An Efficient System for Gene Perturbation in Embryonic Hippocampal Progenitors Using Ex Vivo Electroporation Followed by In Vitro Dissociated Cell Culture

SARS-CoV-2 has spread across the globe with an astonishing velocity and lethality that has put scientist and pharmaceutical companies worldwide on the spot to develop novel treatment options and reliable vaccination for billions of people. To combat its associated disease COVID-19 and potentially newly emerging coronaviruses, numerous pre-clinical cell culture techniques have progressively been used, which allow the study of SARS-CoV-2 pathogenesis, basic replication mechanisms, and drug efficiency in the most authentic context. Hence, this review was designed to summarize and discuss currently used in vitro and ex vivo cell culture systems and will illustrate how these systems will help us to face the challenges imposed by the current SARS-CoV-2 pandemic.

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Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus

Viruses ◽

10.3390/v12101102 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1102

Author(s):

Phoebe Stevenson-Leggett ◽

Sarah Keep ◽

Erica Bickerton

Keyword(s):

Cell Culture ◽

Infectious Bronchitis Virus ◽

Ex Vivo ◽

Serial Passage ◽

In Ovo ◽

Infectious Bronchitis ◽

Protease Treatment ◽

Cellular Tropism ◽

Passage Cell

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2′ cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.

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Long-term In Vitro Toxicity Models: Comparisons Between a Flow-cell Bioreactor, a Static-cell Bioreactor and Static Cell Cultures

Alternatives to Laboratory Animals ◽

10.1177/026119290203000505 ◽

2002 ◽

Vol 30 (5) ◽

pp. 515-523 ◽

Cited By ~ 13

Author(s):

Patricia Pazos ◽

Salvador Fortaner ◽

Pilar Prieto

Keyword(s):

Cell Culture ◽

Cell Viability ◽

Flow Cell ◽

Model Systems ◽

In Vitro Toxicity ◽

Protein Determination ◽

Efficient System ◽

Cytotoxicity Studies

In vitro long-term toxicity testing is becoming an important issue in the field of toxicology, and there is a need to develop new model systems that mimic human chronic exposure and its effects. The aim of this work was to test two long-term in vitro toxicity systems which are available, a flow-cell bioreactor (Tecnomouse) and a static cell bioreactor system (CELLine CL 6-well), and to compare them with the use of conventional cell culture flasks. A human cell line, Int 407, was exposed to cadmium chloride (CdCl2; 10–7–10–8M) for 4 weeks. Cell numbers and cell viabilities were determined by the trypan blue (TB) exclusion assay and from exclusion of propidium iodide (PI) as determined by flow cytometry; and cell viability and metabolic activity were determined by the MTT assay. In addition, total protein determination and cadmium uptake measurements were performed. The results obtained with TB and PI exclusion did not show clear differences in cell viability with increasing CdCl2 concentration. However, in the static cell-culture systems, an increase in MTT reduction was found at low concentrations of CdCl2. Expression of heat-shock protein (Hsp27 and Hsp70) increased differently, depending on the CdCl2 concentration applied and the system used. In summary, of the two bioreactors, the CELLine CL 6-well bioreactor was shown to be the more efficient system for performing long-term cytotoxicity studies. It is easy to handle, it permits the assessment of several endpoints, and sufficient replicates can be made available.

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A simple method for ex vivo honey bee cell culture capable of in vitro gene expression analysis

PLoS ONE ◽

10.1371/journal.pone.0257770 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0257770

Author(s):

Kazuyo Watanabe ◽

Mikio Yoshiyama ◽

Gaku Akiduki ◽

Kakeru Yokoi ◽

Hiroko Hoshida ◽

...

