Scalable cell culture and transient transfection for viral vector manufacturing

α1-Microglobulin (A1M) is a small glycoprotein that belongs to the lipocalin protein family. A major biological role of A1M is to protect cells and tissues against oxidative damage by clearing free heme and reactive oxygen species. Because of this, the protein has attracted great interest as a potential pharmaceutical candidate for treatment of acute kidney injury and preeclampsia. The aim of this study was to explore the possibility of expressing human A1M in plants through transient gene expression, as an alternative or complement to other expression systems. E. coli, insect and mammalian cell culture have previously been used for recombinant A1M (rA1M) or A1M production, but these systems have various drawbacks, including additional complication and expense in refolding for E. coli, while insect produced rA1M is heavily modified with chromophores and mammalian cell culture has been used only in analytical scale. For that purpose, we have used a viral vector (pJL-TRBO) delivered by Agrobacterium for expression of three modified A1M gene variants in the leaves of N. benthamiana. The results showed that these modified rA1M protein variants, A1M-NB1, A1M-NB2 and A1M-NB3, targeted to the cytosol, ER and extracellular space, respectively, were successfully expressed in the leaves, which was confirmed by SDS-PAGE and Western blot analysis. The cytosol accumulated A1M-NB1 was selected for further analysis, as it appeared to have a higher yield than the other variants, and was purified with a yield of ca. 50 mg/kg leaf. The purified protein had the expected structural and functional properties, displaying heme-binding capacity and capacity of protecting red blood cells against stress-induced cell death. The protein also carried bound chromophores, a characteristic feature of A1M and an indicator of a capacity to bind small molecules. The study showed that expression of the functional protein in N. benthamiana may be an attractive alternative for production of rA1M for pharmaceutical purposes and a basis for future research on A1M structure and function.

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Spatiotemporal Modeling of Transient Transfection Processes in Mammalian Cell Culture

Chemie Ingenieur Technik ◽

10.1002/cite.201050184 ◽

2010 ◽

Vol 82 (9) ◽

pp. 1503-1503

Author(s):

U. Jandt ◽

S. Shao ◽

S. Binius ◽

M. Wirth ◽

A. P. Zeng

Keyword(s):

Cell Culture ◽

Mammalian Cell ◽

Mammalian Cell Culture ◽

Transient Transfection ◽

Spatiotemporal Modeling

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Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

BMC Biotechnology ◽

10.1186/1472-6750-11-114 ◽

2011 ◽

Vol 11 (1) ◽

Cited By ~ 23

Author(s):

Kamilla Swiech ◽

Amine Kamen ◽

Sven Ansorge ◽

Yves Durocher ◽

Virgínia Picanço-Castro ◽

...

Keyword(s):

Cell Culture ◽

Transient Transfection ◽

Efficient Production ◽

Hek 293 ◽

Serum Free ◽

Serum Free Suspension

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Novel Stable Ebola Virus Minigenome Replicon Reveals Remarkable Stability of the Viral Genome

Journal of Virology ◽

10.1128/jvi.01316-17 ◽

2017 ◽

Vol 91 (22) ◽

Cited By ~ 7

Author(s):

Wanyin Tao ◽

Tianyu Gan ◽

Mingzhe Guo ◽

Yongfen Xu ◽

Jin Zhong

Keyword(s):

Cell Culture ◽

Culture System ◽

Ebola Virus ◽

Transient Transfection ◽

Genome Replication ◽

Mrna Levels ◽

Interferon Treatment ◽

Cell Culture System ◽

Rnase Digestion ◽

Biosafety Level

ABSTRACT Ebola virus (EBOV) causes severe hemorrhagic fever in humans and other primates with a high case fatality rate. No approved drug or vaccine of EBOV is available, which necessitates better understanding of the virus life cycle. Studies on EBOV have been hampered because experimentations involving live virus are restricted to biosafety level 4 (BSL4) laboratories. The EBOV minigenome system has provided researchers with the opportunity to study EBOV under BSL2 conditions. Here, we developed a novel EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The minigenomic RNA harboring a Gaussia luciferase and hygromycin-resistant marker can replicate for months in a helper cell stably expressing viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and L proteins. Quantification of viral RNA (vRNA), cRNA, and mRNA levels of the EBOV minigenome demonstrated that the stable EBOV replicon had much-more-active minigenome replication than previously developed transient-transfection-based EBOV minigenome systems, which recapitulate viral primary transcription more than genome replication. Interestingly, minigenome replication in the stable EBOV replicon cells was insensitive to interferon treatment or RNA interference. Moreover, RNase digestion of the replicon cell lysates revealed the remarkably stable nature of the EBOV minigenomic vRNA ribonucleoprotein complex, which may help improve understanding of EBOV persistence in convalescent patients. IMPORTANCE The scope and severity of the recent Ebola outbreak in Western Africa justified a more comprehensive investigation of the causative risk group 4 agent Ebola virus (EBOV). Study of EBOV replication and antiviral development can be facilitated by developing a cell culture system that allows experimentation under biosafety level 2 conditions. Here, we developed a novel stable EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The replicon system had more-active genome replication than previously developed transient-transfection-based EBOV minigenome systems, providing a convenient surrogate system to study EBOV replication. Furthermore, self-replicating minigenomic vRNA in the replicon cells displayed strong stability in response to interferon treatment, RNA silencing, and RNase digestion, which may provide an explanation for the persistence of EBOV in survivors.

