scholarly journals Cell Banking of HEK293T cell line for clinical-grade lentiviral particles manufacturing

2020 ◽  
Author(s):  
Unai Perpiña ◽  
Cristina Herranz ◽  
Raquel Martin-Ibañez ◽  
Anna Boronat ◽  
Felipe Chiappe ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Hek293t Cell ◽  
Good Manufacturing Practice ◽  
Clinical Grade ◽  
Cell Bank ◽  
Advanced Therapy ◽  
Working Cell ◽  
Cell Banks ◽  
Hek293t Cell Line

Abstract Background: Cell banks are widely used to preserve cell properties as well as to record and control the use of cell lines in biomedical research. The generation of cell banks for the manufacturing of Advanced Therapy Medicinal Products, such as cell and gene therapy products, must comply with current Good Manufacturing Practice regulations. The quality of the cell lines used as starting materials in viral-vector manufacturing processes must be also assessed.Methods: Three batches of a Master Cell Bank and a Working Cell Bank of the HEK293T cell line were manufactured under current Good Manufacturing Practices regulations. Quality control tests were performed according to product specifications. Process validation includes the training of manufacturing personnel by performing simulation tests, and the continuous measurement of environmental parameters such as air particles and microorganisms. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.Results: All batches of HEK293T Master and Working Cell Banks met the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working Cell Banks maintained the defined cell viability and concentration over a 37 month-period after cryopreservation. Conclusions: Manufacturing cell banks under Good Manufacturing Practice regulations for their use as raw materials or final cellular products is feasible. HEK293T cell banks were used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

scholarly journals Cell Banking of HEK293T cell line for clinical-grade lentiviral particles manufacturing

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Unai Perpiñá ◽  
Cristina Herranz ◽  
Raquel Martín-Ibáñez ◽  
Anna Boronat ◽  
Felipe Chiappe ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Hek293t Cell ◽  
Good Manufacturing Practice ◽  
Clinical Grade ◽  
Cell Bank ◽  
Advanced Therapy ◽  
Working Cell ◽  
Cell Banks ◽  
Hek293t Cell Line

Abstract Background Cell banks are widely used to preserve cell properties as well as to record and control the use of cell lines in biomedical research. The generation of cell banks for the manufacturing of Advanced Therapy Medicinal Products, such as cell and gene therapy products, must comply with current Good Manufacturing Practice regulations. The quality of the cell lines used as starting materials in viral-vector manufacturing processes must be also assessed. Methods Three batches of a Master Cell Bank and a Working Cell Bank of the HEK293T cell line were manufactured under current Good Manufacturing Practices regulations. Quality control tests were performed according to product specifications. Process validation includes the training of manufacturing personnel by performing simulation tests, and the continuous measurement of environmental parameters such as air particles and microorganisms. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products. Results All batches of HEK293T Master and Working Cell Banks met the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working Cell Banks maintained the defined cell viability and concentration over a 37 month-period after cryopreservation. Conclusions Manufacturing cell banks under Good Manufacturing Practice regulations for their use as raw materials or final cellular products is feasible. HEK293T cell banks were used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

scholarly journals Cell Banking of HEK293T Cell Line for Clinical-Grade Lentiviral Particles Manufacturing

2020 ◽  
Author(s):  
Unai Perpiña ◽  
Cristina Herranz ◽  
Raquel Martin-Ibañez ◽  
Anna Boronat ◽  
Felipe Chiappe ◽  
...  
Keyword(s):  
Cell Line ◽  
Hek293t Cell ◽  
Cell Number ◽  
Clinical Grade ◽  
Cell Bank ◽  
Master Cell Bank ◽  
Good Manufacturing Practices ◽  
Working Cell ◽  
Cell Banks ◽  
Hek293t Cell Line