Keyword(s):

Gene Expression ◽

Cell Culture ◽

Honey Bee ◽

Cell Lines ◽

Fluorescent Protein ◽

Cultured Cells ◽

Ex Vivo ◽

Plasmid Vector ◽

Simple Method

Cultured cells are a very powerful tool for investigating biological events in vitro; therefore, cell lines have been established not only in model insect species, but also in non-model species. However, there are few reports on the establishment of stable cell lines and development of systems to introduce genes into the cultured cells of the honey bee (Apis mellifera). We describe a simple ex vivo cell culture system for the honey bee. Hemocyte cells obtained from third and fourth instar larvae were cultured in commercial Grace’s insect medium or MGM-450 insect medium for more than two weeks maintaining a normal morphology without deterioration. After an expression plasmid vector bearing the enhanced green fluorescent protein (egfp) gene driven by the immediate early 2 (IE2) viral promoter was transfected into cells, EGFP fluorescence was detected in cells for more than one week from one day after transfection. Furthermore, double-stranded RNA corresponding to a part of the egfp gene was successfully introduced into cells and interfered with egfp gene expression. A convenient and reproducible method for an ex vivo cell culture that is fully practicable for gene expression assays was established for the honey bee.

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From infancy to fancy - A glimpse into the evolutionary journey of podocytes in culture

Kidney360 ◽

10.34067/kid.0006492020 ◽

2020 ◽

pp. 10.34067/KID.0006492020

Author(s):

Shivangi Agarwal ◽

Yashwanth R. Sudhini ◽

Jochen Reiser ◽

Mehmet M. Altintas

Keyword(s):

Cell Culture ◽

Ex Vivo ◽

Model Systems ◽

Glomerular Diseases ◽

Filtration Barrier ◽

Technological Advances ◽

The Status ◽

Pros And Cons ◽

Critical Components

Podocytes are critical components of the filtration barrier and responsible for maintaining healthy kidney function. An assault on podocytes is generally associated with progression of chronic glomerular diseases. Therefore, podocyte pathophysiology is a favorite subject of research for nephrologists. Despite this, podocyte research has lagged behind because of the unavailability of techniques for culturing such specialized cells ex vivo in quantities that are adequate for mechanistic studies. In recent years, this problem was circumvented by the efforts of researchers who successfully developed several in vitro podocyte cell culture model systems that paved the way for incredible discoveries in the field of nephrology. This review embarks us on a journey that provides a comprehensive insight into the groundbreaking breakthroughs and novel technological advances made in the field of podocyte cell culture so far, beginning from its inception, evolution, and progression. Herein, we also describe in detail the pros and cons of different models that are currently being used to culture podocytes. Our extensive and exhaustive deliberation on the status of podocyte cell culture will facilitate the researchers to wisely choose an appropriate model for their own research to avoid potential pitfalls in the future.

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Retention of quiescent hematopoietic cells with high proliferative potential during ex vivo stem cell culture

Blood ◽

10.1182/blood.v87.2.545.bloodjournal872545 ◽

1996 ◽

Vol 87 (2) ◽

pp. 545-556 ◽

Cited By ~ 68

Author(s):

JC Young ◽

A Varma ◽

D DiGiusto ◽

MP Backer

Keyword(s):

