Journal of Immune Based Therapies
and Vaccines

BioMed Central

Original research

Open AccessA new approach for the large-scale generation of mature dendritic
cells from adherent PBMC using roller bottle technology
Ryan E Campbell-Anson1, Diane Kentor1, Yi J Wang1, Kathryn M Bushnell1,
Yufeng Li1, Luis M Vence1 and Laszlo G Radvanyi*1,2
Address: 1Department of Melanoma Medical Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, 77030, USA and
2Department of Breast Medical Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, 77030, USA
Email: Ryan E Campbell-Anson - recampbe@mdanderson.org; Diane Kentor - dhkentor@mdanderson.org;
Yi J Wang - yjwang@mdanderson.org; Kathryn M Bushnell - kbushne@mdanderson.org; Yufeng Li - yufenli@mdanderson.org;
Luis M Vence - lmvence@mdanderson.org; Laszlo G Radvanyi* - lradvanyi@mdanderson.org* Corresponding author

Published: 6 March 2008
Journal of Immune Based Therapies and Vaccines 2008, 6:1

doi:10.1186/1476-8518-6-1

Received: 15 November 2007
Accepted: 6 March 2008

This article is available from: http://www.jibtherapies.com/content/6/1/1© 2008 Campbell-Anson et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Human monocyte-derived DC (mDC) loaded with peptides, protein, tumor celllysates, or tumor cell RNA, are being tested as vaccines against multiple human malignancies andviral infection with great promise. One of the factors that has limited more widespread use of thesevaccines is the need to generate mDC in large scale. Current methods for the large-scale cultivationof mDC in static culture vessels are labor- and time- intensive, and also require many culture
vessels. Here, we describe a new method for the large-scale generation of human mDC fromhuman PBMC from leukopheresis or buffy coat products using roller bottles, never attempted
before for mDC generation. We have tested this technology using 850 cm2 roller bottles compared
to conventional T-175 flat-bottom static culture flasks.
Methods: DC were generated from adherent human PBMC from buffy coats or leukopherisis
products using GM-CSF and IL-4 in T-175 static flasks or 850 cm2 roller bottles. The cells werematured over two days, harvested and analyzed for cell yield and mature DC phenotype by flow
cytometry, and then functionally analyzed for their ability to activate allogeneic T-cell or recall
antigen peptide-specific T-cell responses.
Results: Monocytes were found to adhere inside roller bottles to the same extent as in static
culture flasks. The phenotype and function of the mDC harvested after maturation from both typeof culture systems were similar. The yield of mDC from input PBMC in the roller bottle system
was similar as in the static flask system. However, each 850 cm2 roller bottle could be seeded with
4–5 times more input PBMC and could yield 4–5 times as many mDC per culture vessel than thestatic flasks as a result.
Conclusion: Our results indicate that the roller bottle technology can generate similar numbersof mDC from adherent PBMC as traditional static flask methods, but with having to use fewer
culture vessels. Thus, this may be a more practical method to generate mDC in large-scale cutting
down on the amount of laboratory manipulations, and can save both time and labor costs.

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Background
Dendritic cells (DC) are the most potent antigen-presenting cells (APC) in the immune system that are the key cells
activating T-cell-based immune responses against viraldisease and cancer [1]. Recently, this powerful ability of
DC is being tested as an active vaccine approach to treat
cancer and viral infections such as HIV and CMV [2,3].
Most of these studies use monocyte-derived DC (mDC)
loaded with antigen in vitro and then injected subcutaneously or intravenously [4]. The most commonly usedmethod to generate mDC is to adhere monocytes on toplastic in static flasks from PBMC followed by culture with
GM-CSF and IL-4 and maturation using any one of anumber of cocktails of pro-inflammatory cytokines (IL1β, TNF-α, IL-6) or Toll-like receptor (TLR) agonists suchas LPS [1,2]. Antigen-loaded DC vaccines have been tested
in multiple malignancies, including melanoma, breast
cancer, prostate cancer, renal cancer, and follicular lymphoma, where they have been found to consistently
induce antigen-specific CD4+ and CD8+ T-cell responses
along with some reported clinical response [5-7].
The production of DC vaccines requires the cultivation of
millions of clinical-grade mDC in large-scale. In somecases more than a billion mDC may be required to ensurethat enough vaccine can be produced for multiple patient
immunizations over a number of months. Vaccination
regimens using antigen-pulsed mDC have ranged frommultiple 10–20 × 106 mDC to up to 100 × 106 or more
mDC injected subcutaneously or intravenously, respectively [8,9]. Monocytes differentiate directly into DC in
these cultures and do not divide and, as a result, leukopheresis products containing billions of PBMC are
required as starting material to have enough monoytes
available for the procedure. Current methods for largescale cultivation of mDC in static culture systems can becumbersome, labor- and time- intensive, and require
many repetitive culture vessels or multi-layered systems[10-13]. The numerous manipulations required to set-up
most static culture flasks for large-scale mDC generation
also increases the chances for product variability from culture to culture and sterility being compromised. Although
non-adherent cell culture systems of isolated CD14+
monocytes have been introduced, there is still somedebate on the quality of these mDC versus those derived
from adherent populations. For example, some studies ofhave found decreased yields of mature CD83+ mDC or
reduced IL-12 production capability versus adherent systems [14,15]. Thus, any improvements in the speed andease of generating DC from adherent monocytes in large
scale and better purity for clinical use would be a great
asset.
We describe a novel method of generating mature mDC inlarge-scale using roller bottle culture technology never