Abstract Background: Cell banks have been widely used to preserve cell properties as well as to record and control cell line access in research. However, the generation of cell banks involved in the manufacturing of Advanced therapy medicinal products such as cell or gene therapy products must comply with the current Good Manufacturing Practice regulation. Similarly, the quality of those cell lines used as starting materials in viral-vector manufacturing processes must be also evaluated.Methods: Three batches of both Master Cell Bank and Working Cell Bank of the HEK293T cell line were manufactured under the current Good Manufacturing Practices regulation. Quality control test were performed according to the product specifications. The process validation includes previous qualification of the manufacturing personnel by performing simulation tests as well as the continuous measure of environmental parameters during manufacturing such as air particles and microorganism. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.Results: All batches of Master Cell Bank and Working Cell Bank fulfilled the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working cell bank maintain the defined viability and cell number 37 months after cryopreservation. Conclusions: Manufacturing cell banks under Good Manufacturing Practices regulation for its use as raw material or final cellular product is feasible. HEK293T cell banks have been used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

scholarly journals Cell Banking of HEK293T Cell Line for Clinical-Grade Lentiviral Particles Manufacturing

2020 ◽  
Author(s):  
Unai Perpiña ◽  
Cristina Herranz ◽  
Raquel Martin-Ibañez ◽  
Anna Boronat ◽  
Felipe Chiappe ◽  
...  
Keyword(s):  
Cell Line ◽  
Hek293t Cell ◽  
Cell Number ◽  
Clinical Grade ◽  
Cell Bank ◽  
Master Cell Bank ◽  
Good Manufacturing Practices ◽  
Working Cell ◽  
Cell Banks ◽  
Hek293t Cell Line

Abstract Background: Cell banks have been widely used to preserve cell properties as well as to record and control cell line access in research. However, the generation of cell banks involved in the manufacturing of Advanced therapy medicinal products such as cell or gene therapy products must comply with the current Good Manufacturing Practice regulation. Similarly, the quality of those cell lines used as starting materials in viral-vector manufacturing processes must be also evaluated.Methods: Three batches of both Master Cell Bank and Working Cell Bank of the HEK293T cell line were manufactured under the current Good Manufacturing Practices regulation. Quality control test were performed according to the product specifications. The process validation includes previous qualification of the manufacturing personnel by performing simulation tests as well as the continuous measure of environmental parameters during manufacturing such as air particles and microorganism. Cell number and viability of cryopreserved cells were periodically measured in order to define the stability of these cellular products.Results: All batches of Master Cell Bank and Working Cell Bank fulfilled the acceptance criteria of their specifications showing the robustness and homogeneity of the processes. In addition, both Master and Working cell bank maintain the defined viability and cell number 37 months after cryopreservation. Conclusions: Manufacturing cell banks under Good Manufacturing Practices regulation for its use as raw material or final cellular product is feasible. HEK293T cell banks have been used to manufacture clinical-grade lentiviral particles for Chimeric Antigen Receptor T-cell based clinical trials.

201 Testing of single guide RNAs, optimization of transfection, and selection systems for the generation of SRY knockout foetal fibroblast cells

2019 ◽  
Vol 31 (1) ◽  
pp. 225
Author(s):  
G. Vans Landschoot ◽  
R. J. Bevacqua ◽  
R. Fernandez y Martin ◽  
F. A. Pereyra-Bonnet ◽  
D. F. Salamone
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Fluorescent Microscopy ◽  
Pulse Length ◽  
Hek293t Cell ◽  
Reference Sequence ◽  
Sry Gene ◽  
Gfp Expression ◽  
Electrode Gap ◽  
Hek293t Cell Line

