Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Ex vivo expanded autologous polyclonal regulatory T cells suppress inhibitor formation in hemophilia

doi: 10.1038/mtm.2014.30

Figure Lengend Snippet: Isolation and ex vivo expansion of murine CD4 + CD25 + FoxP3 + Treg. ( a ) Splenocytes are isolated from BALB/c mice with a GFP reporter gene knock-in at FoxP3 locus. GFP + cells are sorted to 95–98% purity from magnetically purified CD4 + splenocytes. ( b ) Expansion of Treg (starting with 1 × 10 6 cells) after two rounds of stimulation with anti-CD3/-CD28 beads. Data are average ±SD from 5 independent experiments. ( c ) Percent GFP + ( i.e. , FoxP3 expressing) cells as a function of days in culture.

Article Snippet: Mouse CD4 + T cell isolation kit, mouse pan DC isolation kit, LD, LS and MS columns, MACSiMag separator and mouse Treg expansion kit (CD3/CD28 paramagnetic beads) were from Miltenyi Biotec (Auburn, CA).

Techniques: Isolation, Ex Vivo, Gene Knock-In, Purification, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Ex vivo expanded autologous polyclonal regulatory T cells suppress inhibitor formation in hemophilia

doi: 10.1038/mtm.2014.30

Figure Lengend Snippet: Phenotype and in vivo persistence of expanded Treg. ( a ) CTLA-4, CD62L, and helios expression in CD4 + CD25 + FoxP3 + Treg (fresh after isolation or after 11 in culture; representative example, cells were stimulated with beads on days 0 and 7). ( b ) CTLA-4, CD62L, and helios expression in CD4 + CD25 + FoxP3 + Treg at days 0, 7, and 14 in culture (representative example, cells were stimulated with beads on days 0 and 7; data for days 0 and 7 are prior to addition of beads). ( c ) In vitro suppression assay for expanded Treg demonstrating Treg dose-dependent suppression of anti-CD3/CD28 bead stimulated BALB/c splenocytes (CellTrace Violet dilution assay). ( d ) CD80 and CD86 expression in CD11c + DC primed for 3 days with either freshly isolated Treg, ex vivo expanded Treg, or CD4 + CD25 − Teff cells. Data are percent DC that stained positive for CD80/86. Plots are representative of two independent experiments.

Article Snippet: Mouse CD4 + T cell isolation kit, mouse pan DC isolation kit, LD, LS and MS columns, MACSiMag separator and mouse Treg expansion kit (CD3/CD28 paramagnetic beads) were from Miltenyi Biotec (Auburn, CA).

Techniques: In Vivo, Expressing, Isolation, In Vitro, Suppression Assay, Dilution Assay, Ex Vivo, Staining

Journal: Blood cancer discovery

Article Title: Scalable Manufacturing of CAR T cells for Cancer Immunotherapy

doi: 10.1158/2643-3230.bcd-21-0084

Figure Lengend Snippet: The manufacturing process of clinical scale autologous CAR T cell therapies. The process starts with the isolation of PBMCs, collected either from whole blood phlebotomy or, more commonly, through a leukapheresis procedure in hospital apheresis units accredited by organizations such as the Foundation for the Accreditation of Cellular Therapy (FACT) and the Joint Accreditation Committee of ISCT-EBMT (JACIE). The PBMCs can be either cryopreserved locally and then shipped to centralized manufacturing facilities or transferred as fresh products to these centers. Often, density gradient centrifugation or elutriation is performed to reduce unwanted contaminating cells such as granulocytes, red blood cells, and platelets. The selection or depletion of specific T-cell types within the PBMCs is then performed. In some protocols, the cells are enriched for CD3+ T cells prior to or concurrent with their activation, at which point the cells are genetically modified using viral vectors or other nonviral gene delivery methods to express the CAR. The cells are then expanded in the presence of cytokines to a dose suitable for patient administration. In most protocols, the expanded cells are then formulated in an appropriate cryopreservation medium containing dimethyl sulfoxide (DMSO) and cryopreserved. After passing quality control and quality assurance lot release requirements, the gene-modified cells are shipped cryopreserved using a validated liquid nitrogen shipper to treatment locations, where the product is thawed under controlled conditions and infused into patients. AAPCs, artifical antigen-presenting cells. Created with BioRender.com.