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Selection of chromatographic methods for the purification of cell culture-derived Orf virus for its application as a vaccine or viral vector

Journal of Biotechnology ◽

10.1016/j.jbiotec.2020.07.023 ◽

2020 ◽

Vol 323 ◽

pp. 62-72

Author(s):

Keven Lothert ◽

Felix Pagallies ◽

Thomas Feger ◽

Ralf Amann ◽

Michael W. Wolff

Keyword(s):

Cell Culture ◽

Viral Vector ◽

Orf Virus ◽

Chromatographic Methods ◽

Selection Of

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Process Development for Newcastle Disease Virus-Vectored Vaccines in Serum-Free Vero Cell Suspension Cultures

Vaccines ◽

10.3390/vaccines9111335 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1335

Author(s):

Julia Puppin Chaves Fulber ◽

Omar Farnós ◽

Sascha Kiesslich ◽

Zeyu Yang ◽

Shantoshini Dash ◽

...

Keyword(s):

Cell Culture ◽

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Viral Vector ◽

Vero Cells ◽

Suspension Cultures ◽

Emerging Diseases ◽

Fine Tuning ◽

Serum Free

The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms—technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID50 and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 108 TCID50/mL for NDV-GFP and 1.33 × 108 TCID50/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 108 TCID50/mL for NDV-GFP and 3.16 × 107 TCID50/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets.

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A T.E.M. study of mouse mammary epithelial cell secretory differentiation cultured on collagen and Matrigel

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100085241 ◽

1991 ◽

Vol 49 ◽

pp. 188-189

Author(s):

W.N. Bentham ◽

V. Rocha

Keyword(s):

Cell Culture ◽

Mammary Epithelial ◽

Secretory Process ◽

Cell Culture System ◽

Protein Maturation ◽

Cell Culture Techniques ◽

Culture Techniques ◽

Mouse Mammary Epithelial Cell ◽

Secretory Differentiation

It has been an interest of our lab to develop a mammary epethelial cell culture system that faithfully duplicates the in vivo condition of the lactating gland. Since the introduction of collagen as a matrix on which cells are cultivated other E.C.M. type matrices have been made available and are used in many cell culture techniques. We have previously demonstrated that cells cultured on collagen and Matrigel do not differentiate as they do in vivo. It seems that these cultures often produce cells that show a disruption in the secretory process. The appearance of large ribosomal studded vesicles, that specifically label with antibody to casein, suggest an interruption of both protein maturation and secretion at the E.R. to golgi transition. In this report we have examined cultures on collagen and Matrigel at relative high and low seeding densities and compared them to cells from the in vivo condition.

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Solid-phase immunoelectron microscopy for rapid diagnosis of enteroviruses

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100111240 ◽

1984 ◽

Vol 42 ◽

pp. 226-227 ◽

Cited By ~ 1

Author(s):

K. Pegg-Feige ◽

F. W. Doane

Keyword(s):

Electron Microscopy ◽

Cell Culture ◽

Immunoelectron Microscopy ◽

Detection Method ◽

Solid Phase ◽

Protein A ◽

Plant Viruses ◽

Rapid Diagnosis ◽

Virus Diagnosis ◽

Antiviral Antibody

Immunoelectron microscopy (IEM) applied to rapid virus diagnosis offers a more sensitive detection method than direct electron microscopy (DEM), and can also be used to serotype viruses. One of several IEM techniques is that introduced by Derrick in 1972, in which antiviral antibody is attached to the support film of an EM specimen grid. Originally developed for plant viruses, it has recently been applied to several animal viruses, especially rotaviruses. We have investigated the use of this solid phase IEM technique (SPIEM) in detecting and identifying enteroviruses (in the form of crude cell culture isolates), and have compared it with a modified “SPIEM-SPA” method in which grids are coated with protein A from Staphylococcus aureus prior to exposure to antiserum.

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Automated cell counting of astrocytes on patterned substrates containing aliphatic and charged properties

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014124x ◽

1995 ◽

Vol 53 ◽

pp. 974-975

Author(s):

W. Shain ◽

H. Ancin ◽

H.C. Craighead ◽

M. Isaacson ◽

L. Kam ◽

...

Keyword(s):

Cell Culture ◽

Cell Attachment ◽

Culture Method ◽

Cell Counting ◽

Data Sets ◽

Nuclear Staining ◽

Double Positive ◽

A Cell ◽

Wafer Test ◽

Cell Densities

Neural protheses have potential to restore nervous system functions lost by trauma or disease. Nanofabrication extends this approach to implants for stimulating and recording from single or small groups of neurons in the spinal cord and brain; however, tissue compatibility is a major limitation to their practical application. We are using a cell culture method for quantitatively measuring cell attachment to surfaces designed for nanofabricated neural prostheses.Silicon wafer test surfaces composed of 50-μm bars separated by aliphatic regions were fabricated using methods similar to a procedure described by Kleinfeld et al. Test surfaces contained either a single or double positive charge/residue. Cyanine dyes (diIC18(3)) stained the background and cell membranes (Fig 1); however, identification of individual cells at higher densities was difficult (Fig 2). Nuclear staining with acriflavine allowed discrimination of individual cells and permitted automated counting of nuclei using 3-D data sets from the confocal microscope (Fig 3). For cell attachment assays, LRM5 5 astroglial cells and astrocytes in primary cell culture were plated at increasing cell densities on test substrates, incubated for 24 hr, fixed, stained, mounted on coverslips, and imaged with a 10x objective.

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