Obtaining male and female cells from a male could have important implications for endangered mammalian species and domestic animal production. Achieving this could constitute a proof of concept of the use of assisted reproduction technologies for the conservation of endangered species. In particular, SRY is the principal male sex determinant gene and is found on the short arm of the Y chromosome. With the introduction of nuclease-mediated genome editing technologies, such as the CRISPR/Cas9 system, it is possible to envision precise DNA targeting in this gene as strategy to manipulate the sex of cell lines. Based on the above, we tested the CRISPR/Cas9 system to later create knockout (KO) cell lines of the SRY gene in bovine. The aim of this work was (1) to test the efficiency of single guide (sg)RNAs designed to target the bovine SRY gene in HEK293T cell line, and (2) to optimize blasticidin concentrations and electroporation conditions in bovine fetal fibroblast (BFF). To test sgRNA efficiency, we used 3 sgRNA designed and tested over the sequence of bovine SRY gene (690bp). The efficiency of each sgRNA was evaluated in a heterologous way by using a modification of pCAG-EGxxFP (plasmid #50716, Addgene, Cambridge, MA, USA) in which we inserted the bovine SRY sequence (NCBI Reference Sequence: NC_016145.1), and that only results in green fluorescent protein (GFP) expression upon cutting by the CRISPR/Cas9 system. We transfected the HEK293T cell line with the following groups to assess the efficiency of 3 sgRNA: (1) btSRY1+Cas9+pCAG-EGSRYFP; (2) btSRY2+Cas9+pCAG-EGSRYFP; (3) btSRY3+Cas9+pCAG-EGSRYFP; (4) PU6-empty+Cas9+pCAG- EGSRYFP; (5) GFP-only control. In addition, 6 blasticidin concentrations (0, 1, 2.5, 5, 10, and 20µg mL−1) and 3 electroporation conditions (Tfx1, Tfx2, and Tfx3) were tested, both in BFF. Electroporation conditions were as follows: Tfx1=1.25 kV cm−1; number of pulses=3; electrode gap=4 mm; interval=100 ms; Tfx2=1.2 kV/cm; number of pulses=4; electrode gap=4 mm; interval=100 ms; and Tfx3=voltage: 350V, LV mode; pulse length=100 µs; number of pulses=4; electrode gap=4 mm; interval=100ms. Statistical analyses were performed using 2-tailed Mann-Whitney tests. Results for sgRNA efficiency, based on GFP expression by counting GFP+ cells under fluorescent microscopy showed that btSRY1 (17%) and btSRY2 (13%) worked significantly better than btSRY3 (1%). The blasticidin selection assay showed that 5, 10, and 20µg mL−1were significantly more lethal than 0, 1 and 2.5µg mL−1, by counting living cells in Neubauer chamber. We chose 5µg mL−1 as the concentration for future experiments. Last, the Tfx2 electroporation protocol (11.34%) was more efficient than the other 2 protocols tested (Tfx1 and Tfx3: 3.48 and 0.86%, respectively), based on the counting of GFP+ cells under fluorescent microscopy. Electroporation of BFF with btSRY1, btSRY2, or both and Cas9 using Tfx2 protocol and molecular characterisation of colonies are currently in progress with the ultimate objective of producing SRY knockout bovine embryos by somatic cell nuclear transfer.

scholarly journals GMP-grade neural progenitor derivation and differentiation from clinical-grade human embryonic stem cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Loriana Vitillo ◽  
Catherine Durance ◽  
Zoe Hewitt ◽  
Harry Moore ◽  
Austin Smith ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Good Manufacturing Practice ◽  
Clinical Use ◽  
Clinical Grade ◽  
Flexible Tool ◽  
Cell Bank

Abstract Background A major challenge for the clinical use of human pluripotent stem cells is the development of safe, robust and controlled differentiation protocols. Adaptation of research protocols using reagents designated as research-only to those which are suitable for clinical use, often referred to as good manufacturing practice (GMP) reagents, is a crucial and laborious step in the translational pipeline. However, published protocols to assist this process remain very limited. Methods We adapted research-grade protocols for the derivation and differentiation of long-term neuroepithelial stem cell progenitors (lt-NES) to GMP-grade reagents and factors suitable for clinical applications. We screened the robustness of the protocol with six clinical-grade hESC lines deposited in the UK Stem Cell Bank. Results Here, we present a new GMP-compliant protocol to derive lt-NES, which are multipotent, bankable and karyotypically stable. This protocol resulted in robust and reproducible differentiation of several clinical-grade embryonic stem cells from which we derived lt-NES. Furthermore, GMP-derived lt-NES demonstrated a high neurogenic potential while retaining the ability to be redirected to several neuronal sub-types. Conclusions Overall, we report the feasibility of derivation and differentiation of clinical-grade embryonic stem cell lines into lt-NES under GMP-compliant conditions. Our protocols could be used as a flexible tool to speed up translation-to-clinic of pluripotent stem cells for a variety of neurological therapies or regenerative medicine studies.