http://www.jibtherapies.com/content/6/1/1

before reported to be used to generate DC before. The
monocytes from the peripheral blood mononuclear cell
(PBMC) or leukopheresis preparations were adhered tothe inside surface of roller bottles on a roller apparatus at
low speed. After removal of the non-adherent cells, DC
cells are generated using culture medium containing GMCSF and IL-4 and matured using any one of a number ofwell-defined defined cytokine cocktails. This resulted in alarge number of floating non-adherent mature DC thatcan be easily harvested and used for vaccines or other purposes. The roller bottle DC had similar phenotypic andfunctional characteristics as those produced in static culture flasks. Overall, the roller bottle system is a self-contained system requiring minimal manipulation during
culture set-up. The result is faster culture set-up times andless labor for lab personnel than traditional static culture
methods in flat-bottom culture flasks.

Methods
Reagents and equipment
Human recombinant cytokines (GM-CSF, IL-4, IL-1β,
TNF-α, and IL-6) were purchased from R&D Systems(Minneapolis, MN). Prostaglandin E2 (PGE2) was purchased from Sigma-Aldrich (St. Louis, MO). Dendritic cell
culture medium (DC-CM) consisted of Iscove's Modified
Dulbecco's Medium (IMDM) containing Glutamax, 20µg/ml gentamycin, 50 µM 2-mercaptoethanol (all from
Invitrogen, Carlsbad, CA), and 2% normal human AB
serum (Valley Biomedical, Winchester, VA). Roller bottles(850 cm2 or 490 cm2) with vented caps were obtained
from Fisher-Costar (Houston, TX). A Stovall Low Profile
Roller apparatus (Stovall Life Science Inc., Greensboro,NC) was used for the roller bottle cultures. Flat-bottom
static T-175 culture flasks (175 cm2 area) with vented caps
were obtained from Nunc (Rochester, NY). All flow
cytometry antibodies and 7-aminoactinomycin D (7AAD) were purchased from BD Biosciences (La Jolla, CA).
Sources of PBMC for DC generation
PBMC were obtained from peripheral blood leukopheresis products obtained from non-mobilized normal
donors (LifeBlood, Memphis, TN), or G-CSF-mobilized
normal donors (AllCells, Berkeley, CA). Products werecollected in the presence of Anticoagulant Citrate Dextrose Formula A (Gambro). In addition, peripheral blood
buffy coats (Gulf Coast Regional Blood Bank, Houston,TX) were also used for some experiments. In some experiments HLA-A*0201+ positive non-mobilized leukopheresis products were used to generate DC (LifeBlood,
Memphis, TN). The HLA-A*0201 status was further confirmed by flow cytometry after receipt of the sample in thelaboratory. All leukopheresis products and buffy coats
were used within 24 hours post-collection. The PBMC
were isolated by diluting with HBSS, centrifuged at 400 ×g for 20 min over Histopaque-1077 (Sigma-Aldrich). The

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interface cells were collected, pooled, and washed withHBSS until the contaminating platelets were removed.
PBMC not used immediately were frozen in human AB
serum with 10% DMSO (33.3 × 106) cells/ml and stored
in the vapor phase of liquid nitrogen.

cold FACS Stain Buffer (FSB) consisting of D-PBS, 1%
BSA, and 5% normal goat serum. The cells were stainedusing anti-CD83-PE, anti-CD80-FITC, anti-CD86-APC,
CD11c-FITC and CD14-PE (all from BD Biosciences, La
Jolla, CA) on ice for 20 min and washed with cold FWB
and re-suspended in 0.35 ml cold FWB. 7-AAD (2 µg/ml)
was added 5–10 min before FACS analysis to exclude deadcells and enumerate mDC viability. The samples were run
on a FACScalibur or FACScanto flow cytometer and analyzed using FlowJo 7.2.2 software (Tree Star Inc., Ashland,OR).

