Blood samples of 100 mL were collected after confirming diagnosis and before initiation of treatment. PBMCs were isolated by centrifugation over Histopaque 1077 solution (Sigma-Aldrich) according to the product instructions. The cells were frozen in 90% FBS plus 10% DMSO by slow cooling at approximately 1°C/minute using a freezing container (Thermo Scientific) and stored in liquid nitrogen until the time of experimental evaluation. Prior to each experiment, the cells were rapidly thawed at 37°C and PBMCs with ≥ 90% viability were used for the experiments [30 (link)].
Freezing container
Manufactured by Thermo Fisher Scientific
Sourced in Canada, United States
The Freezing container is a laboratory equipment designed to store and preserve samples at ultra-low temperatures. It provides a controlled environment for the storage of materials that require cryogenic conditions.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Cryotube, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Vacutainer cpt system, by BD (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Cryovial, by Thermo Fisher Scientific (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Phase contrast microscope, by Olympus (1 mentions)
Cryogenic vials, by Merck Group (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
8 protocols using freezing container
1
Cryopreservation of Human PBMCs
2
PBMC Isolation and Cryopreservation Protocol
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Blood from study participants was drawn with the Vacutainer CPT™ system into sodium citrate CPT tubes (Becton Dickinson Biosciences), and the tubes were mixed five times before storing them upright at room temperature. Within 2 h of blood collection, CPT tubes were centrifuged in a horizontal rotor (swing-out head) (1,800 g, 20 min, RT). Next, plasma was removed and peripheral blood mononuclear cells (PBMCs) were transferred to 50 ml polystyrene Falcon tubes and mixed with 10 ml of PBS by gently inverting the tubes five times and filled up to 45 ml with PBS. PBMCs were centrifuged (300 g, 10 min, RT) twice in PBS. Tuerk solution staining (Morphisto GmbH) was used for cell counting under the microscope. PBMCs were cryopreserved per vial in 1.8 ml cryotubes (Thermo Scientific) at a concentration of 1 × 107 PBMCs per 1 ml of freezing medium/[fetal calf serum (FCS) (Life Technologies)], supplemented with 10% DMSO (Sigma-Aldrich), using a freezing container (Thermo Scientific) and stored at −80°C. After 24 h, PBMCs were stored in the vapor phase of a liquid nitrogen tank until further use.
3
Isolation and Cryopreservation of Stromal Vascular Fraction
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The remaining tissue was digested using type I collagenase (Worthington Biochemical, Lakewood Township, NJ, USA) at 250 U ml−1 in phosphate-buffered saline and 2% bovine serum albumin (pH 7.4) for ~45 min in a shaking water bath (220 r.p.m.) at 37 °C. The suspension was filtered and subject to brief, gentle centrifugation (10 s at 100 g) before aspiration of adipocytes. After further centrifugation (10 min at 300 g), the remaining pellet was resuspended in erythrocyte lysis buffer for 10 min before a final centrifugation step (5 min at 300 g).3 (link) SVF pellets were stored in 1 ml phosphate-buffered saline:foetal bovine serum:dimethyl sulphoxide (5:4:1) and frozen at a rate of −1 °C min−1 using a freezing container (Thermo Scientific) to −80 °C and stored before subsequent analysis by flow cytometry.
4
Cryopreservation and Resuscitation of Tumour Samples
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Tumours were placed in cryovials, covered with freezing medium (DMEM/F12 GlutaMAX (Gibco, 41965–039), 40% FBS (Gibco, 10500064), 6% DMSO), placed immediately into a freezing container (ThermoFisher) followed by −80 °C overnight incubation. Cryovials were then stored in liquid nitrogen until required. For resuscitation cryovials were from hand warmed. Once thawing was first observed, room temperature medium was added. Tumours were decanted into tubes containing fresh tumour medium (DMEM/F12, 10% FCS, 1x Penicillin/Streptomycin (Invitrogen, 15140122), 5 μg/ml Insulin (Sigma, 11882), 10 ng/ml Epidermal Growth Factor (Sigma, E4127)) making sure not to carry across any freezing medium. Decanting was repeated to wash away any remaining freezing medium. Tumours were mechanically fragmented using a scalpel into 1–3 mm3 pieces for tumour fragment seeding.
