, the running cost of the expansion process
(C; expressedin $) can be calculated fromthe definitionof the
dimensionless process cost (Fig. 2), although this calculation
would require first the knowledge of C
PA
(cost of any single
passage expressed in $/cm
2
).
MELERO-MARTIN ET AL.: OPTIMAL EXPANSION OF CHONDROPROGENITOR CELLS 525
Although the qualitative interpretations of the results
presented are considered valid, from a quantitative point of
view, the accuracy of these results necessarily relies on the
reproducibility of the viable cell numbers during a serial
passaging process. To evaluate this reproducibility, chon-
droprogenitor cells were passaged under the same conditions
(batch cultures seeded at 10
4
cells/cm
2
using 0.3 mL/cm
2
of
DMEMþsupplemented with 40%FCSand 1 ng/mLof TGF-
b1) and the value of viable cell density evaluated at day three
of each passage (Fig. 6). The results depicted in Figure 6A
showed that the viable cell density observed after 3 days of
culture for each passage (frompassage 2–7) were reasonably
reproducible. Although some variations were accounted,
these discrepancies were attributed to the actual time at
which the cultures were harvested. Observing the pattern of
the growth curve corresponding to the culture conditions
used (Fig. 6B), and taking out a variation of Æ6 h from the
selected passage length of 72 h, it can be observed that the
values of viable cell density for all the passages evaluated
were in the expected range (Fig. 6A). This reproducibility of
the growth behavior of chondroprogenitor cells with passage
number was found consistent with unpublished results
provided by Smith and Nephew (Smith and Nephew
Research Centre, York). Although the culture conditions
used in their study differed from ours, they observed how
chondroprogenitor cells have similar growth rates when
maintained for 28 passages and only after 35 passages cell
growth gradually decline.
Feeding Strategies: Comparison of Three Cases
Medium composition for improved performance in batch
operation was already established in previous work (Martin
et al., 2005), where the proliferation enhancement produced
by the use of 40% FCS and 0.3 ng/cm
2
TGF-b1 was evident.
However, batch cultures of chondroprogenitor cells supplied
at 0.3 mL/cm
2
were still partially controlled by substrate
limitation. To review whether another feeding operation
Figure 5. Effect of passage length on process and cost parameters. Total expansion time (A), total expansion factor (B), total passage number (C), and
normalized dimensionless expansion cost (D) were evaluated for different values of final population doubling (n
f
) as a function of passage length (t
p
). Cultures
conditions corresponded to 6-well tissue culture plates seeded at 10
4
cells/cm
2
(passage 2) using 0.3 mL/cm
2
of DMEMþ, 40% FCS and 1 ng/mL of TGF-b1.
For the calculations, viable cell densities were previously evaluated in triplicates at intervals and data points fitted by Gompertz functions (Fig. 3).
526 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 93, NO. 3, FEBRUARY 20, 2006
mode could be more beneficial than the batch operation mode
three culture conditions (or operation modes) were selected
corresponding to (I) Standard Culture Conditions, (II) High
FCS Feeding Culture Conditions, and (III) Optimized Batch
Culture Conditions. The growth curves for each of these
culture conditions are depicted in Figure 7. As it was done
previously for the analysis of different seeding densities, the
experimental data obtained for the growth curves were fitted
to a four coefficients Gompertz function (Fig. 7), and the
values of each coefficient are summarized in Table III.
Important differences in growth kinetics were found
between the high FCS feeding condition (II) and the
optimized batch condition (III), with the latter presenting
both shorter lag phase (characterized by lower values of t
o
)
and sharper growth curve (lower values of b). These
differences in growth kinetics were also observed by the
evaluation of the specific growth rates (Fig. 8B). Again, it was
observed how the optimized batch condition (III) presented
the shortest lag phase (characterized in this case by the time at
which the specific growth rate abruptly raises). However, this
batch operation mode (III) also presented a lower maximum
of specific growth rate (Fig. 8B) than the high FCS feeding
one (II). Finally, both the poor growth kinetics and the
relatively long lag phase observed with the standard culture
conditions (I) were expected due to the presence of a severe
nutrient limitation condition.