Cell Culture ◽

Single Cell ◽

Ex Vivo ◽

Hematopoietic Cells ◽

Proliferative Capacity ◽

Quiescent Cells ◽

Cell Divisions ◽

Single Cell Culture ◽

Bulk Culture

Human CD34+/Thy-1+/Lin- hematopoietic cells purified from bone marrow (BM) or mobilized peripheral blood (MPB) are highly enriched for pluripotent stem cells. Ex vivo expansion of this population is proposed as a means of providing accelerated short-term, as well as long-term, engraftment after myeloablative therapy. Here we demonstrate that primitive quiescent cells are retained in bulk expansion cultures of CD34+/Thy-1+/Lin- cells and that the cell production capacity of the expanded cell product can largely be attributed to cells exhibiting quiescent behavior during culture. CD34+/Thy-1+/Lin- cells from adult BM or MPB were labeled with the fluorescent membrane dye PKH26, followed by in vitro culture of 10(4) cells on a murine stromal layer in the presence of interleukin (IL)-3, IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF). With each subsequent cell division, PKH26 fluorescence is reduced by roughly half, which allows tracking of the number of cell divisions. Progenitor cells present after a 2-week expansion period were sorted into CD34+/Lin-/dyebright and CD34+/Lin- /dyedim fractions and then cultured in a 4-week single-cell proliferation assay to characterize the proliferative capacity of each group. Fifty-nine percent of progenitors remaining dyebright after bulk culture (four or fewer cell divisions) were observed to proliferate in single cell culture, and produced an average of 1,780 cells per plated cell. In contrast, only 26% of dyedim (more than four divisions) progenitors were observed to proliferate and displayed a lower average proliferative capacity of 225 cells per plated cell. Similar behaviors were observed after a second consecutive cycle of bulk culture, indicating that quiescent cells with high proliferative capacity existed in culture for at least 4 weeks. Single CD34+/Lin-/dyebright progenitors purified from bulk cultures were observed to produce as many as 1,000 CD34 positive progeny during single cell culture, and these progeny included multilineage colony forming cells. These data demonstrate that among CD34 positive cells recovered after in vitro bulk culture, a higher proliferative capacity correlated with quiescent behavior. The described culture method provides quantitation of the cell producing capacity of individual cells in hematopoietic cell mixtures and may prove useful for predicting engrafting potential in products intended for cellular therapy.

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Toxicity of food flavorings to ex-vivo, in vitro and in vivo bioassays

Acta Scientiarum Technology ◽

10.4025/actascitechnol.v42i1.44867 ◽

2019 ◽

Vol 42 ◽

pp. e44867

Author(s):

Fabelina Karollyne da Silva dos Santos ◽

Márcia Maria Mendes Marques ◽

Maurício Fraga Van Tilbulrg ◽

Maria Izabel Florindo Guedes ◽

Paulo Agenor Alves Bueno ◽

...

Keyword(s):

Cell Culture ◽

Ex Vivo ◽

Artemia Salina ◽

Significant Inhibition ◽

Pure Form ◽

Root Cells ◽

Significant Toxicity ◽

Cellular Alterations

This study evaluated the toxicity of food flavorings of mint, cinnamon and lemon in meristem root cells of Allium cepa, in pure form (as marketed) and in the concentrations of 12.5, 25, and 50%, after 24 and 48 hours of exposure; in Vero cell culture evaluated by MTT test and in nauplii of Artemia salina, both tests used flavorings in pure form and in the concentrations of 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50%, after 24 hours of exposure. The three flavorings, in all treatments and times of analysis considered, caused significant inhibition of cell division. However, the flavorings did not cause cellular alterations to the evaluated meristems. All evaluated treatments significantly reduced the viability of the evaluated cell line and promoted 100% lethality of A. salina nauplii. The evaluated flavorings, under the established study conditions, promoted wide and significant toxicity.

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Direct Current Stimulation in Cell Culture Systems and Brain Slices—New Approaches for Mechanistic Evaluation of Neuronal Plasticity and Neuromodulation: State of the Art

Cells ◽

10.3390/cells10123583 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3583

Author(s):

Nadine Euskirchen ◽

Michael A. Nitsche ◽

Christoph van Thriel

Keyword(s):

Cell Culture ◽

Direct Current ◽

Neuronal Plasticity ◽

Intracellular Signaling ◽

Ex Vivo ◽

Brain Slices ◽

Field Orientation ◽

Direct Current Stimulation

Non-invasive direct current stimulation (DCS) of the human brain induces neuronal plasticity and alters plasticity-related cognition and behavior. Numerous basic animal research studies focusing on molecular and cellular targets of DCS have been published. In vivo, ex vivo, and in vitro models enhanced knowledge about mechanistic foundations of DCS effects. Our review identified 451 papers using a PRISMA-based search strategy. Only a minority of these papers used cell culture or brain slice experiments with DCS paradigms comparable to those applied in humans. Most of the studies were performed in brain slices (9 papers), whereas cell culture experiments (2 papers) were only rarely conducted. These ex vivo and in vitro approaches underline the importance of cell and electric field orientation, cell morphology, cell location within populations, stimulation duration (acute, prolonged, chronic), and molecular changes, such as Ca2+-dependent intracellular signaling pathways, for the effects of DC stimulation. The reviewed studies help to clarify and confirm basic mechanisms of this intervention. However, the potential of in vitro studies has not been fully exploited and a more systematic combination of rodent models, ex vivo, and cellular approaches might provide a better insight into the neurophysiological changes caused by tDCS.

