Continuous Cell Line

Continuous cell lines or human diploid cells used to produce vaccines must be demonstrated to be free of human infectious and/or pathogenic viruses (Monographs on extraneous agents and cell substrates in Pharmacopoeias, ICH, WHO and regulatory agencies' guidance on cell substrate, e.g., [ai]).

From: Biopharmaceutical Processing , 2018

Cardioviruses☆

C. Billinis , O. Papadopoulos , in Reference Module in Biomedical Sciences, 2014

Virus Propagation

Cardioviruses replicate in primary or continuous cell lines originating from a variety of species, including murine, bovine, porcine, human, primate, guinea pig, and hamster. Baby hamster kidney (BHK-21) and Vero cells are most commonly used. The virus also replicates in baby mice and chicken embryos and is pathogenic to many laboratory animals. EMC virus hemagglutinates guinea pig, rat, horse, and sheep erythrocytes. Serial passages of EMC viruses in cell culture can alter in vitro growth characteristics, reduce virulence, and affect hemagglutinating activity.

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Novel Nanotechnology Based Delivery Systems for Chemotherapy and Prophylaxis of Tuberculosis

Rajesh Jain , ... Mala Menon , in Handbook of Nanomaterials for Industrial Applications, 2018

33.8.2.1 In Vitro Lung Epithelial Cell Culture Models

In vitro cell culture models have various advantages, like continuous cell lines, easy handling, and availability in large numbers. These feasibilities lead to improved and multiple designing of experiments simultaneously, this limits the use of live animals to a great extent. Different cell models derived from pulmonary epithelium from murine and human tissues have been established. The Caco-2 cell line can predict the transporter mechanisms across the pulmonary epithelium and has been standardized for transporter mechanism studies in the gastrointestinal tract. For this precise reason, many studies were performed using pulmonary epithelial cell lines derived from both human and murine sources as a lung-equivalent to Caco-2. A human bronchial epithelial cell line (16HBE14o) has been used for a long time for studying the drug transport mechanism in airways and drug permeation. Higher permeability of hydrophilic molecules in the 16HBE14o cell line is observed as compared to the typical alveolar epithelium cell models. Furthermore, the permeability of lipophilic molecules is found to have a sigmoidal relationship between permeability and lipophilicity.

Another pulmonary epithelium cell model Calu-3, a human subbronchial gland cell line has been extensively used to study drug permeation and transport mechanisms of small drug molecules and xenobiotics to be administered via the pulmonary route. The Calu-3 cells can be cultivated in transwell plates with two compartments: a basolateral compartment with cell culture medium and an apical compartment, which is normally empty, representing air.

Other cell lines derived from human pulmonary epithelium (pneumonocytes Type-2) include the A549 cell line. Murine-derived cells include rat tracheobronchial cell lines and primary cells, rabbit primary alveolar-type cells and cell lines.

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VIRUSES | Norovirus

J.L. Cannon , ... E. Papafragkou , in Encyclopedia of Food Microbiology (Second Edition), 2014

Surrogates

Since the discovery of Norwalk virus, numerous attempts to culture huNoVs in vitro using continuous cell lines of human and animal origin, as well as in primary tissues, have not been successful. Even though murine norovirus (MNV) replicates in macrophages and dendritic cells, the same type of cells are not permissive to human strains. Although recent efforts to cultivate huNoVs have been reported using a three-dimensional organoid model of human intestine epithelial cells attached on collagen-microcarrier beads in a rotating bioreactor, reproduction has not been successful despite attempts by several independent laboratories. Recent findings of three-dimensional microorgan cultures of human intestines from pluripotent stem cells supporting replication of several huNoV GII strains was demonstrated by detection of viral RNA, immunochemistry, confocal microscopy, and immune electron microscopy, providing a promising approach for further development. To date, there is no large-scale cell culture or small animal model available for huNoV, and hence, much information about the virus's infection cycle, pathogenesis, and response to chemical and environmental assaults remains unknown. In recent years, reverse genetic approaches using porcine, bovine, and murine models has advanced the understanding of some critical characteristics of huNoV replication.