scholarly journals EFFECT OF EDEM1 OVEREXPRESSION ON THE GENERATION AND ASSEMBLY OF MAJOR HISTOCOMPATIBILITY COMPLEXES

2016 ◽  
Vol 25 (4) ◽  
pp. 181-186
Author(s):  
Alexandra Circiumaru ◽  
◽  
Gabriela Chiritoiu ◽  
Livia Sima ◽  
Mihai Bojinca ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endoplasmic Reticulum ◽  
Western Blot ◽  
Autoimmune Diseases ◽  
Cell Line ◽  
Hek293t Cell ◽  
Western Blot Assay ◽  
Mhc I ◽  
Transfected Cells ◽  
Hek293t Cell Line

Background. A better understanding of the role of endoplasmic reticulum degradation-enhancing alpha-mannosidase – like protein 1 (EDEM1) in endoplasmic reticulum associated degradation (ERAD) may open new therapeutic approaches in autoimmune diseases. Aim. To study ERAD and EDEM1 in the generation and assembly of MHC I and the potential role in the pathophysiology of autoimmune diseases. Materials and methods. HEK293T cell line (human embrionic kidney cells), A375 cell line (amelanotic melanoma cells) and THP-1 cell line (leukemic monocytes used both as undifferentiated and differentiated) underwent transient transfection with EDEM1 and mock transfection with pTriEx. Western blot experiments assessed the total cellular MHC I levels in cell lysates, while expression on the cellular surface was quantified by flow cytometry of fixed cells. Results were analysed using the FACS Calibur and CellQuest Pro dedicated software. Experiments were done twice with duplicate probes for the Western blot assay and triplicate probes were used for flow cytometry. GraphPad Prism was used for data analysis. Results. MHC I plasma membrane routing and expression was similar in HEK293T and A375 both in mock transfected and non-transfected cells. Western blot assay for EDEM1 transfected cells showed bands corresponding to the total MHC I that migrated at 42kDa mass in non-transfected and mock transfected Hek293T, A375 and undifferentiated THP-1 cells. Mock transfected differentiated THP-1 cells showed a reduction of total MHC I. EDEM1 transfected Hek293T, A375 and undifferentiated THP-1 cells displayed higher levels of total MHC I, while differentiated THP-1 cells showed a marked reduction. Flow cytometry assay showed significantly reduced cell surface MHC I levels in Hek293T cell line. We observed a modest reduction of MHC I complexes on the cellular surface in undifferentiated THP-1 EDEM1 transfected cells, while there was no significant change in the A375 EDEM1 transfected cell line, as well as the differentiated THP-1 EDEM1 transfected cells. Conclusion. The impact of ERAD’s EDEM1 in MHC I reduction may have an important role in autoimmune disease, making ERAD an interesting therapeutic target.

scholarly journals No evidence for genome editing in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo)

2016 ◽  
Author(s):  
Nay Chi Khin ◽  
Jenna L. Lowe ◽  
Lora M. Jensen ◽  
Gaetan Burgio
Keyword(s):  
Cell Line ◽  
Genome Editing ◽  
Sanger Sequencing ◽  
Hek293t Cell ◽  
Human Cell Line ◽  
Blastocyst Stage ◽  
Editing Efficiency ◽  
Stage Of Development ◽  
Mouse Zygotes ◽  
Hek293t Cell Line

AbstractA recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5’- phosphorylated guide DNA (gDNA) from 2.5 ng/μl to 50 ng/μl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that we failed to edit using NgAgo.

scholarly journals Heterogeneity of acetylcholine receptor autoantibody-mediated complement activity in patients with myasthenia gravis.

2021 ◽  
Author(s):  
Abeer H Obaid ◽  
Chryssa Zografou ◽  
Douangsone D Vadysirisack ◽  
Bailey Munro-Sheldon ◽  
Miriam L Fichtner ◽  
...  
Keyword(s):  
Myasthenia Gravis ◽  
Cell Line ◽  
Acetylcholine Receptor ◽  
Disease Burden ◽  
Hek293t Cell ◽  
Live Cell ◽  
Complement Activity ◽  
Regulator Genes ◽  
Complement Regulator ◽  
Hek293t Cell Line

