- Original research
- Open Access
A new approach for the large-scale generation of mature dendritic cells from adherent PBMC using roller bottle technology
© Campbell-Anson et al; licensee BioMed Central Ltd. 2008
- Received: 15 November 2007
- Accepted: 06 March 2008
- Published: 06 March 2008
Human monocyte-derived DC (mDC) loaded with peptides, protein, tumor cell lysates, or tumor cell RNA, are being tested as vaccines against multiple human malignancies and viral infection with great promise. One of the factors that has limited more widespread use of these vaccines is the need to generate mDC in large scale. Current methods for the large-scale cultivation of 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 from human 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.
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 were matured 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.
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 type of 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 the static flasks as a result.
Our results indicate that the roller bottle technology can generate similar numbers of 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.
- Dendritic Cell
- Peripheral Blood Mononuclear Cell
- Culture Vessel
- Buffy Coat
- Mature Dendritic Cell
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 viral disease and cancer . 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 . The most commonly used method to generate mDC is to adhere monocytes on to plastic in static flasks from PBMC followed by culture with GM-CSF and IL-4 and maturation using any one of a number of cocktails of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) or Toll-like receptor (TLR) agonists such as 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 some cases more than a billion mDC may be required to ensure that enough vaccine can be produced for multiple patient immunizations over a number of months. Vaccination regimens using antigen-pulsed mDC have ranged from multiple 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 large-scale cultivation of mDC in static culture systems can be cumbersome, 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 some debate on the quality of these mDC versus those derived from adherent populations. For example, some studies of have found decreased yields of mature CD83+ mDC or reduced IL-12 production capability versus adherent systems [14, 15]. Thus, any improvements in the speed and ease 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 in large-scale using roller bottle culture technology never before reported to be used to generate DC before. The monocytes from the peripheral blood mononuclear cell (PBMC) or leukopheresis preparations were adhered to the 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 GM-CSF and IL-4 and matured using any one of a number of well-defined defined cytokine cocktails. This resulted in a large number of floating non-adherent mature DC that can be easily harvested and used for vaccines or other purposes. The roller bottle DC had similar phenotypic and functional 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 and less labor for lab personnel than traditional static culture methods in flat-bottom culture flasks.
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, 20 μg/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 (7-AAD) 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 were collected 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 the laboratory. 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 interface cells were collected, pooled, and washed with HBSS 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.
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 to loosen 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 concentration of 10 ng/ml IL-1β, 10 ng/ml TNF-α, 15 ng/ml IL-6, and 1 μg/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 . In some experiments, 450 cm2 roller bottles (Fisher-Costar) were used 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 as above 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 for mature DC content and function as above.
Determination of mDC yield and phenotype
Isolated cells were washed in DC-CM and viable cell recovery 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 cold FACS Stain Buffer (FSB) consisting of D-PBS, 1% BSA, and 5% normal goat serum. The cells were stained using 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 dead cells 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).
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 HLA-A2-binding epitopes from flu, CMV, and EBV . 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 numbers of DC or PBMC stimulators (50,000, 25,000, 10,000, 5,000, 1,000, 500, 200, or 100 cells). On day 6, 1 μCi/well of 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 . The plates were incubated overnight and processed as described before .
Monocytes adhere similarly in roller bottles and static flasks
Adherence of peripheral blood CD14+ monocytes to roller bottles and static flasks*
CD14- and CD3- (%)
Roller bottle #1: Post-adherence
Roller bottle #2: Post-adherence
Similar degree of DC maturation in roller bottles as in static flasks
Efficiency in generating large numbers of mature DC in roller bottles
Yield of mature DC from roller bottle cultures and static flask cultures*
PBMC seeded per vessel
Average floating cells recovered
Average mature DC (CD83+, CD86hi) recovered
% yield of mature DC
850 cm2 roller bottles
900 × 106
106 × 106
85 × 106
T-175 static flasks
180 × 106
20 × 106
19.2 × 106
850 cm2 roller bottles
900 × 106
100 × 106
82 × 106
T-175 static flasks
180 × 106
16 × 106
10.2 × 106
850 cm2 roller bottles
800 × 106
84 × 106
23 × 106
T-175 static flasks
200 × 106
29 × 106
6 × 106
Thus, 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 those from static flasks
The generation of large numbers of mature DC in a large-scale 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 and labor 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 phenotypic and 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 the loading of much higher numbers of input PBMC per single vessel with a comparable level of monocyte adherence and mature DC yield, thereby generating much higher numbers of DC per vessel. A number of benefits arise out of this approach, including the need for up to 5 times less culture 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 of flasks, and overall saves technician time and potential labor costs. In addition, the less manipulation required to generate 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 emerged with the use of static culture flasks to generate DC for over 15 years. However, in our loading step the PBMC are rolled 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 to roll along and stay in close contact with the surface and then 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 each other 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 to evenly distribute the cell and culture medium over all the stacked 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 of mature DC allowing even novice technicians to set-up the cultures with ease and higher reproducibility.
Roller bottles have been used in vaccine manufacture to culture strongly adherent fibroblast producer lines and have 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 are functionally equivalent in terms of their ability to activate T-cell responses as mDC generated in static flasks.
The roller bottle method described here is an new and more practical way to generate large numbers of mature DC with potent APC activity for vaccine applications or large 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 can also be easily translated into a GMP environment where roller bottles have been used for other applications.
This work was supported by a NIH grant P30 CA016672 31. We thank Karena Fernandez and Jacqueline Page for technical assistance.
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