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Changes in the number of chick ciliary ganglion neuron processes with time in cell culture.

The Journal of Cell Biology ◽

10.1083/jcb.104.2.363 ◽

1987 ◽

Vol 104 (2) ◽

pp. 363-370 ◽

Cited By ~ 20

Author(s):

L W Role ◽

G D Fischbach

Keyword(s):

Cell Culture ◽

Time Course ◽

Culture Conditions ◽

Ciliary Ganglion ◽

Intact Cells ◽

Dissociated Cell Culture ◽

Ciliary Ganglion Neurons ◽

Ganglion Neurons

The purpose of this study was to describe the shape of chick ciliary ganglion neurons dissociated from embryonic day 8 or 9 ganglia and maintained in vitro. Most of the neurons were multipolar during the first three days after plating, with an average of 6.0 processes extending directly from the cell body. The neurons became unipolar with time. The remaining primary process accounted for greater than 90% of the total neuritic arbor. This striking change in morphology was not due to the selective loss of multipolar cells, or to an obvious decline in the health of apparently intact cells. The retraction of processes was neither prevented nor promoted by the presence of embryonic muscle cells. Process pruning occurred to the same extent and over the same time course whether the cells were plated on a monolayer of embryonic myotubes or on a layer of lysed fibroblasts. Process retraction is not an inevitable consequence of our culture conditions. Motoneurons dissociated from embryonic spinal cords remained multipolar over the same period of time. We conclude that ciliary ganglion neurons breed true in dissociated cell culture in that the multipolar-unipolar transition reflects their normal, in vivo, developmental program.

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Molecular characterization of hematopoietic stem cells after in vitro amplification on biomimetic 3D PDMS cell culture scaffolds

Scientific Reports ◽

10.1038/s41598-021-00619-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lisa Marx-Blümel ◽

Christian Marx ◽

Jürgen Sonnemann ◽

Frank Weise ◽

Jörg Hampl ◽

...

Keyword(s):

Cell Culture ◽

Signaling Pathways ◽

Metabolic Flux ◽

Ex Vivo ◽

Proteome Analysis ◽

Flux Analysis ◽

Molecular Signaling ◽

Hematopoietic Stem ◽

Molecular Signaling Pathways

AbstractHematopoietic stem cell (HSC) transplantation is successfully applied since the late 1950s. However, its efficacy can be impaired by insufficient numbers of donor HSCs. A promising strategy to overcome this hurdle is the use of an advanced ex vivo culture system that supports the proliferation and, at the same time, maintains the pluripotency of HSCs. Therefore, we have developed artificial 3D bone marrow-like scaffolds made of polydimethylsiloxane (PDMS) that model the natural HSC niche in vitro. These 3D PDMS scaffolds in combination with an optimized HSC culture medium allow the amplification of high numbers of undifferentiated HSCs. After 14 days in vitro cell culture, we performed transcriptome and proteome analysis. Ingenuity pathway analysis indicated that the 3D PDMS cell culture scaffolds altered PI3K/AKT/mTOR pathways and activated SREBP, HIF1α and FOXO signaling, leading to metabolic adaptations, as judged by ELISA, Western blot and metabolic flux analysis. These molecular signaling pathways can promote the expansion of HSCs and are involved in the maintenance of their pluripotency. Thus, we have shown that the 3D PDMS scaffolds activate key molecular signaling pathways to amplify the numbers of undifferentiated HSCs ex vivo effectively.

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