Additionally, the passage expansion factors (calculated as
1þa/X
0
; see Fig. 1) achievable for each of the operation
modes investigated corresponded to 10, 60, and 30 for I, II,
and III respectively. Consequently, despite the batch mode
(III) showed a shorter lag phase, it also had a lower value of
the expansion factor achievable in a single passage than that
Figure 6. Reproducibility of cell density with passage number. Chon-
droprogenitor cells were serially culture in batch for six passages. For each
passage, T-25 tissue culture flasks were seeded at 10
4
cells/cm
2
using 0.3 mL/
cm
2
of DMEMþ supplemented with 40% FCS and 1 ng/mL of TGF-b1.
Viable cell densities were evaluated at day 3 of each passage (A). Each bar
represents the mean of three separate cultures ÆSD. B: represents the viable
cell density expected at 72 Æ6 h from the Gompertz function calculated for
the seeding density used.
Figure 7. Comparison of growth curves for different feeding strategies.
Cultures were plated at 10
4
cells/cm
2
using 0.3 mL/cm
2
of medium. Three
different culture conditions were compared corresponding to medium
composed of DMEMþsupplemented with (I) 10% FCS, (II) 40% FCS, and
(III) 40%FCSþ1ng/mLof TGF-b1. Mediumreplacements were carried out
every (I) 48 h, (II) 12 h, or (III) no mediumreplacement. Viable cell densities
were evaluated at intervals. Each data point represents the mean of three
separate cultures ÆSD. Data points fitted by Gompertz functions (lines).
Table III. Coefficients estimation for the Gompertz functions at different feeding.
Culture conditions
Gompertz, four coefficients (value ÆSE)
X ¼ Xo þa Á exp Àexp
ÀðtÀtoÞ
b
_ _ _ _
X
0
(cell/cm
2
) to (h) b (h) a (cell/cm
2
) r
2
I—standard (10% FCS, 48 h) 10,600 Æ2,600 86 Æ5 40 Æ8 97,000 Æ10,000 0.9947
II—high FCS feeding (40% FCS, 12 h) 9,500 Æ5,500 105 Æ1 41 Æ2 560,000 Æ13,000 0.9978
III—optimized batch(40%FCS, TGF-b1, batch) 10,500 Æ2,600 83 Æ1 39 Æ1 304,000 Æ4000 0.9994
MELERO-MARTIN ET AL.: OPTIMAL EXPANSION OF CHONDROPROGENITOR CELLS 527
of the high FCS feeding condition (II). Since in a serial
passaging operation, a higher value of passage expansion
factor could compensate for the detrimental effect of a longer
lag phase, it was clear again that the determination of an
optimal operation mode necessarily involve a compromise
between growth kinetics (time) and expansion factor
achievable. The optimization approach followed implied
the maximization of the final cell number (F) achievable for a
given total expansion time (T). The optimal passage length
(t
p
) was found by plotting (Ln(E
p
)/t
p
) against t
p
and
determining the maximum of the resulting curve. The results
of this analysis for each of the three culture conditions
investigated are depicted in Figure 8A. Interestingly, both the
optimized batch operation (III) and the high FCS feeding
operation (II) presented similar optimal proliferation perfor-
mance (maximum of their respective curves in Fig. 8A). The
detrimental effect produced by the lower passage expansion
factor observed in the batch operation mode (III) was
compensated by the presence of a shorter lag phase through
the selection of a shorter passages length (t
p
¼73 h). On the
contrary, in the high FCS feeding operation (II), the detri-
mental effect produced by its longer lag phase was com-
pensated by its higher passage expansion factor, in this case
through the selection of a longer passages length (t
p
¼96 h).
To quantify these findings, calculations concerning both
passage and expansion process performance for each of the
operation modes (I, II, and III) were carried out (Table IV).
