T cell spinoculation protocol. After washing the wells with PBS (step 6 above), add .

T cell spinoculation protocol 5-FU is a nucleotide analogue that is incorporated into DNA during the S-phase of the cell cycle. Representative flow plots of the expression of CD44 and CD62L in CD8 and CD4 T cells following transduction by spinoculation in the presence of The results indicated that viral infection and transduction rates were more efficient and effective for T cells in spinoculation with retronectin compared to other groups. The following protocol has been developed from the literature 1 for use in a 6-well plate with Hav- ing shown that spinoculation enhances HIV-1 IIIB particle ad- sorption to CEM-SS cells, we wished to confirm the generality of this mechanism by testing two additional T-cell lines, CD4 This protocol is for infection of adherent cells (such as HEK293t) with prepackaged lentivirus. The transduction efficiency is extremely variable and donor There is a growing need to close and automate manufacturing of chimeric antigen receptor (CAR)-T cell therapies. T-cells were stimulated with CD3/28 dynabeads before being divided between 6 different conditions: Bag centrifuge, 2 i. Edit and publish protocols, collaborate in communities, share insights through comments, and track progress with run records. For our clinical immunogene therapy study for the treatment of renal cell carcinoma (RCC) patients, we had developed a protocol for gene transduction and expansion of human T cells in compliance with good manufacturing practice (GMP) criteria. For experimental details see Table 2. Dilute the cells in media to the following concentrations: a. Learn how to streamline this step while keeping flexibility during process development. Check the cells under the microscope over the next 2-4 days and note any significant cell death and/or debris in any of the conditions. Chimeric antigen receptor (CAR) T cell therapy is undoubtedly leading a revolution in cancer therapy. Particularly, cost-effectiveness considerations of cell production protocols are of importance for conducting clinical studies. The following protocol has been developed from the literature 1 for use in a 6-well plate with The MISSION ® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. Producing dry macroporous alginate scaffolds to mediate transduction is relatively simple and is a suitable low-cost replacement for the conventional method. 44% of cells exposed to The MISSION ® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. 05 between all groups) (A) and relative change in transgene expression (p < . 3,4 Cell and gene therapies are finally becoming viable patient treatment options, with both T cell- and hematopoietic stem cell (HSC)-based therapies being approved to market in Europe. c. Signaling Pathway Reporters; Bioluminescent Imaging Vectors; Protocol: Spinoculation of Suspension Cells Protocol: TransDux Protocol: MC-Easy Minicircle Production Protocol: Validate Cas9 Expression We have used this protocol to generate CAR T cells with multiple specificities, including EGFRvIII and CD19, and have used the Thy1. One of the most popular ways is spinoculation. 1 single-use kit, offers a flexible, automated, and closed solution for T-cell lentiviral Moreover, the upstream preparation of T cells, as well as the method and reagents used to expand transduced T cells, can impact performance. The protocol was carried out in parallel with an overnight incubation of virus with cells in the presence of polybrene and with transductions conducted on fibronection-coated plates. 30 Although this strategy has not been further validated in preclinical (murine) T cell gene transfer studies, spinoculation may increase the transduction efficiency. SpinOculation C The MISSION ® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. Author links open overlay panel Léa Rosselle 1 8 9, Thibault Leray 1 2 8, Sandy Joaquina 1 2, Benjamin Caulier 1 3 4, Pre-warm the centrifuge to 32°C before performing the spinoculation of the plate with the T cell/virus mix. 27 However, selection of complex phenotypes would require MOIs using either a RetroNectin reagent + spinoculation protocol or the transduction sponge for 24 hr. 4. However, standardized protocols for their generation are limited. After washing the wells with PBS (step 6 above), add . After the centrifugation step, transduction media were replaced with Count the cells and seed them in a concentration of 1 × 10 6 cells per ml using T-cell activation medium (see Note 6) in a 24-well plate. Note: This density is optimal for TCR-mediated T cell activation in our experiments. Critical to our successful clinical-scale transductions of patient T cells was the use of Retronectin in combination with of T cells in a functionally closed system Katharina Drechsel, Constanze Radek, Carolin Kolbe, Daniela Mauer, Kristina Schweizer, Nadine Mockel-Tenbrinck, Thomas Schaser, Ian C. Why do most CAR-T cell CAR-T cell manufacture has remained consistent over the years, enhance antitumor responses. Protocol for production of tonic CAR T cells with dasatinib. Materials and Methods To rule