Estimating huNoV infectivity, until now, has required the use of representative surrogate viruses. Alternative culture models for huNoVs have been examined considerably in previous years and have included feline calicivirus (FCV), murine norovirus (MNV), poliovirus (PV), and male-specific coliphage (MS2). FCV historically has been utilized as a huNoV surrogate, as it was one of the first cultivable members of the Caliciviridae family, sharing similarities with huNoV in terms of its size, shape, and genome organization. The environmental persistence, transferability, and response to disinfection have been studied extensively with FCV. There have been concerns about its validity as a surrogate, however, due to its sensitivity to extremes in pH and its different behavior to other decontamination treatments (e.g., heat, alcohol) compared with the huNoVs. After the discovery of MNV in 2003, this virus was incorporated in the list of suitable model viruses as it can replicate in the intestine and share several physicochemical properties with huNoV. Despite enteric shedding, however, MNV does not cause gastroenteritis and does not depend on HBGAs for infection. Application of dual surrogates, FCV in conjunction with MNV, thus has been recommended. In recent years, thorough investigation of the culture-adapted, enteropathogenic Cowden strain of porcine sapovirus as well as the Tulane virus, cultivable in monkey kidney cells, has begun to determine their promise as huNoV surrogates.

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Modern Production Strategies in the Vaccine Industry

Hari Pujar , Mats Lundgren , in Biopharmaceutical Processing, 2018

Viral Vaccine Purification

Although traditional whole-virus vaccines did not undergo much purification, newer vaccines, especially those that are produced in continuous cell lines and those that are inactivated undergo extensive purification ( Fig. 43.4).

Fig. 43.4. Example of a typical viral vaccine production process.

The broad diversity of the purification challenge—in virus size, enveloped-nature, stability, and the cell line, contrasts with purification of monoclonal antibodies where a platform process is established in many firms. The key downstream processing goal is to reduce host-cell derived impurities, with residual DNA of particular concern in the case of continuous cell lines [34a], while keeping the usually fragile virus active. Furthermore, the route of administration, oral versus intramuscular, also matters. This is compounded by the lack of robust assays for purity and potency of the virus, thus making downstream process development especially challenging.

Modern downstream purification starts with harvesting of the virus by centrifugation and/or filtration, purification via chromatography, inactivation as needed with chemical additives, and final conditioning with tangential flow filtration [35,36]. Some of the steps are not performed, as driven by the target product profile. Inactivation, if needed, may occur at several different steps, depending on the process design. It is usually advisable to perform inactivation after purification is completed so as to not cross-link host-cell proteins and the target virus, although it makes most of the downstream process BSL-2 or higher.

Purification of whole virus vaccines in the early days sometimes used density gradient ultracentrifugation in combination with filtration techniques. Ultracentrifugation is time-consuming, poorly scalable, and requires extensive maintenance. Thus the transition to chromatographic methods is necessary. Although size-exclusion chromatography can provide efficient purification at laboratory scale, the low throughput [37] restricts its use in processing at an industrial scale. The next approach was to use chromatography resins designed for protein separations; however, the large size of viruses resulted in low binding capacity, leading to the introduction of wide-pore resins. More recently, flow-through chromatography, either in the fixed packed-bed format or in the membrane format, whereby impurities in the feed are adsorbed to the resin while the target virus passes through the column unretained, provides significantly higher throughput with minimal dilution. Innovations in resin design such as a new core bead resin type [38] will continue to advance purification platforms. The resin is bi-functional, consisting of an inactive outer layer and an active core with ligands for capture of the contaminants (Fig. 43.5). The bead pores are specifically designed to exclude larger molecular entities such as viruses and big protein complexes while allowing smaller entities such as proteins, peptides and small DNA fragments, into the internal space where adsorption to the ligands can occur.