Dendritic cell culture in roller bottles
Washed PBMC from leukopheresis products or buffy coats
were diluted to 30 × 106 cells/ml in DC-CM and 30 ml(900 × 106 cells) were seeded into 850 cm2 roller bottles
with vented caps (Fisher-Costar, Houston, TX). The bottles were placed on the roller bottle apparatus in a 37°C,
5% CO2 incubator and rolled at low speed (1 rpm) for 2
to 3 h. The bottles were then taken out and agitated toloosen any non-adherent cells and the floating cells
removed. The bottles were then washed 2 times with 80–
100 ml warm DC-CM by rolling the bottle inside a laminar flow hood. After removal of the second wash, 150–
180 ml of DC-CM containing 1,000 U/ml GM-CSF and
1,000 U/ml IL-4 was added to each bottle. The bottles
were placed back on the roller bottle apparatus in the
incubator and rolled at 2 rpm for 4–5 days. A dendritic
cell maturation cocktail consisting of a final concentrationof 10 ng/ml IL-1β, 10 ng/ml TNF-α, 15 ng/ml IL-6, and 1µg/ml PGE2 (ITIP) [13,16]. After 20–24 h the floating cells
were harvested in all bottles and analyzed for mature DC
content. In some experiments, an alternative maturation
cocktail called the "Pittsburgh Protocol" (25 ng/ml IL-1β,
50 ng/ml TNF-α, 1,000 U/ml IFN-γ, 20 µg/ml poly I:C,and 3,000 U/ml IFN-α) was used to generate so-called α
Type-1DC (α DC1) was added on day 4 or 5 [17]. In someexperiments, 450 cm2 roller bottles (Fisher-Costar) wereused with PBMC seeded at 250 to 450 × 106 cells per bottle.
Dendritic cell generation in flat-bottom static T-175 flasks
Washed PBMC from leukopheresis products or buffy coats
were seeded into T-175 culture flasks in 15 ml of DC-CM(175 × 106 cells per flask). The flasks were incubated asabove for 2 to 3 h and non-adherent cells were removed.
The flasks were then washed with 50 ml of warm DC-CM
and 60 ml of DC-CM containing 800 U/ml GM-CSF and
1,000 U/ml IL-4 was added. The cells were incubated for
4–5 days and matured for 20–24 h and analyzed formature DC content and function as above.
Determination of mDC yield and phenotype
Isolated cells were washed in DC-CM and viable cellrecovery determined with Trypan Blue staining and counting live cells on a hemocytometer using a light microscope. The total floating cells isolated were divided by the
number of culture vessels to determine the yield per flask
or per bottle. For cell surface staining, the cells were
washed 2 times in cold FACS Wash Buffer (FWB) consisting of D-PBS, 1% BSA and re-suspended at 10 × 106/ml in

Functional analysis of isolated mDC
DC isolated from roller bottles and static flask cultures
were assayed for their ability to induce allo-antigen T-cell
responses and CD8+ T-cell recall responses against HLAA2-binding epitopes from flu, CMV, and EBV [18]. For
allo-antigen responses, 50,000 monocyte-depleted PBMC
(2-hour plastic-non-adherent PBMC) from a normal
donor other than that used to generate the DC were incubated in U-bottom 96-well plates with different numbersof DC or PBMC stimulators (50,000, 25,000, 10,000,
5,000, 1,000, 500, 200, or 100 cells). On day 6, 1 µCi/wellof 3H-thymidine was added to each well and the cells harvested the next day and total cpm/well determined. Recall
antigen CD8+ T-cell responses were done in ELISPOT
plates (Millipore) using 5 × 105 monocyte-depleted autologous PBMC incubated with peptide-pulsed mDC harvested from roller bottles or static flask cultures. The mDC
were pulsed with 5 µg/ml of the HLA-A2-binding epitopes
from influenza A matrix (GILGFVFTL), CMV pp65 (NLVPMVATV), and EBV BMLF1 (GLCTLVAML) for 90 min,washed and added to the responder cells in the ELISPOT
plates [18]. The plates were incubated overnight and processed as described before [19].