out a possibility that the enhancement of HBV infectivity by spinoculation is limited to this particular HepG2-NTCP12 cell line, we tested spinoculation protocol in another HepG2/NTCP cell line established recently by Seeger et al , and found that the HBV infection efficiency in HepG2/NTCP cells was markedly enhanced by spinoculation as For spinoculation (optional), centrifuge the plates with cells and virus at low speed (800-1000 g) for 1-2 hours at 32°C, then incubate at 37°C with 5% CO₂. 1. Some of these modifications include spinoculation,2 magnetic transduction (see MISSION® ExpressMag® This protocol is for infection of adherent cells (such as HEK293t) with prepackaged lentivirus. It assumes that the four factor retrovirus being used has a high titer and that the BJ Fibroblasts being used are growing robustly. 26 Pre-enrichment of CD62L+ cells has been included in some protocols and can be easily achieved with the CliniMACS system. SpinOculation C With the use of this protocol, CB CD8 + T cells are more susceptible to LV transduction compared to PB CD8 + T cells. , 2019; Yang and Jiang, 2020). The following protocol has been developed from the literature 1 for use in a 6-well plate with Transducing cells while centrifuging (‘spinfection’ or ‘spinoculation’), is a method to achieve efficient transduction of a large number of cells. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. 5E6 cells/mL in 6 mL total (low-density) STAR Protocols is an open access, peer-reviewed journal from Cell Press. less T cell enrichment process Protocol software parameters (Fig 2) Patient dependent SpinOculation C-Pro protocol software dependent Lentiviral vector (LVV) stability SpinOculation C-Pro protocol software, combined with Sepax C-Pro instrument and CT-60. Close and automate T cell transduction using SpinOculation C-Pro software with the Sepax C-Pro instrument and single-use kit. virus are used by standard protocols to overcome the limitations of hard-transduced cells like primary T cells. In T cells, spinoculation for The spinoculation protocol is very flexible, and allows for user adjustment of volume, cell concentration, duration of spin, and g-force at every step. e process has proven to decrease techni-cian hands-on time, decrease the overall processing time, In preparation for a clinical phase I/II adoptive immunogene therapy study for renal cell carcinoma (RCC) patients, we developed a protocol that meets GMP criteria regarding the gene transduction and expansion of primary human T lymphocytes [3, 10]. 3. After centrifugation, the viral medium was removed and the cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 200 IU/ml Results: In this study, activated T cells were transduced using different transduction methods such as spinoculation, retronectin, polybrene, spinoculation + retronectin, and spinoculation + polybrene after selecting the most efficient transfection method to produce recombinant viral particles containing MUC1 CAR. Re-suspend the cells thoroughly in fresh, warm media and count. 26 Pre-enrichment of CD62L+ cells has been included in some protocols and can be easily achieved with the transduce isolated cells by spinoculation) Rotea low-volume recovery small- to mid-scale cell processing T cell, NK cell, and MSC wash The protocol presented here introduces a new method for viral gene transfer of T cells without the need for spinoculation of retronectin-coated plates. • To enable gamma Lentivirus-mediated gene transfer is an efficient method to introduce a variety of transgenes to human T cells. × Please change the country on your profile in order to switch to another country store. The following protocol has been developed from the literature 1 for use in a 6-well plate with NY-ESO-1-specific T cells have shown promising activity in the treatment of soft tissue sarcoma (STS). Initial evaluation of this method The MISSION ® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. The use of high concentrations of the virus activated T cells transduced by spinoculation + retronec-tin showed that 63. T cells obtained from four patients and three healthy individuals were transduced in X-fold bags and in CultiLife bags according to our static transduction protocol, and in CultiLife bags according to the Takara spinoculation transduction protocol, as recommended for CultiLife bags. The following protocol has been developed from the literature 1 for use in a 6-well plate with concentration that will yield the highest percentage of successfully infected cells. ) or hard-to-transduce cell types. In this study, two different NY-ESO-1-specific T cell production However, the same optimised protocol and using the same VLPs failed to transduce primary human CD4 + T cells. For the spinoculation, T cells were mixed and incubated with the LV for 30 min at room temperature and centrifuged for 30 min at 800 × g at 32°C. To streamline this step while keeping flexibility during process development, we introduced a stand-alone protocol software that uses existing Sepax C-Pro technology. The protocol was performed in parallel with an overnight incubation of virus with cells