Fig. 43.5. Schematic cross sectional view of a core bead surrounded by virus particles.

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Bioartificial Pancreas

Athanassios Sambanis , in Principles of Tissue Engineering (Fourth Edition), 2014

β cell lines

Recognizing the substantial difficulties involved with the procurement and amplification of pancreatic islets, several investigators have pursued the development of continuous cell lines, which can be amplified in culture, yet retain key differentiated properties of normal β cells. One of the first successful developments in this area was the generation of the βTC family of insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene; these cells retained their differentiated features for about 50 passages in culture [15]. The hypersensitive glucose responsiveness of the initial βTC lines was reportedly corrected in subsequent lines by ensuring expression of glucokinase and of the high Km glucose transporter Glut2, and no or low expression of hexokinase and of the low Km transporter Glut1 [16,17]. A similar approach was used to develop the murine MIN-6 cell line that exhibits glucose-responsive secretion of endogenous insulin [18]. Subsequently, Efrat and co-workers developed the βTC-tet cell line, in which expression of the SV40 T antigen (Tag) oncoprotein is tightly and reversibly regulated by tetracycline. Thus, cells proliferate when Tag is expressed, and shutting-off Tag expression halts cell growth [19]. Such reversible transformation is an elegant approach in generating a supply of β cells via proliferation of an inoculum, followed by growth arrest when the desirable population size is reached. In any case, when retained in capsules, proliferating cells do not grow uncontrollably, since the dissolved oxygen concentration in the surrounding milieu supports up to a certain number of viable, metabolically active cells in the capsule volume. This number of viable cells is maintained through equilibration of cell growth and death processes [20–22]. Thus, growth arrest is primarily useful in preventing the growth of cells that have escaped from broken capsules in vivo and in reducing the cellular turnover in the capsules. The latter reduces the number or accumulated dead cells in the transplant and thus the antigenic load to the host due to antigens shed by dead and lysed cells, which pass through the capsule material.

In a different approach, Newgard and co-workers [23] carried out a stepwise introduction of genes related to β cell performance into a poorly secreting rat insulinoma (RIN) line. In particular, RIN cells were iteratively engineered to stably express multiple copies of the insulin gene, the glucose transporter Glut2, and the glucokinase gene, which are deemed essential for proper expression of β cell function. Although this is an interesting methodology, identifying and expressing all the genes necessary for reproducing β cell function in a host cell is a daunting task. Also, significant progress has been made towards establishing a human pancreatic β cell line that appears functionally equivalent to normal β cells [24]. This was accomplished through a complicated procedure involving retroviral transfection of primary β cells with the SV40 large T antigen and cDNAs of human telomerase reverse transcriptase. This resulted in a reversibly immortalized human β cell clone, which secreted insulin in response to glucose, expressed β cell transcriptional factors, prohormone convertases 1/3 and 2 that process proinsulin to mature insulin, and restored normoglycemia when transplanted in diabetic immunodeficient mice [24].

With regard to β cell lines capable of proliferation under appropriate conditions, key issues that remain to be addressed include their long-term phenotypic stability, their potential tumorigenicity, especially if cells escape from the encapsulation device, and their possible recognition by the immune system of the host.

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PRODUCTION AND ASSAY OF HUMAN LYMPHOCYTE INTERFERON INDUCED BY ANTIGENS

Jon A. Green , Sidney R. Cooperband , in In Vitro Methods in Cell-Mediated Immunity, 1971

2 Interferon Assay

The CPE of vesicular stomatitis virus is better visualized in human amnion cell cultures than in HeLa cell cultures. However, the technical difficulties involved in the preparation of amnion cells recommends against their use. When continuous cell lines are employed for interferon assay they must be used within a few hours of becoming confluent. If these cells are allowed to overgrow, the many dividing and dying cells will obfuscate the viral induced CPE. In addition, cultures of high cell density are easily detached from the glass by manipulations necessary to perform the assay. The quantity of interferon detected by this assay is partly related to the challenge dose of virus. Increases in the concentration of challenge virus will result in a lower titre of interferon.