Results
Monocytes adhere similarly in roller bottles and static
flasks
We first tested whether human monocytes can adhere
inside roller bottles as in traditional static flat-bottom
flasks. PBMC from normal donor buffy coats were seeded
into 490 cm2 roller bottles or T-175 culture flasks andadhered for 2.5 h (1 rpm for the roller bottles) in the incubator. The non-adherent cells were collected and stained
for CD14 and CD3 expression. Adherence of monocytes
will deplete the CD14+ population in the non-adherent
cell suspension. As shown in Table 1, the CD14+ monocytes adhered in roller bottles with similar efficiency as
flat-bottom T-175 flasks, as indicated by the drop in percentage of CD14+ cells in the suspended cell fraction.

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Table 1: Adherence of peripheral blood CD14+ monocytes to roller bottles and static flasks*

Condition
Pre-adherent PBMC
Flask: Post-adherence
Roller bottle #1: Post-adherence
Roller bottle #2: Post-adherence

CD14+ (%)

CD3+ (%)

CD14- and CD3- (%)

14.6
2
2.2
2.1

45.4
59.5
55.2
56

40
38.4
36.4
41.9

*PBMC isolated from a normal donor buffy coat donor were incubated in T-175 culture flask or 450 cm2 roller bottles for 2.5 h. The roller bottles
were rolled at low speed (1 rpm). The non-adherent cells were isolated and stained along with a sample of the original PBMC for CD14 and CD3
expression. The percent CD14+, CD3+, or CD14-CD3- cells are shown before (pre-adherent PBMC) and after the adherence protocol.

ITIP Maturation

StaticStaticflasks
flasksRollerRollerbottles
bottles

Generation
static flask
Figure1 culturesof phenotypically mature mDC from adherent monocytes using ITIP in roller bottle cultures in comparison toGeneration of phenotypically mature mDC from adherent monocytes using ITIP in roller bottle cultures incomparison to static flask cultures. PBMC from a normal donor leukopheresis product was seeded into 850 cm2 roller
bottles or into T-175 flasks and the monocytes adhered for 2.5 h as described in the Methods section. After washing out the
non-adherent cells in both systems, the cells were cultured for 4 days with 1,000 U/ml GM-CSF and 1,000 U/ml IL-4 and thenmatured using ITIP. The floating cells were harvested after 24 h and stained for CD11c, CD14, HLA class II DP, DQ, DR,
CD83, CD86, and CD80. The unstained and stained populations in the histograms are shown in grey and red, respectively. In
the case of CD86 and CD83 staining, the surface expression on cells from non-matured cultures (in blue) is shown as a comparison to verify that maturation was induced in both systems. The results of one out of 3 similar experiments are shown.

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Similar degree of DC maturation in roller bottles as instatic flasks
Next, we generated monocyte-derived DC in 850 cm2
roller bottles versus T-175 static flasks after monocyte
adherence and assessed the phenotype and viability of theDC generated from each culture type after maturation
with 10 ng/ml IL-1β, 10 ng/ml TNF-α, 15 ng/ml IL-6, and1 µg/ml PGE2 (ITIP). The floating cells isolated from both
culture types 24 h after addition of the maturation cocktail were stained for CD83, CD86, CD80, CD11c, andCD14 and analyzed by FACS. Both types of culturesinduced comparable levels of DC maturation, as indicatedby the similar percentages of CD83+, CD80+, CD86hi,
CD11c+, CD14-/lo generated using two separate methods,
ITIP maturation (Fig. 1) and Pittsburgh Protocol maturation (Fig. 2). The viability of the harvested mature DC
from the roller bottles and static flasks was also assessedusing 7-AAD staining of the cells prior to FACS analysis. In
both cases, the CD83+ DC were > 90% viable, as shown in
the two separate experiments shown in Fig. 3.

B

Pittsburgh Protocol Maturation

Static flasks

Roller bottles

Figurecomparison
Generation
2 ofto phenotypicallystatic flask culturesmature mDC from adherent monocytes using the Pittsburgh Protocol in roller bottle cultures inGeneration of phenotypically mature mDC from adherent monocytes using the Pittsburgh Protocol in roller
bottle cultures in comparison to static flask cultures. PBMC from a normal donor leukopheresis product was seeded
into 850 cm2 roller bottles or into T-175 flasks and the monocytes adhered for 2.5 h as described in the Methods section. Afterwashing out the non-adherent cells in both systems, the cells were cultured for 4 days with 1,000 U/ml GM-CSF and 1,000 U/
ml IL-4 and then matured using the Pittsburgh Protocol combination of cytokines. The floating cells were harvested after 24 hand stained for CD11c, CD14, HLA class II DP, DQ, DR, CD83, CD86, and CD80. The unstained and stained populations in
the histograms are shown in grey and red, respectively. In the case of CD86 and CD83 staining, the surface expression on cells
from non-matured cultures (in blue) is shown as a comparison to verify that maturation was induced in both systems. The
results of one out of 3 similar experiments are shown.

