Single-cell suspensions obtained from SLNs were resuspended in X-VIVO™ 15 serum-free cell culture medium (LONZA) at a density of 4 × 106 cells/ml in the presence of 1000 IU/ml recombinant human interleukin-2 (Shuanglu, China). These cells were plated in flasks or plates and maintained in a humidified atmosphere containing 5 % CO2 at 37 °C. The autologous tumor lysate was added to the initial culture at a dilution of 1/100 (v/v) as described previously [15 (link)]. To induce highly tumor-specific SLN-T cells, re-stimulation was performed by adding autologous tumor lysate together with irradiated autologous PBMCs during SLN-T cell cultures. One week before transfusion, 5 ml of culture medium was removed for a bacterial and fungal contamination test using BACTEC 9120 (Becton–Dickinson), and the endotoxin levels were measured based on the Limulus reaction. On the day of transfusion, these assays were repeated to detect any bacterial, fungal or endotoxin contamination. The lymphocyte subsets of SLN-T cells were analyzed. Furthermore, 1 × 106 cells were used for flow cytometry analysis of the tumor surface marker epithelial cell adhesion molecule (EpCAM) to exclude the presence of tumor cells.
X vivo 15 serum free cell culture medium
Manufactured by Lonza
Sourced in Switzerland
X-VIVO 15 is a serum-free cell-culture medium. It is formulated to support the growth and maintenance of a variety of cell types in vitro.
Automatically generated - may contain errors
6 protocols using x vivo 15 serum free cell culture medium
1
Generating Tumor-Specific SLN-T Cells
2
Isolation and Culture of UCB-MNC
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Human UCB samples were obtained upon signed informed consent, in compliance with Portuguese legislation. Donations were approved by the ethical committees of Centro Hospitalar e Universitário de Coimbra, Portugal. Samples were stored and transported to the laboratory in sterile bags with anticoagulant solution (citrate‐phosphate‐dextrose) and processed within 48 hours after collection.
Two million UCB‐MNC/mL were cultured in X‐VIVO 15 serum‐free cell‐culture medium (Lonza Group, Ltd, Basel, Switzerland), supplemented with 0.5 μg/mL of FMS‐like tyrosine kinase‐3 and 0.5 μg/mL of stem‐cell factor, under ischemia (0.5% O2). After 18 hours, conditioned media (CM) was collected for small extracellular vesicle (sEV) purification.
Two million UCB‐MNC/mL were cultured in X‐VIVO 15 serum‐free cell‐culture medium (Lonza Group, Ltd, Basel, Switzerland), supplemented with 0.5 μg/mL of FMS‐like tyrosine kinase‐3 and 0.5 μg/mL of stem‐cell factor, under ischemia (0.5% O2). After 18 hours, conditioned media (CM) was collected for small extracellular vesicle (sEV) purification.
3
Isolation and Purification of Extracellular Vesicles from Umbilical Cord Blood
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Human UCB samples were obtained upon signed informed consent, in compliance with Portuguese legislation. The collection was approved by the ethical committee of Centro Hospitalar e Universitário de Coimbra, Portugal. Samples were stored and transported to the laboratory in sterile bags with an anticoagulant solution (citrate-phosphate-dextrose) and processed within 48 h after collection. UCB units were processed in an accredited cryobank (Crioestaminal, Cantanhede, Portugal) using an automated system AXP, according to the manufacturer’s recommendations.
After at least one week of storage, UCB-MNC were thawed and cultured at 2 million cells/mL in X-VIVO 15 serum-free cell-culture medium (Lonza, Basel, Switzerland), supplemented with 0.5 μg/mL of FMS-like tyrosine kinase-3 and 0.5 μg/mL of stem-cell factor, under ischemia (0.5% O2) conditions. Following 18 h of secretion, conditioned media were cleared by centrifugation and filtration, followed by ultrafiltration at 3 bar with a 100 kDa filter (Sartorius, Goettingen, Germany). Finally, the concentrated conditioned medium underwent size exclusion chromatography, and EV-enriched fractions were collected, concentrated, and stored at −80 °C until further use. A more detailed description of the EV purification process is published elsewhere (19).