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Engineering Fundamentals of Biotechnology

S. Sood , ... A. Kumar , in Comprehensive Biotechnology (Second Edition), 2011

2.13.8.6 Inoculation of Cell Lines

There are three categories of cell monolayers normally used for viral cultures: primary, semi-continuous, and continuous. Primary cell lines are obtained after dissociating tissues and organs but have a short life due to contact inhibition of the cell monolayer. These are used for inoculation of Orthomyxoviruses and echoviruses. The subculture of primary cell monolayers yields semi-continuous cell lines, which survive up to 50–100 population doublings before dying. These include fibroblast cell lines such as MRC5 and WI38. Continuous cell lines are subcultured indefinitely in glass, plastic surfaces, or suspensions as they are obtained usually from carcinogenic cells. These cell lines are used most often because of faster and longer growth, less nutritional requirement, and high plating efficiency. Some common examples are HeLa (human cervical carcinoma), HEp2 (human epithelial), and BHK 21 (baby hamster kidney). After the inoculation of viruses, the cell lines are incubated at 37  °C for 12–24   h to allow viruses to adsorb to the cell monolayer. To reduce the toxicity further, the inoculated cell lines are incubated at 37   °C for 12–24   h to allow viruses to adsorb to cell monolayer. The inoculum and the culture medium is removed and replaced with fresh maintenance medium. This yields maximum virus recovery. The above viral culture constitutes the inoculum for passage into a second set of tubes. This is repeated to reduce nonspecific toxicity and to enhance the cytopathic effects of the virus. This helps to generate maximum yield and diagnosis of the virus [43].

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Cell sources and methods for producing organotypic in vitro human tissue models

Patrick J. Hayden , in Organ-on-a-chip, 2020

Continuous (immortal) cell lines

Human somatic cells are in general capable of only a limited number (40–60) of cell divisions in culture before they become senescent and lose their ability to divide (Hayflick and Moorhead, 1961). However, immortal somatic cell lines with a capacity for unlimited division potential can be obtained by a number of processes. Genetically mutated cells derived from cancerous tissues are a common source for establishing immortal cell lines. In rare cases, cultured normal (noncancer-derived) cells may spontaneously acquire genetic mutations that provide the ability for unlimited growth. Normal cells may also be transformed into immortalized cells by the introduction of viral oncogenes such as EBV, SV40LT, HPV16 E6/E7, and Ad5 E1A (Honegger, 2001; Freshney, 2016). Induction of telomerase activity by transduction of human Telomerase reverse transcriptase (hTERT) into cells can induce immortal transformation while retaining more normal cell phenotypes than viral-induced transformations (Freshney, 2016).

The development of methods for establishing and maintaining continuous cell lines in culture marked a revolutionary advancement in biology. Since the establishment of HeLa cells as the first immortal human cell line in 1952 (Gey et al., 1952), continuous cell lines have become widely used as indispensable and inexpensive tools for basic biological research, chemical metabolism and toxicity tests, and production of biological compounds such as vaccines, antibodies, and therapeutic proteins. Numerous immortal cell lines derived from a wide variety of tissues are now readily available. Key advantages of immortal cell lines are that they are affordable, well characterized, and easy to culture. However, immortal cell lines generally exhibit significant genotypic and phenotypic abnormalities that may limit their ability to reproduce normal cell behavior and may undergo additional genotypic or phenotypic drift with continued long-term passaging. Furthermore, many continuous cell lines have been misidentified or have become contaminated with mycoplasma or other cell lines over time (Geraghty et al., 2014; Lorsch et al., 2014). Authentication of cell lines is now recommended or required for publication or submission of research results to regulatory authorities (Geraghty et al., 2014).