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Expt. #1

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Expt. #2

FlasksRollerbottlesFigure3
Roller bottle
cultures yield mature CD83+ DC with high viability
Roller bottle cultures yield mature CD83+ DC with high viability. Mature mDC were generated in 850 cm2 roller bottles or in T-175 static flasks as before using ITIP maturation. The floating cells were harvested and stained for DC maturation
markers without fixation. Immediately before FACS analysis 2 µg/ml 7-ADD was added as viability indicator. The dot plots
shown the total cells in the floating fractions with the CD83+, 7-AAD- and CD83+, 7-AAD+ cells gated. In both cases, theCD83+ cells exhibited 94–96% viability. The results of two separate experiments are shown.

Efficiency in generating large numbers of mature DC inroller bottles
One of the benefits of using culture vessels with increased
surface area such as roller bottles is maximizing the scalein which DC cells can be generated while minimizing thenumber of separate culture vessels needed to achieve
high-throughput production. Using 850 cm2 roller bottles
we found that up to 900 × 106 PBMC could be loaded during the monocyte adherence step, while up to 180 × 106

cells could be loaded in T-175 flasks. We determined theyield of total floating cells and mature DC recovered inboth culture systems. In these experiments, PBMC from
G-CSF-mobilized or non-mobilized leukopheresis products were loaded into the culture vessels and adherent
cells treated with GM-CSF and IL-4 for 4 to 5 days followed by treatment with the ITIP maturation cocktail or α
DC1 maturation cocktail (not shown) for 24 h. Table 2
shows the results of three separate experiments comparing

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Table 2: Yield of mature DC from roller bottle cultures and static flask cultures*

Expt #

Culture system

PBMC seeded per
vesselAverage floating cells
recovered

Average mature DC(CD83+, CD86hi)
recovered% yield of mature DC1**

850 cm2 roller bottles
T-175 static flasks
850 cm2 roller bottles
T-175 static flasks
850 cm2 roller bottles
T-175 static flasks

900 × 106
180 × 106
900 × 106
180 × 106
800 × 106
200 × 106

106 × 106
20 × 106
100 × 106
16 × 106
84 × 106
29 × 106

85 × 10619.2 × 106
82 × 10610.2 × 106
23 × 106
6 × 106

9.4%
10.7%
9.1%
5.7%
2.9%
3%

2**
3***

*Human peripheral blood leukopheresis products were seeded and monocytes adhered in the two types of culture vessels, as described in the
Methods section. After 4 to 5 days of culture with GM-CSF and IL-4, ITIP cocktail was added to induce DC maturation. On average, 2–3 roller
bottles or static flasks were set up for each experiment. The floating cells were harvested one day later and viable cellrecovery was determined by
Trypan Blue staining with a hemocytometer followed by FACS staining for CD83 and CD86. The number of mature DC was calculated from thepercent CD83+, CD86hi cells using the total number of viable floating cells. The results of three separate experiments are shown.
**Experiment was done directly with normal donor non-G-CSF-mobilized leukopheresis products.
***Experiment was done directly with a G-CSF-mobilized normal donor leukopheresis product, accounting for the lower yield of mature DC.

the yield of mature DC in both systems and the percentage
of mature DC relative to the original PBMC load. On average, from normal donor non-GSF-mobilized leukopheresis products a single 850 cm2 roller bottle culture yielded
80–85 × 106 CD83+ CD86hi DC and the static T-175 flasks
yielded up to 10–20 × 106CD83+ CD86hi DC; this represent an average 5 to 6-fold more mDC per single culture
vessel (Table 2). In addition, DC from both types of cultures were able to be cryopreserved in 90% AB serum,
10% DMSO with > 80% viability after thawing (data notshown). The roller bottle system could also generate mDCfrom G-CSF-mobilized leukopheresis products with asimilar yield as static flasks (Table 2; Experiment #3). In
this case, the yield of mDC per input cells was lower
because of the lower percentage of mature CD14+ monocytes in these products than in non-mobilized PBMC.
Similar relative results were obtained with the Pittsburgh
Protocol maturation protocol (data not shown). Thus, theroller bottle approach allows for the efficient scale-up forNot matured
StaticflasksRollerbottlesFigure 4 analysis of DC purity in non-matured DC cultures from roller bottle and static flask cultures
Phenotypic
Phenotypic analysis of DC purity in non-matured DC cultures from roller bottle and static flask cultures. DC
were generated as before in 850 cm2 roller bottles or T-175 static flasks for 4 days and then incubated for an additional 24 hwithout any additional cytokines ("Not matured"). The floating cells were harvested after this additional 24 h incubation andstained for CD11c, CD13, CD14, CD83 and CD86 and analyzed by flow cytometry. In each case all the isolated floating cells
were analyzed without gating and phenotype of DC compared between the roller bottles and static flask system. The numbersin the dot plots indicate the percentage of cells out of the total population of floating cells having the indicated phenotype. The
results of one out of 4 similar experiments are shown.

