Supplementary MaterialsAdditional document 1. boost R&D throughput. DC surface area marker profiles, cytokine creation, DDX3-IN-1 and capability to activate antigen-specific cytotoxic T cells had been characterized. Outcomes Monocyte differentiation into Mo-DCs Cdh5 resulted in the increased loss of Compact disc14 appearance with concomitant upregulation of Compact disc80, CD86 and CD83. Considerably increased degrees of IL-12 and IL-10 were observed after maturation in day 9. Antigen-pulsed Mo-DCs turned on antigen-responsive Compact disc8+ cytotoxic T cells. No significant distinctions in surface area marker appearance or tetramer-specific T cell activating strength of Mo-DCs had been noticed between TCPS and FEP lifestyle vessels. Conclusions Our results demonstrate that viral antigen-loaded Mo-DCs stated in downscaled FEP luggage can elicit particular T cell replies. In view from the dire scientific need for shut system DC processing, FEP luggage represent a nice-looking option to speed up the translation of appealing rising DC-based immunotherapies. , the changeover from functionally-open TCPS plates to shut systems such as for example FEP or polyolefin luggage leads to a concurrent transition in material properties including gas permeability, mechanical properties, surface topography, surface chemistry and surface wettability. This may affect protein adsorption profiles and resulting changes in the cell microenvironment which may impact Mo-DC cell fate decisions, as observed with other therapeutic cells . A number of groups have reported successful production of Mo-DCs in FEP bags based on the upregulation of DC markers and on the capacity DDX3-IN-1 to stimulate T cells [15C19]. The number of direct comparative studies between TCPS plates and FEP bags is however much more limited [9, 11, 12]. Most studies comparing TCPS flasks with FEP or other types of hydrophobic culture bags report no marked changes in Mo-DC differentiation [7C9, 11, 12, 20, 21]. However, subtle differences in cytokine production  and the expression levels of certain surface markers such as CD1a [7, 22] have been reported. The impact of these differences on antigen-specific T cell activation, a key function of Mo-DC, and hence product potency has not been thoroughly assessed . The lack of commercially available research-scale culture bags limited the throughput of past comparative studies, and hence the dynamics of the DC differentiation process in FEP bags have not been reported. Together, these limitations result in a gap in our understanding of cell-material interactions early in the upscaling process and thus, in bag usage in the clinical setting. The main objective of this study was to compare the phenotype and functional capacities of Mo-DCs cultured in open TCPS-based plates to the closed fluorinated ethylene propylene (FEP) culture bag systems. Research-scale FEP bags were tested, providing a novel platform for translational studies using cell culture materials more similar to clinical-scale cultures. Mo-DCs generated in FEP bags and TCPS plates showed comparable levels of antigenic expression and cytokine production and were able to efficiently induce tetramer-specific effector T cell response upon viral antigen stimulation. Methods Culture surfaces Immature as well as mature Mo-DCs were cultured in Nunclon? Delta-treated TCPS 24-multiwell plates (Nunc, ThermoFisher) or untreated VueLife? FEP culture bags (Saint-Gobain) of 1 1?mL (1PF-0001), 2?mL (2PF-0002) and 7?mL (1PF-0007) volumes. The respective internal dimensions of the bags were approximately 3.8?cm??2?cm, 2.5?cm??8.6?cm or 3.4?cm??5.8?cm with a single Luer-lock cell seeding and medium exchange port. These bags are commercialized for cell cryopreservation applications but can also be used for cell culture. Generation of Mo-DCs using lipopolysaccharides to induce maturation CD14-positive monocytes were freshly isolated from peripheral whole blood of healthy human donors. Peripheral blood was subjected to gradient density centrifugation using Histopaque?-1077 DDX3-IN-1 (Sigma Aldrich) in SepMate?-50 tubes (STEMCELL). CD14-positive cells were isolated via magnetic cell sorting using MACS? Technology with CD14 Microbeads, LS columns and a MidiMACS? Separator (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturers instructions. CD14-positive cells were then seeded at a density of 1 1.5??106 cells/ml (~?0.5?mL/cm2 bottom surface area) onto TCPS or FEP culture surfaces in GMP DC medium (CellGenix GmbH, Freiburg, Germany) supplemented with 1000?IU/ml of GM-CSF and 1000?IU/ml of IL-4 (both from Miltenyi Biotec) and cultured at 37?C, 5% CO2 for 7?days to obtain immature DCs. Adherent and non-adherent cells from TCPS plates were harvested via incubation with TrypLE Express (Thermo Fisher Scientific) for 5?min at 37?C, 5% CO2, followed by centrifugation at 300for 5?min. FEP bags display a non-adherent surface and did not require trypsin exposure to recover cells. To induce maturation, Mo-DCs were cultured 2 additional days in CellGenix? GMP DC medium supplemented with 1000 U/mL GM-CSF, 100 U/mL IL-4 and 100?g/ml of Toll-like receptor-validated.
Supplementary MaterialsAdditional document 1