These calculations were done for a target value of final
population doubling (n
f
) between 28 and 32 doublings, and a
starting initial cell number of one million cells. In Table IV, it
can be observed for instance how the high FCS feeding
operation (II) requires seven passages (each of themof 95 h),
while the batch operation (III) needs nine passages (each of
them of 72 h). However, both operation modes results in the
final desired cell number in a similar process time of 28 and
27 days. Apart from these similarities in cell expansion
performance, an equally interesting comparison resides in
the analysis of the consumption values related to each
operation mode. These consumption values (culture area,
DMEMþ, FCS, and TGF-b1) were expressed per unit of final
cell achieved to deal with the fact that the calculations for
each operation mode corresponded to slightly different final
population doubling. Comparing to the optimized batch
operation (III), the high FCS feeding condition (II) presents a
438% increase in media consumption per unit of cell (both
DMEMþ and FCS) and a 48% reduction in surface area
required (as expected from the less number of passages).
Consequently, in the feeding operation mode, while the
culture area is used by the cells more efficiently, the media is
used much less efficiently. These differences in consumables
are obviously translated into differences in process cost.
Although the final confirmation will require the real
quantification of the costs (consumable prices, evaluation
of the cost impose by the use of TGF-b1, the labor and
operational costs associated with the feeding, etc.) it seems
that the optimized batch operation results in a more
economical option as an operation mode for monolayer
cultures of chondroprogenitor cells. In addition, Table IV
proves that an increase in the value of passage length from73
to 120 h (culture conditions III and IV in Table IV) would
significantly reduce the process cost, although this increase
also imposes a higher value of the total expansion time
required (from 27 to 35 days).
Monolayer Expansion Process Summary
A quantification of the different passage and process para-
meters involved in a six passages operation was estimated.
The selected culture conditions corresponded to 10
4
cell/cm
2
as the seeding density, 0.3 mL/cm
2
as the medium concen-
tration, DMEMþsupplemented with 40%FCS, and 1 ng/mL
of TGF-b1 as the medium composition, 120 h as the passage
length and batch as the operation mode. Figure 9 summarizes
Figure 8. Optimization of passage length for different feeding strategies.
Cultures were plated at 10
4
cells/cm
2
using 0.3 mL/cm
2
of medium. Three
different culture conditions were compared corresponding to medium
composed of DMEMþsupplemented with (I) 10% FCS, (II) 40% FCS, and
(III) 40%FCSþ1ng/mLof TGF-b1. Mediumreplacements were carriedout
every (I) 48 h, (II) 12 h, or (III) no medium replacement respectively. Viable
cell densities were evaluated in triplicates at intervals and the data points
fitted by Gompertz functions (Fig. 7). A: Optimal passage length was
determined by the maximum of (Ln (E
p
)/t
p
). B: Specific growth rate
expressed as a function of the passage length.
528 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 93, NO. 3, FEBRUARY 20, 2006
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MELERO-MARTIN ET AL.: OPTIMAL EXPANSION OF CHONDROPROGENITOR CELLS 529
the results of this evaluation for a six passages operation. In
term of cell expansion, starting with one million cells, the
proposed culture conditions would lead to a final cell number
of 1.1 Â10
14
cells (which represents 1.1 Â10
8
fold expan-
sion) in just 30 days of operation. Despite this outstanding
expansion factor, attention should also be played to the
requirements that each passage would impose in terms of
surface area (5.4 Â10
8
cm
2
) and medium consumption
(1.6 Â10
8
mL). Large scale monolayer systems would be
required to feasibly cope with this scale of operation.
DISCUSSION
Among all the process parameters affecting the final expan-
sion performance, special attention was given to the selection
of both an optimal seeding density and passage length and
their influence on the proliferation kinetics of the cells.