Background: Autoantibodies targeting the acetylcholine receptor (AChR) in the serum of myasthenia gravis (MG) patients are broadly polyclonal and heterogeneous in their pathogenic capacity. Specifically, AChR autoantibody-mediated pathology occurs through three mechanisms that include complement-directed tissue damage, blocking of the acetylcholine binding site on the AChR, and modulation (internalization) of the AChR. Clinical assays used for diagnosis and prognosis measure only AChR autoantibody binding and they provide weak association with disease burden, thereby limiting understanding of mechanistic heterogeneity, and monitoring therapeutic response. Objective: To develop an in-vitro cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation. Methods: A HEK293T cell line, which is commonly used for live cell-based AChR autoantibody binding assays, was modified such that the expression of the complement regulator genes (CD46, CD55 and CD59) were disrupted using CRISPR/Cas9 genome editing. This modified cell line was used to measure serum AChR autoantibody-mediated complement MAC formation via flow cytometry. Results: AChR autoantibody-mediated MAC formation required the use of a modified HEK293T cell line in which the surface expression of three complement regulator genes was absent. Serum samples (n=155) from 97 clinically confirmed AChR patients were tested along with 32 healthy donor (HD) samples; the MG cohort included a wide range of disease burden and AChR autoantibody titer. AChR autoantibodies were detected in 139 of the 155 (89.7%) AChR patient samples via a live cell-based assay. Of the 139 AChR positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), while no autoantibodies or MAC formation was detected in samples from the HD group. Autoantibody-mediated MAC formation positively associated with autoantibody binding in most MG patient samples. However, a subset displayed a disassociation between binding and MAC formation. Conclusions: We demonstrate the development of a novel assay for evaluating AChR autoantibody-mediated complement activity. It is anticipated that this assay will afford a deeper understanding of the heterogeneous disease pathology and allow for the identification of MG patients who may benefit from complement inhibitor therapy.

scholarly journals Screening for 15 pathogenic viruses in human cell lines registered at the JCRB Cell Bank: characterization of in vitro human cells by viral infection

2018 ◽  
Vol 5 (5) ◽  
pp. 172472 ◽  
Author(s):  
Setsuko Shioda ◽  
Fumio Kasai ◽  
Ken Watanabe ◽  
Kohei Kawakami ◽  
Azusa Ohtani ◽  
...  
Keyword(s):  
Viral Infection ◽  
Cell Line ◽  
Cell Lines ◽  
Human Cell ◽  
The Other ◽  
Pcr Analysis ◽  
Human Cell Lines ◽  
Cell Bank

Human cell lines have been used in a variety of research fields as an in vitro model. These cells are all derived from human tissue samples, thus there is a possibility of virus infection. Virus tests are routinely performed in clinical practice, but are limited in cell lines. In this study, we investigated 15 kinds of viruses in 844 human cell lines registered at the Japanese Collection of Research Bioresources (JCRB) Cell Bank. Our real-time PCR analysis revealed that six viruses, EBV, HTLV-1, HBV, B19V, HHV-6 and HHV-7, were detected in 43 cell lines. Of them, 20 cell lines were transformed by intentional infection in vitro with EBV or HTLV-1. Viruses in the other 23 cell lines and one EBV transformed cell line are derived from an in vivo infection, including five de novo identifications of EBV, B19V or HHV-7 carriers. Among them, 17 cell lines were established from patients diagnosed with virus-associated diseases. However, the other seven cell lines originated from in vivo cells unrelated to disease or cellular tropism. Our approach to screen for a set of 15 viruses in each cell line has worked efficiently to identify these rare cases. Virus tests in cell lines contribute not only to safety assessments but also to investigation of in vivo viral infection which can be a characteristic feature of cell lines.

scholarly journals Cost-Effective Master Cell Bank Validation of Multiple Clinical-Grade Human Pluripotent Stem Cell Lines From a Single Donor

2014 ◽  
Vol 3 (10) ◽  
pp. 1116-1124 ◽  
Author(s):  
Liani Devito ◽  
Anastasia Petrova ◽  
Cristian Miere ◽  
Stefano Codognotto ◽  
Nicola Blakely ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Lines ◽  
Pluripotent Stem Cell ◽  
Cost Effective ◽  
Clinical Grade ◽  
Human Pluripotent Stem Cell ◽  
Cell Bank ◽  
Single Donor ◽  
Master Cell Bank ◽  
Stem Cell Lines
Sign in / Sign up

Export Citation Format

Share Document