Article Snippet: Following transduction, the cells undergo expansion to achieve the necessary clinical dose and, in most cases, are cryopreserved before infusion into patients. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Baylor College of Medicine ( 15 , 22 , 62 ) Memorial Sloan Kettering Cancer Center ( 10 , 65 ) University of Pennsylvania ( 9 , 11 ) Fred Hutchinson Cancer Research Center ( 51 , 53 , 55 ) National Institutes of Health (NIH; refs. ( 12 , 13 , 61 ) Source of the starting material PBMC isolation by density gradient Apheresis product, cell wash by Cytomate cell washer Apheresis product enriched by mononuclear cell elutriation, density gradient separation Apheresis product followed by CD4, CD8, or CD62L selection (CliniMACS) Apheresis product Fresh vs. frozen Either Frozen Either Either Fresh Culture vessels Flasks, plates, G-Rex Bags/rocking platform -WAVE bioreactor system Rocking platform -WAVE bioreactor system Culture bags Culture bags Activation Soluble OKT3, plate/flask-bound anti-CD3/28 CD3/CD28 antibody–coated paramagnetic beads CD3/CD28 antibody–coated paramagnetic beads CD3/CD28 antibodies conjugated to paramagnetic beads Soluble OKT3, anti-CD3/CD28 paramagnetic beads Expansion media Clicks Medium/RPMI1640 X-Vivo 15 X-Vivo 15 RPMI1640 AIMV medium Sera FBS Human serum Human serum Human serum Human serum Transduction enhancer RetroNectin RetroNectin RetroNectin Polybrene RetroNectin Spinoculation No No No Yes No Cytokines IL2, IL7/15 IL2 IL2 IL2 and feeders IL2 Open in a separate window NOTE: When CAR T cells were first explored in clinical trials, primarily single-center studies were conducted in academic settings, with manufacturing relying on open or semiclosed culture systems.

Techniques: Isolation, Gradient Centrifugation, Selection, Activation Assay, Genetically Modified, Modification

Journal: Blood cancer discovery

Article Title: Scalable Manufacturing of CAR T cells for Cancer Immunotherapy

doi: 10.1158/2643-3230.bcd-21-0084

Figure Lengend Snippet: Academic manufacturing protocols for CAR T cell therapy

Article Snippet: Following transduction, the cells undergo expansion to achieve the necessary clinical dose and, in most cases, are cryopreserved before infusion into patients. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Baylor College of Medicine ( 15 , 22 , 62 ) Memorial Sloan Kettering Cancer Center ( 10 , 65 ) University of Pennsylvania ( 9 , 11 ) Fred Hutchinson Cancer Research Center ( 51 , 53 , 55 ) National Institutes of Health (NIH; refs. ( 12 , 13 , 61 ) Source of the starting material PBMC isolation by density gradient Apheresis product, cell wash by Cytomate cell washer Apheresis product enriched by mononuclear cell elutriation, density gradient separation Apheresis product followed by CD4, CD8, or CD62L selection (CliniMACS) Apheresis product Fresh vs. frozen Either Frozen Either Either Fresh Culture vessels Flasks, plates, G-Rex Bags/rocking platform -WAVE bioreactor system Rocking platform -WAVE bioreactor system Culture bags Culture bags Activation Soluble OKT3, plate/flask-bound anti-CD3/28 CD3/CD28 antibody–coated paramagnetic beads CD3/CD28 antibody–coated paramagnetic beads CD3/CD28 antibodies conjugated to paramagnetic beads Soluble OKT3, anti-CD3/CD28 paramagnetic beads Expansion media Clicks Medium/RPMI1640 X-Vivo 15 X-Vivo 15 RPMI1640 AIMV medium Sera FBS Human serum Human serum Human serum Human serum Transduction enhancer RetroNectin RetroNectin RetroNectin Polybrene RetroNectin Spinoculation No No No Yes No Cytokines IL2, IL7/15 IL2 IL2 IL2 and feeders IL2 Open in a separate window NOTE: When CAR T cells were first explored in clinical trials, primarily single-center studies were conducted in academic settings, with manufacturing relying on open or semiclosed culture systems.

Techniques: Isolation, Selection, Activation Assay, Transduction