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the generation of large numbers of mature DC with similar yield of mDC per input cells as in static flasks.

found in the non-matured cultures (Fig. 4), but theselargely disappeared in the ITIP-matured cultures (Fig. 5)
with mostly a minor population CD13-, CD14-, CD11cpopulation making up the FSClo, SSClo population. In thematured cultures from both the flasks and roller bottles,
the FSClo, SSClo, CD13- subset was less than 10% in eachcase (Fig. 5). Lastly, CD14 was down-modulated in cells
obtained from both ITIP-matured static flasks and roller
bottles (Fig. 5), as compared to cells isolated from nonmatured cultures (Fig. 4).

The generation of DC from adherent PBMC from peripheral blood does not yield a 100% pure population of
floating mature DC. The mDC are mixed with other cells
that are carried over from the original PBMC loaded into
the culture vessels during the monocyte adherence step.
We determined the percentage of CD83+, CD11c+, CD13+,
and CD14+ in the high forward scatter (FSChi) and highside scatter (SSChi) population (DC gate), as well as thelow forward scatter (FSClo) and low side scatter (SSClo)
population isolated from non-matured (Fig. 4) and ITIPmatured (Fig. 5) cultures from both the static flask androller bottle systems. The flow cytometry profiles in Fig. 4
and Fig. 5 are all on total (ungated) cells in each sample.
CD83 was highly induced in the FSChi, SSChi populationin the ITIP matured cultures from both the flask and roller
bottle systems (Fig. 5), while none of FSClo, SSClo cells
expressed these high CD83 levels (Fig. 5). In addition,
only the FSChi, SSChi population was CD11c+ in thematured cultures from both system, with only a small
fraction (< 1%) of FSClo, SSClo cells expressing CD11c(Fig. 5). The FSClo, SSClo cells were further analyzed andfound to consist largely of CD13-, CD14- (non-myeloid
origin) and CD11c- cells which by process of elimination
are essentially lymphocytes (T and B cells) or NK cells.
Some FSClo, SSClo cells having low CD13 expression wereThus, both roller bottle and static flask cultures yielded
mature DC of similar purity with a similar minor population of FSClo, SSClo cells having a lymphocyte (CD11c-,
CD13-, CD14-) phenotype.
DC generated in roller bottles function similarly as thosefrom static flasks
In order to determine whether DC generated in roller bottles functioned similarly as antigen-presenting cells (APC)
as those generated in static flasks, we tested both types of
DC for their ability to activate allo-specific and autologous recall antigen peptide-specific T cell responses. DC
were generated in 850 cm2 roller bottles or T-175 static
culture flasks as before and the floating cells were isolated
and tested for APC activity. Fig. 6 shows an example of theallo-stimulatory function of DC generated from a normal
leukopheresis donor (APH 10) in roller bottles versus

Matured - ITIP
StaticflasksRollerbottlesFigure 5 analysis of DC purity in matured DC cultures from roller bottle and static flask cultures
Phenotypic
Phenotypic analysis of DC purity in matured DC cultures from roller bottle and static flask cultures. DC weregenerated as before in 850 cm2 roller bottles or T-175 static flasks for 4 days and then incubated for an additional 24 h withITIP cocktail to induce DC maturation ("Matured-ITIP"). The floating cells were harvested after 24 h after addition of the ITIP
maturation cocktail and stained for CD11c, CD13, CD14, CD83 and CD86 and analyzed by flow cytometry. In each case all
the isolated floating cells were analyzed without gating and phenotype of DC compared between the roller bottles and static
flask system. The numbers in the dot plots indicate the percentage of cells out of the total population of floating cells having theindicated phenotype. The results of one out of 4 similar experiments are shown.

