Wild-type hiPSCs PC056c2 were derived by Phenocell (Grasse, France) from human primary fibroblasts using Sendai vectors [2 (link)] and cultured with StemMACS iPS-Brew XF medium (Miltenyi Biotec) in vitronectin (Gibco-coated dishes). Culture medium was changed three times a week. Cells were passaged following gentle harvesting with EDTA 0.25 mM (Invitrogen every 5–7 days). For CRISPR/Cas9 editing experiments, hiPSCs were pretreated with a rock inhibitor (Y-27632; Tocris) at least one-hour prior transfection and harvested with an enzymatic treatment (Stempro Accutase cell dissociation reagent; Life technologies). Following amplification of single cell-derived clones, cells were banked using CryoStor CS10 (Sigma-Aldrich).
Cryostor cs10
Manufactured by Merck Group
Sourced in United States
CryoStor CS10 is a cryopreservation media product from Merck Group. It is designed for the storage and preservation of cells and tissues at low temperatures.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Roche (3 mentions)
Liberase tl, by Roche (2 mentions)
Facsaria fusion, by BD (2 mentions)
X vivo 15, by Lonza (2 mentions)
Facsaria 2, by BD (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Stempro accutase cell dissociation reagent, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Bio-Techne (1 mentions)
Stemmacs ips brew xf medium, by Miltenyi Biotec (1 mentions)
Recovery cell culture freezing medium, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Ficoll paque density gradient centrifugation, by Merck Group (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Ack buffer, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Worthington (1 mentions)
Raji cell, by American Type Culture Collection (1 mentions)
Clinimacs pbs edta buffer, by Miltenyi Biotec (1 mentions)
Clinimacs plus instrument, by Miltenyi Biotec (1 mentions)
Poch 100i automated hematology analyzer, by Sysmex (1 mentions)
23 protocols using cryostor cs10
1
Derivation and Cryopreservation of Wild-type hiPSCs
2
Clonal Expansion and Cryopreservation of Edited Cells
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FACS-enriched populations of edited cells were seeded at a density of 1 × 104 cells in a 10-cm GFR Matrigel–coated tissue culture plate. After 5–7 d, clones were manually picked with a pipette and transferred into individual wells of 96-well GFR Matrigel–coated tissue culture plates with mTeSR1 supplemented with 1% P/S and 10 µM ROCK inhibitor for 1 d. After 3–4 d of normal maintenance with mTeSR1 supplemented with 1% P/S, colonies were dispersed with Accutase and transferred into a fresh GFR Matrigel–coated 96-well plate. After recovery, the plate was divided into daughter plates for ongoing culture, freezing, and gDNA isolation.
For cryopreservation of clones in a 96-well format, when cells were 60–85% confluent, they were dissociated and pelleted in 96-well V-bottom plates. Cells were then resuspended in 60 µl mTeSR1 supplemented with 1% P/S and 10 µM ROCK inhibitor. Two sister plates were frozen using 30 µl cell suspension per plate, added to 170 µl CryoStor CS10 (Sigma) in non–GFR Matrigel coated 96-well tissue culture plates. Plates were sealed with Parafilm and stored at −80°C.
For cryopreservation of clones in a 96-well format, when cells were 60–85% confluent, they were dissociated and pelleted in 96-well V-bottom plates. Cells were then resuspended in 60 µl mTeSR1 supplemented with 1% P/S and 10 µM ROCK inhibitor. Two sister plates were frozen using 30 µl cell suspension per plate, added to 170 µl CryoStor CS10 (Sigma) in non–GFR Matrigel coated 96-well tissue culture plates. Plates were sealed with Parafilm and stored at −80°C.
3
Psoriatic Arthritis Patient Sample Collection
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Blood and synovial fluid samples were collected with written informed patient consent from PsA patients undergoing intra-articular aspiration at Oxford University Hospitals. The study was performed in accordance with protocols approved by the Oxford Research Ethics committee (Ethics reference number 06/Q1606/139). Synovial tissue was obtained from knee joints of PsA patients prior to commencement of any immunomodulatory therapy, including systemic corticosteroids by a minimally invasive, ultrasound-guided technique with written informed consent30 (link). Protocols for these procedures were approved by relevant research ethics committees (reference number 12/NE/0251 Newcastle and West Midlands Black Country Research Ethics committee 07/H1203/57 (Birmingham)). Upon collection of biopsies, they were immediately cryopreserved in cryopreservation media Cryostor CS10 (Sigma—MERCK)31 (link). Demographics for all patients enrolled in this study are listed in Supplementary Table 1 .
4
Profiling CD8+ T Cells from Synovial Tissue
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Whole blood was collected and PBMCs were isolated using Ficoll-Paque density gradient centrifugation (Sigma Aldrich, GE17-1440-03) for cellular profiling of CD8+ T cells. Cells were cryopreserved in Recovery Cell Culture Freezing Medium (Thermo Fisher Scientific, 12648010). For synovial tissue disaggregation, cryopreserved synovial tissues in Cryostor CS10 (Sigma-Aldrich, C2874-100ML) were obtained from hospital for special surgery. Each synovial tissue specimen was fully thawed at 37 °C and rinsed with RPMI 1640 (Corning Life Science, MT15040CV) containing 10% FBS (ATCC, 30-2020) and 1% glutamine (Gibco, 25030081). The tissue fragments were minced and digested with 100 μg/mL Liberase TL (Roche, 05401020001) and 100 μg/mL DNase I (Roche, 4716728001) in RPMI 1640 for 30 min at 37 °C with inverting to disaggregate the fragments into single cell suspensions. The digested fragments were filtered through a 70 μm cell strainer and washed with cold RPMI 1640 with 10% FBS and 1% glutamine before antibody staining and 10X single cell RNA sequencing.