in the presence of polybrene and with transductions conducted on fibronection-coated plates. The following protocol has been developed from the literature 1 for use in a 6-well plate with T-cells and further cell expansion are challenging. By eliminating magnetic T cell activation beads, sequential To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell In this study, activated T cells were transduced using different transduction methods such as spinoculation, retronectin, polybrene, spinoculation + retronectin, and A combination of spinoculation and poloxamer-based chemical adjuvant increases the transduction of primary T-cells by greater than twofold. The following protocol has been developed from the literature 1 for use in a 6-well plate with A combination of spinoculation and poloxamer-based chemical adjuvant increases the transduction of primary T-cells by greater than twofold. However, 34. Open in a new tab The transfection protocol was performed by three methods; lipofectamine2000™ Reagent (Invitrogen), polyethyleneimine (PEI) (Sigma), and The concentrating effect of spinoculation. The combination of poloxamer-based SpinOculation C-Pro protocol software automates lentiviral vector (LVV) transduction and maintains a functionally closed system within a single-use disposable kit, and Variables found to be critical to high gene transfer and subsequent efficient T cell expansion included CD3/CD28 magnetic bead to cell ratio, time from T cell activation to initial spinoculation, frequency of T cell spinoculation, interleukin-2 concentration in the medium, and the initial purity of the T cell preparation. There is a growing need to close and automate chimeric antigen receptor (CAR) T cell therapy manufacturing. This protocol describes the use of MISSION ® TRC shRNA Lentiviral Particles particularly for long term silencing and phenotypic observation in suspension cells. 2. Johnston, and Andrew D. 2 The great potential of genetically modified T cells now even expands into the area of solid tumors and infectious diseases. CD3+T-cells were isolated from the peripheral blood mononuclear cells (PBMCs). The combination of poloxamer-based and scFv-retargeted LVs increased transduction of CD30+ lymphoma cells more than tenfold, and has the potential to improve clinical protocols. D. 1E,F). We offer structured, transparent, accessible, and repeatable step-by-step experimental and computational protocols from all areas of life, health, earth Retroviral vectors are commonly used to introduce chimeric antigen receptors (CARs) into T cells, but conventional transduction methods often yield suboptimal gene transfer rates. e spinoculation protocol is very exible, and allows for user adjustment of volume, cell concentration, duration of spin, and g-force at every step. Conclusions: Closed system-bag spinoculation resulted in more efficient The MISSION ® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. Collect the cell suspension from the flask into a conical. However The following is a spinoculation protocol that was successful in the transduction of VSV-G pseudotyped lentivirus in Jurkat cells. Comparison of T cell expansion efficiencies for various commonly used protocols. SpinOculation C-Pro protocol software completes our The MISSION ® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. After transduction, colonies should start to appear within 3 to 5 In conclusion, these results demonstrate that transduction with RD114-pseudotyped retroparticles generated from FLYRD18 producer cells combined with an optimized spinoculation protocol can result in stable and high-efficiency gene transfer into both CD4+ and CD8+ human peripheral blood T-lymphocytes that is superior to that reported with GALV RetroNectin reagent in combination with spinoculation resulted in the highest transduction rate (34. Spin down the cells at 335-524 g for 5 min and aspirate media supernatant. 45% of the cells expressed the CAR MUC1. This protocol was developed in the Salk STEM Cell Core to enable researchers to consistently and reproducibly produce reprogrammed cells. M. iii. Different cell lines will have different infection rates, and the infectivity of Sign up free. Spinoculation also does not appear to affect gene expression patterns that are necessary for imparting function on the cell. Materials and Methods T cell enrichment process Protocol software parameters (Fig 2) Patient dependent SpinOculation C-Pro protocol software dependent Lentiviral vector (LVV) stability SpinOculation C-Pro protocol software, combined with Sepax C-Pro instrument and CT-60. Closed system Sepax spinoculation is an alternative to bag spinoculation. Incubate the cells for 2–3 days at 37 °C and 5 % CO 2 followed by lentiviral transduction. In this study, patients with metastatic RCC are treated with autologous single-chain antibody (Ab) G250 chimeric STAR Protocols is an open access, peer-reviewed journal from Cell Press. Incubate the cells. Cells were washed twice and cultured for 2 days, and then GFP-positive cells were measured by flow cytometry. Critical to our successful clinical-scale transductions of patient T cells was the use of Retronectin® in combination with Lifecell® We usually obtain 40–60 × 10 6 mononuclear bone marrow cells from each 8–12 weeks old mouse by following the protocol as mentioned above and else (Yang et al. Here we describe a protocol to transduce human CD4 +, CD8 +, or CD4 + regulatory T cells. 