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3H-thymidine incorporation (cpm)

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70000

APH10 PBM C

60000

APH10 RB mDC

50000

APH10 Flask mDC

40000
30000
20000
10000
0
1:1

1:2

1:5

1:10

1:50

1:100

1:250

1:500

DC to T-cell ratio
Figure 6 cells generated in roller bottle cultures have potent allo-stimulatory capability
Dendritic
Dendritic cells generated in roller bottle cultures have potent allo-stimulatory capability. Dendritic cells weregenerated in 850 cm2 roller bottles or T-175 static flasks as before using ITIP maturation. The floating cells were harvested,irradiated at 20 Gy and mixed with 50,000 allogeneic T-cell-enriched PBMC (plastic non-adherent PBMC) at different stimulator to responder ratios in 96-well plates. After 5 days, 1 µCi/well of 3H-thymidine was added to each well and the plates harvested 18 h later. Irradiated PBMC from the original DC donor were also used as stimulators as a control. The average cpm
and standard deviation of triplicate cultures are shown for each stimulator type.
static flasks. In both cases, the DC induced a similar rateof allo-specific T-cell proliferation at the different DC
doses used in the assay (Fig. 6). Floating cells isolated
from non-matured roller bottle DC cultures as well as theoriginal PBMC population has substantially lower allostimulatory activity on a per cell basis than the mature DC(Fig. 6). In another experiment, we found that both the
ITIP and α DC1 maturation protocols in roller bottles
induced DC of comparable potent allo-stimulatory capacity in comparison to the original starting PBMC population in the leukopheresis product (data not shown). To
test the ability of DC to present peptides and activate
autologous T cells, we used a recall antigen response assay
using HLA-A*0201-binding peptides. In this case, we generated DC in 850 cm2 roller bottles or T-175 static flasks
from HLA-A*0201+ donor leukopheresis products. The
floating DC after the maturation step were isolated andpulsed with 9-mer peptides from flu, CMV, and EBV (seeMaterials and Methods). The peptide-pulsed DC were
washed and incubated with autologous monocytedepleted PBMC in an overnight IFN-γ ELISPOT assay(1:50 or 1:100 DC to responder ratios). As shown in Fig.
7, mature DC generated using either approach yielded
cells of comparable APC activity in terms of the number
of IFN-γ spot-forming cells in the assay. Little or no IFN-γ

production was found in cultures with non-preloaded
DC, or in cultures without added DC (Fig. 7). Thus, DC
generated in roller bottles yield highly competent APC for
T-cell stimulation.

Discussion
The generation of large numbers of mature DC in a largescale culture processes for application in vaccine clinical
trials still remains a challenge using present static flask
technology due to the high number of culture vessels
needed. Although new devices such as multi-level static
culture devices such as Cell Factories™ (Nunc, Rochester,NY) have improved our ability to generate DC in large
scale, most static culture systems are still cumbersome andlabor intensive [4,11,13]. We have developed an alternative approach for the large-scale generation of mature DC
from adherent human monocytes using roller bottle technology. This system can generate DC from plastic-adherent monocytes as traditional static flask cultures. The DC
generated using roller bottles had the same phenotypicand functional attributes as those generated in static flask
cultures. However, given the large surface area in a single
roller bottle (850 cm2), this technology allows for theloading of much higher numbers of input PBMC per single vessel with a comparable level of monocyte adherence

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Journal of Immune Based Therapies and Vaccines 2008, 6:1

B

Nopeptide
peptide50,000 DC No

A2 recallA2 recall
peptidespeptides

10,000 DC
5,000 DC

RB + ITIP

0 DC
50,000 DC
10,000 DC
5,000 DCFlask + ITIP

0 DC

Spot-forming cells/500,000

A

http://www.jibtherapies.com/content/6/1/1

250

RB - peptide
RB + peptide

200

Flask - peptide
Flask + peptide

150

100

50

0

1

1 to 50

2

1 to 100

3No DC

DC to T-cell ratio
Figure 7 cells generated in roller bottles stimulate autologous peptide-specific T-cell responses
Dendritic
Dendritic cells generated in roller bottles stimulate autologous peptide-specific T-cell responses. Dendritic cells
from HLA-A*0201+ normal donor leukopheresis products were differentiated and matured with ITIP in roller bottles or static
flasks as before. The floating cells were harvested, pooled, irradiated (20 Gy), and pulsed with HLA-A*0201 epitopes from flu,
EBV, and CMV (see Materials and Methods for details). The DC were washed and added to 500,000 T-cell-enriched autologous
PBMC in anti-IFN-γ antibody-coated ELISPOT plates in the numbers indicated. Each assay was run in triplicate. The plates were
harvested after overnight culture and developed. Shown is the image taken of the developed ELISPOT plate (A) and corresponding graphical representation of the number of spots per 500,000 input responder cells under the different conditions (B).
Dendritic cells without peptide pre-pulsing were used as controls. The results are representative of two similar experiments.

