Gmp dc medium

Manufactured by CellGenix

Sourced in Germany

CellGenix GMP DC medium is a serum-free and animal-component-free cell culture medium designed for the expansion and differentiation of dendritic cells (DCs) from monocytes or progenitor cells under good manufacturing practice (GMP) conditions.

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Lab products found in correlation

Ficoll paque plus, by GE Healthcare (4 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions) Trypan blue, by Merck Group (2 mentions) Theraflex methylene blue technology, by Macopharma (2 mentions) Celltrace violet, by Thermo Fisher Scientific (2 mentions) Automated cell counter, by Countstar (1 mentions) Cryovial, by Corning (1 mentions) Hanks balanced salt solution (hbss), by Merck Group (1 mentions) Fetal calf serum (fcs), by Merck Group (1 mentions) Fetal calf serum (fcs), by Biosera (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Hanks balanced salt solution (hbss), by Lonza (1 mentions) Il 4 and gm csf, by Immunotools (1 mentions) Lymphoprep, by Abbott (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) 24 well culture plate, by Corning (1 mentions) Macs ls column, by Miltenyi Biotec (1 mentions) 96 well plate, by Corning (1 mentions) Gm csf, by CellGenix (1 mentions)

21 protocols using gmp dc medium

Evaluating MicroDEN Performance with Systematic Trials

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A total of three identical experiments (N1, N2, N3) were systematically
performed to evaluate the performance of MicroDEN. Each experiment consisted of
three MicroDEN cartridges (one cartridge per seeding density) and one or two 6-well
plates (two to three wells per seeding density). MOs from a single donor were used
for each individual experiment (N1, N2, or N3), requiring three total donors for iDC
generation. T cells from a fourth donor were used for all allogeneic functional
assays. CellGenix GMP DC medium was used as the base medium for MO-to-iDC
differentiation. The medium was supplemented with 1% penicillin-streptomycin (Gibco
15140122) and 350 U/mL preclinical IL-4 and GM-CSF (CellGenix) for iDC
generation.
Each MO-to-iDC differentiation experiment was 6 days in duration. All
experiments were performed in a standard cell culture incubator maintained at 37
°C and 5% CO₂ at near saturation humidity. All work was performed
under aseptic conditions in a laminar flow hood. All cell counts were conducted
using a Countess II Automated Cell Counter.

Kozbial A., Bhandary L, & Murthy S.K. (2019). Effect of Monocyte Seeding Density on Dendritic Cell Generation in an Automated Perfusion-Based Culture System. Biochemical engineering journal, 150, 107291.

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Cryopreservation and Thawing of Dendritic Cells

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DC were cryopreserved in pre-cooled GMP DC medium (CellGenix) supplemented with 40% Human Serum Albumin (HSA)(Biotest) and 10% DMSO (Sigma) in a cryovial (Corning). cryovials were immediately placed in a cool cell in a -80°C freezer for 24 hours before transfer into a storage box. cryovials were stored short-term (less than one month) in a -80°C freezer or in liquid nitrogen long-term (more than one month). Cells were thawed in a 37°C water bath until ice crystals dissolved then transferred to an appropriate centrifuge tube where at least 5 ml of pre-warmed HBSS (Lonza) with 10% FCS (Biosera) was slowly added. The tubes were then topped up with HBSS with 10% FCS and washed in HBSS with 10% FCS by centrifugation twice at 400 xg for 8 minutes at room temperature before being counted and viability assessed by trypan blue (Sigma).

Cooke F., Neal M., Wood M.J., de Vries I.J., Anderson A.E., Diboll J., Pratt A.G., Stanway J., Nicorescu I., Moyse N., Hiles D., Caulfield D., Dickinson A.M., Blamire A.M., Thelwall P., Isaacs J.D, & Hilkens C.M. (2022). Fluorine labelling of therapeutic human tolerogenic dendritic cells for 19F-magnetic resonance imaging. Frontiers in Immunology, 13, 988667.

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Generation of Tolerogenic Dendritic Cells

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Peripheral blood mononuclear cells (PBMC) were isolated by gradient density centrifugation using Lymphoprep (Alere). Monocytes were further separated from PBMC by positive selection using CD14⁺ magnetic microbeads and a MACS LS column (Miltenyi) according to manufacturer’s instructions. Monocytes were seeded into 24-well culture plates (Corning) at 0.5 x 10⁶/ml (total of 1 ml per well) in GMP DC medium (CellGenix) containing IL-4 and GM-CSF (both at 50 ng/ml; Immunotools) and cultured for 7 days at 37°C, 5% CO₂. On day 3, IL-4 and GM-CSF were refreshed at 50 ng/ml final concentration, and half of the medium was replaced with fresh GMP DC medium. To generate tolDC, dexamethasone (Sigma) was added at 10^-8 M on days 3 and 6 of culture, and 10^-10 M 1,25-dihydroxyvitamin D3 (either Calcitriol from Tocris or Decostriol from Mibo) and 1 µg/ml of the TLR4 agonist monophosphoryl A (MPLA; Avanti) on day 6. To generate mature DC (matDC), cells were treated with 1 µg/ml MPLA (Avanti) on day 6 of culture only. In addition, on day 6 tolDC were labelled with ¹⁹F-NP (1.1, 2.2 or 4.4 mg/ml) or left unloaded as a control. DC were harvested on day 7, resuspended in HBSS + 1% FCS in a 30 ml universal tube (Starlab) and placed on ice for further processing (see sections below). Viability and cell numbers were determined by trypan blue (Sigma).