Mathematical expressions of the growth curves provided a
detailed analysis of the effects that the seeding density has on
both cell growth kinetics and expansion process perfor-
mance. Despite most authors still use the exponential growth
approach to characterize the behavior of proliferative cells in
culture, this assumption was not considered accurate for
chondroprogenitor cells. The concept of exponential growth
by mammalian cells in culture is based upon the apparent
linearity of semi logarithmic data plots. With exponential
growth, data points should fall on a horizontal straight line
when specific growth rate is plotted against time. However,
the majority of the cell lines exhibits non-exponential growth
patterns (Skehan and Friedman, 1984). The kinetics of
chondroprogenitors consisted of an initial period of growth
acceleration followed by a later phase of deceleratory
growth, and therefore their growth in culture is predomi-
nantly non-exponential. The kinetics of decelerating growth
can be described by diverse equations such as Gompertz,
logistic, inverse cube root and power functions (Buchanan
et al., 1997; Deakin, 1970; Skehan and Friedman, 1982). In
particular, Gompertz functions have been proposed to model
the growth of a diversity of biological systems, including
tumors (Heegaard et al., 2003; Lloyd, 1975), human breast
cancer (Spratt et al., 1993), rat glial tumor cell lines (Skehan
and Friedman, 1982), colon and lung carcinoma cell lines
(Castro et al., 2003), insect cell lines (Marteijn et al., 2000),
bacterial systems (Buchanan et al., 1997; Kayombo et al.,
2003), and algae systems (Kayombo et al., 2003). One of
these functions, a four coefficients Gompertz equation, was
found to fit the experimental data for chondroprogenitor cell
growth with high accuracy.
Two questions were considered essential before proceed-
ing with the establishment of an optimal culture condition for
monolayer systems. Firstly, it was necessary to determine the
seeding density for optimal cell expansion. Secondly, for any
selected seeding density, it was necessary to establish the
value of the passage length that makes the expansion process
optimal in a serial operation. Since chondroprogenitor cells
have the ability to form colonies and proliferate at very low
densities (Dowthwaite et al., 2004), it could be tempting to
inoculate the cultures at lowdensities as a way to enhance the
Figure 9. Monolayer expansion process outline. Summary of the passage and process parameters estimation for a serial passaging expansion of
chondroprogenitor cells in monolayer culture (six passages). Culture conditions corresponded to batch cultures seeded at 10
4
cells/cm
2
using 0.3 mL/cm
2
of
DMEMþ supplemented with 40% FCS and 1 ng/mL of TGF-b1. Passage length was selected at 120 h.
530 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 93, NO. 3, FEBRUARY 20, 2006
expansion factors achievable in a single passage. However
we have shown how in addition to this expansion enhance-
ment, seeding density significantly affects the growth
kinetics of chondroprogenitor cells. Although lower seeding
densities obviously led to superior multiplication of the cells
by the end of a single passage, the slower growth kinetics
imposed by low seeding densities significantly increased the
time necessary to achieve the desired expansion factor. The
determination of an optimal seeding density is a compromise
between expansion factor achievable and growth kinetics,
which also imposes a significant role for the length of each
monolayer passage. The determination of the optimal
seeding density and the corresponding optimal passage
length was carried out using a mathematical approach, and
the value of 10
4
cell/cm
2
for seeding density with 73 h of
passage length were found to be the optimal conditions for
cell expansion in a serial passaging operation.
Although this optimal value of initial cell density (10
4
cell/
cm
2
) was found similar to those frequently reported for
chondrocyte monolayer cultures elsewhere, very few reports
mention the reasons for the selection of certain seeding
density or the influence that such selection would have on the
optimization of the expansion process. One study concerning
the effect of seeding density on the expansion of articular
chondrocytes has been recently reported (Mandl et al., 2004).
The authors suggested that expansion culture of chondro-
cytes with low densities is preferable based on the fact that
high seeding densities require more passages and more time
to produce sufficient amount of cells. Moreover, despite low
densities can be detrimental for the dedifferentiation capacity
of articular chondrocytes (Watt, 1988), Mandl et al. (2004)
claimed that ‘‘enough’’ expansion (defined as the number of
cells necessary for autologous treatment of a typical size
defect) can be achieved using low seeding densities without
loosing the ability of the cells to redifferentiate. However,
comparing with the analysis presented in this paper, Mandl’s
study was found incomplete for our purpose. As we have
shown, higher seeding densities impose lower expansion
factors when considering a single passage, but the key to
optimize the expansion process in a serial passaging
operation is the selection of a suitable passage length for
each seeding density, even though this would eventually
mean more passages. Also ‘‘sufficient expansion’’ (Mandl
et al., 2004) for autologous treatments cannot be considered
appropriate in allogeneic applications, where intensive
expansion processes will be required for the generation of
large cell banks and/or tissue engineering constructs. In
addition, the issue of phenotype preservation with chondro-
progenitors is less sensitive than with adult differentiated
chondrocytes. Chondroprogenitor cells ability to synthesize
hyaline-like cartilage after extensive expansion have been
already reported under both standard culture conditions and
high FCS concentrations in the presence of TGF-b1
(Dowthwaite et al., 2004; Martin et al., 2005).