After at least one week of storage, UCB-MNC were thawed and cultured at 2 million cells/mL in X-VIVO 15 serum-free cell-culture medium (Lonza, Basel, Switzerland), supplemented with 0.5 μg/mL of FMS-like tyrosine kinase-3 and 0.5 μg/mL of stem-cell factor, under ischemia (0.5% O2) conditions. Following 18 h of secretion, conditioned media were cleared by centrifugation and filtration, followed by ultrafiltration at 3 bar with a 100 kDa filter (Sartorius, Goettingen, Germany). Finally, the concentrated conditioned medium underwent size exclusion chromatography, and EV-enriched fractions were collected, concentrated, and stored at −80 °C until further use. A more detailed description of the EV purification process is published elsewhere (19).
4
Isolation and Purification of Small Extracellular Vesicles
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hUCBMNCs, isolated and stored as described above, were thawed in warm X-VIVO medium and immediately treated with DNAse I to degrade DNA arising from death cells (due to the freeze-thawing process). Subsequently, cells were centrifuged at 300 × g for 5 min and 2 × 106 cells/mL were cultured in X-VIVO 15 serum-free cell-culture medium (Lonza) supplemented with 100 ng/mL Flt-3 and 100 ng/mL stem cell factor (both from PeproTech) under hypoxia conditions (0.5% O2) for 18 h. sEVs were purified from the conditioned medium (CM) by differential centrifugation as previously described.58 In brief, CM was collected and centrifuged at 300 × g, for 10 min at 4°C to remove cells, followed by a centrifugation at 2,000 × g for 20 min at 4°C to deplete cellular debris. Subsequently, samples were ultracentrifuged twice at 10,000 × g for 30 min at 4°C, the pellet was discarded, and the supernatant was submitted to an ultracentrifugation at 100,000 × g, for 2 h at 4°C to pellet sEVs. Finally, the pellet from the last step was washed with cold PBS, ultracentrifuged again at 100,000 × g for 2 h at 4°C, resuspended in 150 μL of cold PBS, and stored at −80°C. Ultracentrifugation steps were performed using a swinging bucket rotor SW 32 Ti in an Optima XPN 100K ultracentrifuge (Beckman Coulter, CA, USA) and 28.7-mL polyallomer conical tubes (Beckman Coulter).
5
Primary Human CD4+ T-cell Culture
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1-mL vials of naïve CD4+ T-cells from a human donor were purchased (Astarte Biologics, Bothell, WA, USA) and stored in the vapor phase of liquid nitrogen until ready for culture. The cells were thawed in a 37 °C water bath for 1–2 min or until pipettable, transferred to a 15-mL conical tube, and resuspended in 9 mL of 37 °C X-VIVO 15 serum-free cell culture medium (Lonza, Basel, Switzerland). The cells were pelleted via centrifugation at 200× g for 10 min, the supernatant was removed, and the pellet was resuspended in 10 mL of 37 °C culture medium before determining live cell concentration with Trypan Blue (Gibco Laboratories, Gaithersburg, MD, USA) and manual hemocytometer counts. The cell concentration was adjusted to 166,667 cells/mL with additional 37 °C medium to achieve desired seeding number (50,000 cells/well) before addition of Dynabeads Human T-Activator CD3/CD28 activation beads (Gibco Laboratories) in a 1:1 (bead:cell) ratio. The solution was then plated in 300 µL volumes on two round-bottom polypropylene 96-well plates from Corning Life Sciences (Corning, NY, USA) and incubated at 37 °C for three days.
6
Isolation and Culture of UCB-MNCs
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Human UCB samples were obtained upon signed informed consent, in compliance with Portuguese legislation. Donations were approved by the ethical committees of Centro Hospitalar e Universitário de Coimbra, Portugal. Samples were stored and transported to the laboratory in sterile bags with anticoagulant solution (citrate-phosphate-dextrose) and processed within 48 hours after collection.
2 million UCB-MNC/mL were cultured in X-VIVO 15 serum-free cell-culture medium (Lonza Group Ltd, Basel, Switzerland), supplemented with 0.5 g/mL of FMS-like tyrosine kinase-3 and 0.5 g/mL of stem-cell factor, under ischemia (0.5% O2). After 18 hours, conditioned media (CM) was collected for sEV purification.
2 million UCB-MNC/mL were cultured in X-VIVO 15 serum-free cell-culture medium (Lonza Group Ltd, Basel, Switzerland), supplemented with 0.5 g/mL of FMS-like tyrosine kinase-3 and 0.5 g/mL of stem-cell factor, under ischemia (0.5% O2). After 18 hours, conditioned media (CM) was collected for sEV purification.
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