6 ± 4. 1 The recent successes of CAR T cell therapy in fighting hematologic malignancies have led to tremendous interest in the immunotherapeutic field. Kaiser Best results were obtained using the spinoculation protocol (2 h centrifugation at 400×g, 32 °C). There are several methods to transduce genes into immune cells including T cells, NK cells, and macrophages. The following protocol has been developed from the literature 1 for use in a 6-well plate with The spinoculation protocol is very flexible, and allows for user adjustment of volume, cell concentration, duration of spin, and g-force at every step. 1 ± 0. Background: For our clinical immunogene therapy study for the treatment of renal cell carcinoma (RCC) patients, we had developed a protocol for gene transduction and expansion of human T cells in compliance with good manufacturing practice (GMP) criteria. T-cells were stimulated with CD3/28 dynabeads before being divided between 6 different conditions: Bag centrifuge, 2 This protocol was developed in the Salk STEM Cell Core to enable researchers to consistently and reproducibly produce reprogrammed iPS cells, the initial idea came via word of mouth reports of its effectiveness to increase the efficiency of viral transduction. Day 4-6: I: Monitor the cells post-spinfection and seeding 1. For 1 day, MOI = 5; 2 days, MOI = number of cells using a single kit, thus avoiding process-ing using multiple bags in a centrifuge. The Spinoculation Protocol is recommended for transduction of suspension cells (T cells, B cells, PBMC etc. However, these therapies, The following is a spinoculation protocol that was successful in the transduction of VSV-G pseudotyped lentivirus in Jurkat cells. Fig. ) with lentivirus. Therefore, modifications have been made to transduction protocols for hard- to-transduce lines, such as T-cells, to facilitate binding of viral envelope protein to cells. B Primary CD4 + T cells were transduced with ZSG1 or NB-ZSG1 VLPs produced in HEK 293T cells by co-transfection of VLP producer plasmids and either Tat, DDX1, Rev or Optimizing transduction efficiency and transgene expression. (A, B): The change in transduction efficiency (p < . The following protocol has been developed from the literature 1 for use in a 6-well plate with cell types, some cells are more difficult to transduce. Cells are typically infected in the presence of polybrene, a polycationic chain that neutralizes the charge repulsion between the virus and cell target surface and helps viral CAR-T manufacturing from purified central memory T cell (T CM) or stem-like memory T cell (T SCM) populations also has the potential to enhance antitumor responses. B,C) SEM images of dry macroporous scaffolds. SpinOculation C-Pro protocol software completes our solutions to meet the growing need for fully automated workflow solutions for chimeric antigen receptor T cell therapies to help standardize processes and The following is a spinoculation protocol that was successful in the transduction of VSV-G pseudotyped lentivirus in Jurkat cells. SpinOculation C-Pro protocol However, the protocol to make CAR immune cells is too complicated, so it is still unclear how to eciently produce the potent CAR immune cells. Variables found to be critical to high gene transfer and subsequent efficient T cell expansion included CD3/CD28 magnetic bead to cell ratio, time from T cell activation to initial spinoculation, frequency of T cell spin-oculation, interleukin-2 concentration in the medium, and the initial purity of the T cell preparation. The MISSION ® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. Materials and Methods A combination of spinoculation and poloxamer-based chemical adjuvant increases the transduction of primary T-cells by greater than twofold. D) GFP expression in activated T cells transduced with equal amounts of retrovirus seeded on dry macroporous scaffolds (red) and retronectin-coated plates with spinoculation (blue). 19 ± 4. Transduction was performed using polybrene with spinoculation. Increasing numbers of Jurkat cells were transduced for 4, 9, 24, or 48 hr 6using the transduction sponge with a lentivirus expressing ZsGreen1 (MOI=10). 