The analysis of both seeding density and passage length
was considered crucial in the optimization of expansion
processes, and their correct selection should be taken as an
essential requisite to establish culture conditions for mono-
layer systems. To our knowledge, a ‘‘gold standard’’ for the
multiplication of either chondrocytes or chondroprogenitor
cells in monolayer, with respect to seeding density and
passaging, is lacking, and the results presented in this paper
constitute the first reported work concerning a mathematical
approach to optimize the seeding density and the passage
length of a serial monolayer expansion process.
Additional considerations concerningthe costs of the process
were introduced in this paper. Independent of the seed-
ing density, we have shown how the selection of the passage
length had a direct influence on the final cost of the
expansion process. Although the optimal passage length will
always give the desired expansion factor in a minimum
process time, longer values of the passage length could
eventually create a situation where the same desired
expansion factor is achieved with a lower total process cost
by reducing the total number of passages required. This
analysis led to the selection of an alternative optimal value of
120 h for the length of each passage. Although this value will
lead to a suboptimal proliferation criteria and process time,
the running cost of the process will nevertheless be reduced
by more than 60%.
Despite the proliferation enhancement produced by the use
of 40% FCS and 1 ng/mL of TGF-b1, batch cultures of
chondroprogenitor cells supplied at 0.3 mL/cm
2
are still
partially controlled by substrate limitation (Martin et al.,
2005). To overcome the presence of substrate limitation the
culture medium was supplemented with 40% FCS and
medium replenished every 12 h (referred to as feeding
operation mode). At this condition, a threshold value of
5.3 Â10
5
cell/cm
2
was achieved after 10 days (Martin et al.,
2005). Comparing the proliferative behavior of the cultures,
the batch operation mode presented a less pronounced lag
phase and consequently required shorter passage lengths and
imposed higher passage numbers during the overall expan-
sion process. On the other hand, the feeding operation mode
required longer passage lengths to achieve similar expansion
factor and consequently less overall passage numbers. These
results may appear unexpected at first examination, since it
could be expected that a high FCS feeding condition should
impose both better growth kinetics and shorter lag phase.
While the reason for a higher maximum specific growth rate
could be that no substrate limitation was present under those
culture conditions, the reason for a longer lag phase was less
evident. An explanation could reside in the fact that medium
was refreshed very often. This operation mode could
potentially lead to a ‘‘continuous’’ elimination of relevant
enzymes that may be produced during the lag phase of the
culture (Bailey and Ollis, 1986), making the concentration of
such enzymes suboptimal. Nevertheless, we have shown in
this paper how both operation modes have similar expansion
potential (in terms of cells achieved in a given time) if
suitable passage lengths are selected (73 and 95 h for the
batch and the feeding operation, respectively).
Despite these similarities in expansion capabilities, the
analysis of the operational costs associated with each
MELERO-MARTIN ET AL.: OPTIMAL EXPANSION OF CHONDROPROGENITOR CELLS 531
operation mode led to the recommendation of the optimized
batch operation mode as the preferred culture condition for
cell expansion. However, additional investigations are
recommended since they could lead to further improvements.