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Potency Assay for Tolerogenic DCs

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As part of the formal release criteria of therapeutic tolDC, we optimised a ‘potency assay’ as an in-vitro surrogate for in-vivo activity to confirm that tolDC lacked the ability to induce IFNγ production by allogeneic cells. Allogenic PBMC from 4 different donors (10⁵ cells/well for each donor) were thawed (see above) and co-cultured with each of the DC populations (mature DC and/or tolDC; 10⁴ cells/well each) in a 96 well plate (Corning) in 200 µl of GMP DC medium (CellGenix) supplemented with 4% HSA (Biotest) in duplicate. After 4 days the supernatants from duplicates were combined, divided into aliquots and stored at -80°C for assessment of IFNγ by ELISA.

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Tumor Organoid Recognition Assay

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Tumor or healthy organoids were isolated from Geltrex using 10 mg/mL dispase type II (Sigma) and cultured in organoid medium for three days with 10 μM Y-27632 (Cayman Chemicals) in 6-well plates. In some cases, two days later, 100 μL/well Geltrex was added to organoids. After three days, organoids were used in tumor recognition assays as described under flow cytometry.
Monocytes were sorted on the basis of CD14 on a MoFlo Astrios flow cytometer and cultured in GMP DC medium (CellGenix) with 800 U/mL GM-CSF and 400 U/mL IL-4 (CellGenix). Five days after differentiation, DCs were matured with GMC-CSF, IL-4, IL-6 (1 ng/mL), IL-1β (1 ng/mL), TNFα (1 ng/mL) and PGE1 (0.5 μg/mL). DCs were seeded at 2*10⁴ cells / well and loaded with Geltrex or 40 Gy dissociated and irradiated tumor or healthy organoid cells (2*10⁴ cells/well). Two days later, 1*10⁵ T cells were added to each well and cultured for 24h. T cells were assayed for CD137 expression as described under flow cytometry.

Dijkstra K.K., Cattaneo C.M., Weeber F., Chalabi M., van de Haar J., Fanchi L.F., Slagter M., van der Velden D.L., Kaing S., Kelderman S., van Rooij N., van Leerdam M.E., Depla A., Smit E.F., Hartemink K.J., de Groot R., Wolkers M.C., Sachs N., Snaebjornsson P., Monkhorst K., Haanen J., Clevers H., Schumacher T.N, & Voest E.E. (2018). Generation of tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids. Cell, 174(6), 1586-1598.e12.

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HexaBody-CD38 Opsonized Daudi Cell Phagocytosis

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Calcein AM-labelled Daudi cells were opsonized with HexaBody-CD38 for 15 min, after which macrophages were added at an E:T ratio of 2:1. Macrophages were derived from healthy donor PBMC-derived CD14⁺ monocytes that were cultured in GMP DC medium (CellGenix, cat# 20801-0500) supplemented with 50 ng/mL macrophage colony-stimulating factor (M-CSF, GIBCO, cat# PHC9501) for 6 or 7 days. Co-cultures were incubated for 24 h at 37 °C. Flow cytometry was used to monitor cell viability (using TO-PRO-3) and macrophages that had phagocytosed Daudi cells (CD11b⁺/Calcein⁺ cells). The percentage loss of Daudi cells was determined from the % non-phagocytosed Daudi cells (CD11b⁻/calcein AM⁺/CD19⁺ cells) relative to the average number of CD11b⁻/calcein AM⁺/CD19⁺ cells in the no antibody samples. The % macrophages with phagocytic activity was determined by evaluating the % of CD11b⁺/calcein AM ⁺ CD19^low cells.

Hiemstra I.H., Santegoets K.C., Janmaat M.L., De Goeij B.E., Ten Hagen W., van Dooremalen S., Boross P., van den Brakel J., Bosgra S., Andringa G., van Kessel-Welmers B., Verzijl D., Hibbert R.G., Frerichs K.A., Mutis T., van de Donk N.W., Ahmadi T., Satijn D., Sasser A.K, & Breij E.C. (2023). Preclinical anti-tumour activity of HexaBody-CD38, a next-generation CD38 antibody with superior complement-dependent cytotoxic activity. eBioMedicine, 93, 104663.

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Skin Wound Healing with PBMC Secretome

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Starting on day 29 after skin wounding, mice were injected with 100 µL 0.9% NaCl, medium (phenol red-free CellGenix GMP DC medium), or PBMCsec, which was prepared as described above, every second day for two weeks. Subsequently, half of the mice from each group (n = 2) were sacrificed and analyzed, while the other half (n = 2) were left for another two weeks without further intervention and then sacrificed.