5
Cryopreservation of Human PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated from heparinised samples of whole blood via density gradient centrifugation over Lymphoprep (Stem Cell Technologies, Cambridge, UK). Aliquots of 5 × 106 to 20 × 106 PBMCs/ml per vial were stored in liquid nitrogen after cooling overnight to −80°C at a controlled rate of −1°C/min in a Cryostor CS10 (Sigma-Aldrich, St Louis, MO, USA).
6
Cryopreserved Synovial Tissue Immune Cell Isolation
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For this study, patients were recruited and consented through the Accelerating Medicines Partnership (AMP) Network for RA and SLE (Zhang et al. 2022 (link)). Synovial tissue samples and matched peripheral blood mononuclear cells were cryopreserved after collection as described (Donlin et al. 2018 (link)). Stored synovial tissue samples were then thawed and disaggregated into single-cell suspensions by mincing and digesting with 100 μg/mL LiberaseTL (Roche) and 100 μg/mL DNaseI (Roche) in RPMI (Life Technologies) for 15 min, with occasional inversion during disaggregation. Disaggregated cells were passed through a 70 μm cell strainer and washed prior to antibody staining with anti-CD235a antibodies (clone 11E4B-7–6 (KC16), Beckman Coulter) and Fixable Viability Dye eFluor 780 (eBioscience/ThermoFisher). Live non-erythrocyte cells (viability dye- CD235−) were collected by fluorescence-activated cell sorting (BD FACSAria Fusion) and were initially cryopreserved in Cryostor CS10 (Sigma-Aldrich). The disaggregated synovial tissue cells and matched cryopreserved peripheral blood mononuclear cells were then thawed in batches, and both T and B cells were collected by fluorescence-activated cell sorting (BD FACSAria II) (Figure S1B , Table S4 ).
7
Tissue Dissociation Protocol for Single-Cell Suspension
8
In Vitro Cytotoxicity Assay for CAR T Cells
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At 24 h post-delivery of CD19 CAR mRNA, cells were cryopreserved in CryoStor CS10 (Sigma-Aldrich, St Louis, MO, USA). In vitro cytotoxicity was measured with an impedance assay using the xCELLigence real-time cell analyzer single plate instrument (ACEA Biosciences). Wells of the electronic microtiter plates were coated with 4 µg/mL CD40 (ACEA Biosciences) for 3 h. Raji cells (American Type Culture Collection, Manassas, VA, USA) were seeded at 5 × 104 cells/well and allowed to adhere overnight. At 19–21 h later, CAR T cells were thawed and added to the Raji cells at E:T ratios of 2.5:1, 1.25:1, 0.6:1 and 0.1:1. Impedance was monitored every 1 min for 4 h, every 5 min for 8 h and then every 15 min for at least 92 h. Cell indexes were normalized to a cell index of the time point when CAR T cells were added and specific lysis was calculated in comparison with control effector cell-only cultures.
9
Isolation and Cryopreservation of CD3+ T Cells
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CD3+ cells were purified from healthy donors’ Leukopaks received from Hemacare (Northridge, CA, USA). Upon reception, the leukapheresis material was analyzed using the Sysmex pocH-100i Automated Hematology Analyzer (Sysmex, Milton Keynes, UK), diluted with CliniMACS PBS/EDTA buffer (Miltenyi, Bisley, UK), supplemented with 0.5% human AB serum (Seralab, Hayward Heath, UK) and centrifuged at 300 × g for 15 min at room temperature to remove platelets. The CD3+ fraction was purified from the plasma on the CliniMACS Plus instrument (Miltenyi) using the CliniMACS CD4 and CD8 reagents (Miltenyi) anti-CD4 and anti-CD8 monoclonal antibodies conjugated to superparamagnetic iron dextran particles. The viability of the final CD3+ product was assessed using the Vi-CELL XR (Beckman Coulter, High Wycombe, UK) and Nucleocounter NC-200 (ChemoMetec, Allerod, Denmark). The number of CD45+ cells, CD3+ cells, CD4+ cells, and CD8+ cells was evaluated before and after the cell separation on the CliniMACS by flow cytometry on the Fortessa (BD Biosciences, San Jose, CA, USA). The acceptance criteria for the CD3+ cell purification were set as follows: (1) viability over 95% and (2) number of CD3+ cells over 95%. Following purification, a working cell bank of CD3+ cells was cryopreserved in CryoStor CS10 (Sigma, Gillingham, UK) until use.
10
Cryopreserved PBMC Isolation and Revival
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Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from heparinized syringes using Ficoll density gradient centrifugation. All isolated PBMCs were cryopreserved in freezing media (CryoStor CS10, Sigma-Aldrich) and stored in liquid nitrogen until use. Revived cells were incubated at 37°C in humidified air containing 5% CO2. A fresh batch of culture media was made every 7 days. PBMCs were obtained under ethical approvals and with informed consent as described in the Human Subjects section.
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