and mature DC yield, thereby generating much higher
numbers of DC per vessel. A number of benefits arise outof this approach, including the need for up to 5 times lessculture vessels to generate the equal number of DC versus
static T-175 flasks. The roller bottle method is also easy to
perform, more practical than handling large numbers offlasks, and overall saves technician time and potentiallabor costs. In addition, the less manipulation required togenerate DC products in large scale will also help ensure
less chance of error and contamination with infectious
agents that would destroy the product.
In our initial experiments, we found that monocytes had
a similar capacity to adhere to the plastic inside the roller
bottles as in the static flasks. Initially, this was a surprise
to us, considering the dogma in the field that has emergedwith the use of static culture flasks to generate DC for over
15 years. However, in our loading step the PBMC arerolled in the bottles at sufficiently low speed (1 rpm)
allowing the monocytes to adhere just as well as in static
flasks. The low volume per vessel surface area used during
the loading process in the bottles allows the monocytes toroll along and stay in close contact with the surface andthen eventually attach. This is akin to the attachment of
monocytes rolling along the walls of blood vessels in the
body during extravasation into tissues.

Recently, newer static flat-bottom culture systems for DC
have been developed such as the Cell Factories™ (Nunc,
Rochester, NY) [11,13]. These systems consist of two or
more flat-bottom culture surfaces stacked on top of eachother in a single large flask format. The cells are seeded
into a main port and distributed over the multiple stacked
surface areas. We have found however that these vessels
are cumbersome to handle and it is not straightforward toevenly distribute the cell and culture medium over all thestacked surfaces in the culture vessel (unpublished observations). In addition, feeding additional growth factors
and DC maturation agents so that they are evenly distributed in each culture level also requires additional manipulation and is not straightforward. In contrast, the roller
bottle system offers a simpler and more fool-proof
method to generate the same or even greater number ofmature DC allowing even novice technicians to set-up thecultures with ease and higher reproducibility.
Roller bottles have been used in vaccine manufacture toculture strongly adherent fibroblast producer lines andhave never been tested for their ability to generate monocyte-derived DC in large scale. Thus, this approach is a
novel application that increases the versatlity of this technology and broadens its application in vaccine manifacturing. In addition, the mDC generated in roller bottles

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are functionally equivalent in terms of their ability to activate T-cell responses as mDC generated in static flasks.

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Felzmann T, Witt V, Wimmer D, Ressmann G, Wagner D, Paul P,
Huttner K, Fritsch G: Monocyte enrichment from leukapharesis products for the generation of DCs by plastic adherence,or by positive or negative selection.
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5(5):391-398.
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G, Schuler-Thurner B: Large-scale generation of mature monocyte-derived dendritic cells for clinical application in cell factories. J Immunol Methods 2002, 268(2):131-140.
Buchler T, Kovarova L, Musilova R, Bourkova L, Ocadlikova D, Bulikova A, Hanak L, Michalek J, Hajek R: Generation of dendritic cells
using cell culture bags--description of a method and review
of literature. Hematology 2004, 9(3):199-205.
Kurlander RJ, Tawab A, Fan Y, Carter CS, Read EJ: A functional
comparison of mature human dendritic cells prepared influorinated ethylene-propylene bags or polystyrene flasks.
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Mailliard RB, Wankowicz-Kalinska A, Cai Q, Wesa A, Hilkens CM,
Kapsenberg ML, Kirkwood JM, Storkus WJ, Kalinski P: alpha-type-1
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12.

Conclusion
The roller bottle method described here is an new andmore practical way to generate large numbers of mature
DC with potent APC activity for vaccine applications orlarge scale laboratory studies where > 100 million mDC
are routinely needed. The method is easy to perform, saves
time, and generates mDC of equal potency and similar
purity as traditional static flask methods. The method canalso be easily translated into a GMP environment whereroller bottles have been used for other applications.

Competing interests
The author(s) declare that they have no competing interests.

13.

14.

15.

16.

17.

Authors' contributions
REC performed experiments and wrote the manuscript.
DK, YJW, YF, and LMV helped perform experiments. KMB
processed and provided human PBMC materials forexperiments. LGR supervised the work and helped write
the manuscript.

Acknowledgements

18.

19.

This work was supported by a NIH grant P30 CA016672 31. We thankKarena Fernandez and Jacqueline Page for technical assistance.

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