Vorstandlechner V., Copic D., Klas K., Direder M., Golabi B., Radtke C., Ankersmit H.J, & Mildner M. (2023). The Secretome of Irradiated Peripheral Mononuclear Cells Attenuates Hypertrophic Skin Scarring. Pharmaceutics, 15(4), 1065.

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Expansion of HIV/EBV-specific CD8+ T cells

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Thawed PBMCs from HIV-infected individuals were labeled with CellTrace Violet (Thermo Fisher Scientific) at 1μM according to the manufacturer's protocol. The cells were then plated at 5 × 10⁶ cells/well in 24 well plate in CellGenix GMP DC Medium (CellGenix) containing recombinant human (rh) IL-7 (1ng/ml, PeproTech) to support minimal T cell survival and rh FLT3L (50ng/ml, PeproTech) to mobilize primary DCs. After 24 hours (day 1), cognate CD8 epitope peptides from HIV or EBV were added to the culture at 1μg/ml. On day 2, human serum (Access Biologicals) and rh IL-2 (Miltenyi Biotec) were added at final concentrations of 8% (volume/volume) and 20U/ml, respectively. Half of the medium was replaced with RPMI-1640 containing 8% human serum, rhIL-2 (20U/ml), and penicillin-streptomycin (100 U/ml, Quality Biological) on days 5, 7 and 9. HIV and EBV-specific CD8⁺ T cells in the culture were analyzed by flow cytometry on days 6 and 12.

Takata H., Kakazu J.C., Mitchell J.L., Kroon E., Colby D.J., Sacdalan C., Bai H., Ehrenberg P.K., Geretz A., Buranapraditkun S., Pinyakorn S., Intasan J., Tipsuk S., Suttichom D., Prueksakaew P., Chalermchai T., Chomchey N., Phanuphak N., de Souza M., Michael N.L., Robb M.L., Haddad E.K., Crowell T.A., Vasan S., Valcour V.G., Douek D.C., Thomas R., Rolland M., Chomont N., Ananworanich J, & Trautmann L. (2022). Long-term antiretroviral therapy initiated in acute HIV infection prevents residual dysfunction of HIV-specific CD8+ T cells. eBioMedicine, 84, 104253.

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GMP-Compliant Secretome Preparation from Irradiated PBMCs

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PBMCsec was produced in compliance with good manufacturing practice by the Austrian Red Cross, Blood Transfusion Service for Upper Austria (Linz, Austria) as previously described [30 (link),34 (link)]. Briefly, the PBMCs were enriched using Ficoll-Paque PLUS (GE Healthcare, Chicago IL, USA) density gradient centrifugation. Cell suspensions were adjusted to 2.5 × 10⁷ cells/mL and exposed to 60 Gy γ-irradiation (IBL 437, Isotopen Diagnostik CIS GmbH, Dreieich, Germany). Subsequently, cells were cultured in phenol red-free CellGenix GMP DC medium (CellGenix, Freiburg, Germany) for 24 h. Cells, as well as cellular debris, were removed by centrifugation. The conditioned supernatants containing the secretome were filtered through 0.22 µm filters followed by viral clearance using Theraflex methylene blue technology (MacoPharma, Mouvaux, France). The secretomes were lyophilized and sterilized by high-dose γ-irradiation (25,000 Gy, Gammatron 1500, Mediscan, Seibersdorf, Austria). CellGenix GMP DC medium without cells was used as vehicle control. The GMP batches A000918399131, A00918399136, and A000918399132 were used in this study. The stock concentration of one vial lyophilized secretome equals to 25 units/mL.

Klas K., Ondracek A.S., Hofbauer T.M., Mangold A., Pfisterer K., Laggner M., Copic D., Direder M., Bormann D., Ankersmit H.J, & Mildner M. (2022). The Effect of Paracrine Factors Released by Irradiated Peripheral Blood Mononuclear Cells on Neutrophil Extracellular Trap Formation. Antioxidants, 11(8), 1559.

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Lipidomic Analysis of Dendritic Cells

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Commercially available OVA (N = 3; Sigma-Aldrich, Cat. #A5503–1G, Lot #SLCB8249), nAFP (N = 3; Cell Sciences, Cat. #CSI10379, Lot #4111714), and tAFP (N = 3; Bio-Rad, Cat. #13752600, Lot #64110896) were submitted diluted in PBS (Gibco, Cat. #20–012–050) at 1,000 μg/mL on dry ice. CellGenix GMP DC Medium (N = 1; CellGenix, Cat. #20801–0500) media and supernatants of mDCs from an HD (N = 1) differentiated in the presence of 5 μg per mL of OVA, nAFP, or tAFP were tested. Lipid analysis (Supplementary Table S1) was performed at the UCSD Lipidomics Core (46 (link)).

Munson P.V., Adamik J., Hartmann F.J., Favaro P.M., Ho D., Bendall S.C., Combes A.J., Krummel M.F., Zhang K., Kelley R.K, & Butterfield L.H. (2023). Polyunsaturated Fatty Acid–Bound α-Fetoprotein Promotes Immune Suppression by Altering Human Dendritic Cell Metabolism. Cancer Research, 83(9), 1543-1557.

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