For instance, from a therapeutic application perspective, it
can be envisaged that the ideal culture conditions should be
either at a reduced-serum or serum-free condition. Addition-
ally, a feeding operation mode involving the use of TGF-b1
could reduce both the substrate limitation identified in the
batch operation and the high FCS demand of the feeding
operation. As a result, optimization of the medium formula-
tion would improve the performance of batch operations,
allowing better growth kinetics and overcoming any possible
substrate limitation situation. Although these potential
improvement of media formulation would impose different
growth kinetics than the one reported in this paper, the
approach presented to optimize the seeding density and the
passage length would be still valid.
The implications that the preferred culture conditions
would have in a large scale operation were estimated by the
quantification of the different passage and process para-
meters involved. If large scale monolayer expansion of
chondroprogenitor cells is finally considered, it would be first
necessary to determine what kind of culture systems can cope
with the desired scale of operation. There are currently
available several alternatives of large scale culture vessels
and systems from different manufacturers. One of these
alternatives is based on the roller bottle technology. The
RollerCell 40 (CELLON S.A., Luxembourg) is a self-
contained, automated roller bottle processing system, and
its use has been recently reportedfor large scale production of
retroviral vectors (Wikstrom et al., 2004). Up to ten
RollerCell 40 units can be linked together in series with a
single CPU (RollerCell Max; CELLON S.A., Luxembourg),
providing a surface area of 3.5Â10
5
cm
2
(equivalent to 200
roller bottles of 1,750 cm
2
each). This kind of system could
cope for instancewith the surface requirements estimated in a
six passages operation for chondroprogenitor cells by the use
of 1, 22, and 484 RollerCell Max units during the fourth, fifth
and sixth passage respectively. An alternative to roller bottles
is the Corning CellCube System (Corning Incorporated,
USA), an integrated modular bioreactor systemthat has been
successfully used in a variety of large scale culture
applications (Aunins et al., 2003; Kotani et al., 1994; Merten
et al., 2001; Ozuer et al., 2002; Wikstrom et al., 2004). This
systemallows continuous perfusion of fresh media and can be
scaled up to 3.4Â10
5
cm
2
of growth surface using the same
control package. Again, 1, 22, and 484 CellCube units would
be necessary during the fourth, fifth and sixth passage
respectively. Finally, the choice of static systems may
be beneficial if high rates of medium replenishment are
required. In this case, multitray battery systems would
be recommended. This system is designed for large scale
operations, and its use has been reported in a variety of
culture processes, including the production of human
fibroblast interferon (Pakos and Johansson, 1985; Utsumi
et al., 1984). Two commercially multitray systems are
available: the Corning CellSTACK (Corning Incorporated,
USA) and the Nunclon
TM
D Cell Factory (Nunc, Denmark).
Both products allow assembling up to 40 trays, providing a
surface area of 2.5 Â10
4
cm
2
. Consequently, in this occasion
13, 282, and 6,195 multitray units would be necessary during
the fourth, fifth, and sixth passage respectively. The intro-
duction of these large scale monolayer systems may impose
different growth kinetics than the one reported in this paper,
and it would be therefore necessary to estimate the detailed
growth curves for each specific operation mode. However,
the approach presented to optimise the seeding density and
the passage length would be still valid once the growth
kinetics associated with each condition are determined.
Nevertheless, all these large scale monolayer culture
vessels and systems present inherent limitations in term of
scalability due to their lowratios of surface to volume, which
could finally compromise the suitability of these systems in
very large scale operation. Current work is being focused on
the analysis of alternative expansion systems based on
microcarrier cultures and how the model presented in this
manuscript would have to be modified for microcarrier cell
expansion of chondroprogenitor cells. These systems could
potentially provide the improved ratio of surface to volume
necessary to cope with the scale of chondroprogenitors
expansion require for allogeneic tissue engineering applica-
tions. Finally, it should be noted that the data used in this
manuscript was based on bovine chondroprogenitor cells.
Current work at Smith and Nephew (Smith and Nephew
Research Centre, York, UK) is evaluating whether the human
equivalent would have similar in vitro growth kinetics,
nutritional requirements and culture conditions. These
potential differences in growth kinetics will not invalid
however the approach presented in this manuscript to
optimise the seeding density and the passage length.
JMMMis funded by BBSRCIndustrial Co-operative Award in Science
and Engineering.
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