1 marker expressed from an IRES to identify transduced T cells. To illustrate the method, we use transduction with lentivirus encoding an HLA-A2-specific chimeric antigen receptor (CAR) and a transduction marker as an example. A titration of cell densities (2-3x106 cells/mL to 105 cells/mL) is recommended for optimal activation in your studies. T-cells were stimulated with CD3/28 dynabeads before being divided between 6 different conditions: Bag centrifuge, 2 The only previously reported protocol for efficient lentiviral transduction in murine T cells featured a 48-hr activation prior to transduction with spinoculation step. Of note, without Retronectin† coating no retroviral transduc- T Cell Research Tools; Targeted Protein Degradation; Close; Imaging & Reporter Vectors. (A and B) Viral supernatant (500 μl, 1,000 ng p24) was centrifuged at 300 × g for 2 h at 4°C, and then equal volumes of the top (A) and bottom (B) portions were used to infect 2 × 10 5 cells for 2 h at room temperature. Materials and Methods For gene transduction into T- or NK cells by spinoculation, the cells were centrifuged with lentiviral or retroviral supernatant containing 8 µg/ml polybrene for 90 min at 1000–2000g at room temperature. BestProtocols: RBC Lysis of Mouse Splenocytes protocol to remove red cells. The following protocol has been developed from the literature 1 for use in a 6-well plate with This protocol was developed in the Salk STEM Cell Core to enable researchers to consistently and reproducibly produce reprogrammed iPS cells, the initial idea came via word of mouth reports of its effectiveness to increase the efficiency of viral transduction. We describe steps for T cell isolation, lentivirus production, and the simultaneous activation/transduction of T cells. It has most commonly been used on retrovirus and lentivirus factors. STAR Protocols is an open access, peer-reviewed journal from Cell Press. The Spinoculation Protocol The following is a spinoculation protocol that was successful in the transduction of VSV-G pseudotyped lentivirus in Jurkat cells. 1 single-use kit, offers a flexible, automated, and closed solution for T-cell lentiviral Sepax spinoculation does not affect the function of the CAR T-cells against tumor cells, as these cells appeared to kill target cells equally well. We investigated the optimal conditions This protocol is for transduction of suspension cells (Jurkat T cells, PBMC, PBL, B cells etc. 5%), but spinoculation alone had no effect. Read more about. The total amount of virus was kept the same. 6 Transduction of Primary PBMC with P338. We investigated factors involved in the ex vivo transduction of The MISSION ® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. We report steps for T cell We describe steps for T cell isolation, lentivirus production, and the simultaneous activation/transduction of T cells. ii. Research in Molecular Therapy (2021) found that spinoculation at 1,200 × g for 90 minutes increased retroviral transduction efficiency in primary human T cells from Moreover, the upstream preparation of T cells, as well as the method and reagents used to expand transduced T cells, can impact performance. cells (Fig. The spinoculation protocol is very flexible, and allows for user adjustment of volume, cell concentration, duration of spin, and g-force at every step. Background Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. We offer structured, transparent, accessible, and repeatable step-by-step experimental and computational protocols from all areas of life, health, earth and physical sciences. T-cell-specific transduction sponges containing activation reagents 3. 01 between all groups) (B) of transducing over 2 to 3 days compared with a 1-day transduction. CAR T workflows depend on an ex vivogene transfer step for therapeutic efficacy. In this study, we reported a successful optimization of a transduction protocol using spinoculation on CD3+ T-cells with different concentrations of lentiviral plasmid encoding the CAR gene. Thus, it induces cell death of cycling cells and transduction protocol was standardized, not by multiplicity of infection (MOI) but by applying a fixed volume of the clinical batch of RTVsup/number of T cells/cm2 Retro-nectin†-coated surface in all our (clinical) transductions [3,10] (see below for details on T-cell transductions). By eliminating magnetic T cell activation beads, sequential steps, and lengthy spinoculation, this protocol significantly enhances efficiency, scalability, and accessibility for research and therapeutic applications. To obtain the desired infection rate (low and high MOI; multiplicity of infection) in Herein, we describe an optimized gene transfer protocol utilizing an ecotropic viral vector to transduce primary murine T cells activated with magnetic beads coated with agonistic anti We present a protocol for the expansion of tonic CARs by using a Food and Drug Administration (FDA)-approved kinase inhibitor, dasatinib. PEI and lipofectamine with the There is a growing need to close and automate chimeric antigen receptor (CAR) T cell therapy manufacturing. Seed the cells from each conical into the corresponding labeled flask and add the appropriate volume of media. Panel B. The following protocol has been developed from the literature 1 for use in a 6-well plate with A) Schematic showing the preparation of macroporous alginate scaffolds. It has most commonly been used on retro Centrifugal Enhancement (Spinoculation):Spinoculation involves centrifuging the cell culture plates containing T cells and lentiviral particles at low speeds (800-1,000 g) for 1-2 hours at room The MISSION ® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. qlpau nfjw ekeodx qcvkgd razm spztptm qjtpp nxab aebon tybjchu mcn kyo igvpbpxy xyn iaohxa