5
Cryopreservation of Keratinocyte Populations
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Keratinocyte populations s1 and s2 were isolated and primocultured until they reached 70–95% confluence. Keratinocytes were then trypsinized and resuspended at 2 × 106 cells/mL (s1) and 4 × 106 cells/mL (s2) in cryopreservation medium (See Appendix B ). Cryovials (Thermo Fisher Scientific) containing 1 mL aliquots of the cell suspensions were kept on ice during handling and were rapidly transferred into a freezing container (Thermo Fisher Scientific) filled with 4 °C 100% isopropyl alcohol (Commercial Alcohols, Boucherville, QC, Canada). The container was then placed at −80 °C overnight and Cryovials were transferred into liquid nitrogen. Keratinocytes were kept frozen in liquid nitrogen for 14 (s1) and 11 (s2) years prior to this study.
Keratinocyte populations s1 and s2 were thawed by placing the Cryovials in a 37 °C water bath for no more than 1 min. The cell suspensions were then transferred into 4 °C cAMP inducer-free ckDME-Ham and centrifuged at 300× g for 10 min. Keratinocytes were then resuspended in cAMP inducer-free ckDME-Ham and seeded in P1. Mortality rates were estimated with trypan blue (Sigma-Aldrich) staining and a hemacytometer under a phase-contrast microscope (Olympus, Toronto, ON, Canada). Mortality never exceeded 7%.
Keratinocyte populations s1 and s2 were thawed by placing the Cryovials in a 37 °C water bath for no more than 1 min. The cell suspensions were then transferred into 4 °C cAMP inducer-free ckDME-Ham and centrifuged at 300× g for 10 min. Keratinocytes were then resuspended in cAMP inducer-free ckDME-Ham and seeded in P1. Mortality rates were estimated with trypan blue (Sigma-Aldrich) staining and a hemacytometer under a phase-contrast microscope (Olympus, Toronto, ON, Canada). Mortality never exceeded 7%.
6
Cryopreservation of Human PBMCs
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Human peripheral blood mononuclear cells (PBMCs) were isolated from 7 mL of venous blood, which was drawn before surgery, using the density gradient separation (Ficoll‐Paque™ Plus; GE Healthcare, Chicago, IL, USA). PBMCs were separated by low‐speed centrifugation of 400 g for 30 min, and this step was completed with the centrifuge ‘brake off’. PBMCs were then collected from the interphase layer and washed with RPMI 1640 medium (Gibco, Life Technologies, Carlsbad, CA, USA). Cell pellets were suspended in 1 mL freezing medium composed of 200 μL dimethyl sulfoxide (DMSO; Sigma‐Aldrich, St. Louis, MO, USA) and 800 μL foetal bovine serum (FBS, Gibco, Life Technologies) and transferred into 1.2 mL cryogenic vials (Sigma‐Aldrich), which were subsequently transferred into a freezing container (Thermo Fisher Scientific, Waltham, MA, USA) that had a cooling rate of −1°C per min. After 24 h at −80°C, cryogenic vials were transferred to liquid nitrogen (−196°C) for long‐term storage.
7
PBMC Isolation and Cryopreservation
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Methodology used for PBMC preparation is identical to what we have previously published.42 PBMCs were isolated from blood samples (100 mL) from each subject at each timepoint pre- and post-vaccination using cell preparation tubes with sodium citrate (CPT™), as previously published.42 Purified PBMCs were resuspended at a concentration of 1×107/mL in RPMI-1640 medium containing L-glutamine (Invitrogen), supplemented with 10% dimethyl sulfoxide (DMSO) and 20% fetal calf serum (Hyclone). Cells were frozen overnight at 80°C in freezing containers (Thermo Fisher Scientific) to reach an optimal rate of cooling and then transferred for storage to liquid nitrogen.
8
PBMC Isolation and Cryopreservation
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