Despite their shortcomings, immortal cell lines remain in widespread use and will continue to be important biological tools that may be suitable or advantageous for specific OoC applications. Table 2.1 presents a list of commonly used immortal human cell lines derived from a variety of organs. These cell lines, as well as others, are also available as authenticated and quality-controlled resources from a number of nonprofit repositories (Table 2.2).

Table 2.1. Commonly used continuous (immortal) human cell lines.

Cell type Origin Name Reference
Endothelial Liver SK HEP-1 Heffelfinger et al. (1992)
Hepatic Liver HepaRG Parent et al. (2004), Guillouzo et al. (2007), and Takahashi et al. (2015)
Epithelial Liver HepG2 Diamond et al. (1980)
Epithelial Breast MCF-7 Brooks et al. (1973)
Epithelial Breast ZR-75-1 Engel et al. (1978)
Keratinocyte Epidermis HaCaT Boukamp et al. (1988)
Type I pneumocyte Lung hAELVi Kuehn et al. (2016)
Type II pneumocyte Lung A549 Giard et al. (1973)
Type II pneumocyte Lung NCI-H441 Brower et al. (1986)
Epithelial Lung BEAS-2B Reddel et al. (1988)
Epithelial Lung Calu-3 Fogh and Trempe (1975)
Epithelial Kidney HEK-293 Graham et al. (1977)
Epithelial Ovary A2780 Tsuruo et al. (1986)
Epithelial Colon Caco-2 Fogh et al. (1977)
Epithelial Colon HT-29 Fogh and Trempe (1975)
Epithelial Cervix HeLa Gey et al. (1952)
Glial Glioma MOG-G-CCM Balmforth et al. (1986)
Glial Brain U-251 MG Ponten and Macintyre (1968)
Lymphocytic Blood EB-3 Epstein and Barr (1964)
Myeloid Blood K562 Andersson et al. (1979)
Myeloid Blood HL-60 Olsson et al. (1981)
Myeloid Blood THP-1 Tsuchiya et al. (1980)
Myeloid Blood U937 Sundström and Nilsson (1976)

Adapted from Freshney, R.I. (2016). Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (7th ed.). NJ: Wiley-Blackwell, with additions.

Table 2.2. Nonprofit immortal cell line repositories.

Institution Headquarters Website
ATCC Manassas, VA, United States www.atcc.org
CellBank Australia, Sydney, Australia, www.cellbankaustralia.com
ECACC Salisbury, Wiltshire, United Kingdom www.phe-culturecollections.org.uk/collections/ecacc.aspx
The Leibniz Institute DSMZ: German Collection of Microorganisms and Cell Cultures GmbH (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) Braunschweig, Germany www.dsmz.de
JCRB Cell Bank Ibaraki City, Osaka, Japan www.cellbank.nibiohn.go.jp

ATCC, American Type Culture Collection; ECACC, The European Collection of Authenticated Cell Cultures; JCRB, Japanese Collection of Research Bioresources.

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TISSUE ENGINEERING

Randall McClelland PhD , ... Jeffery M. Macdonald PhD , in Introduction to Biomedical Engineering (Second Edition), 2005

7.4.3 Time Scales of Mass Transfer

The importance of mass transfer in tissue and cellular function is often overlooked. The limitations imposed by molecular diffusion become clear if the average displacement distance with time is plotted for diffusion coefficients that are typical for biological entities of interest in tissue function (Fig. 7.30). The diffusional penetration lengths over physiological time scales are surprisingly short and constrain the in vivo design and architecture of organs. The same constraints are faced in the construction of an ex vivo device, and high mass transfer rates into cell beds at physiological cell densities may be difficult to achieve.

The biochemical characteristics of the microenvironment are critical to obtaining proper tissue function. Much information exists about the biochemical requirements for the growth of continuous cell lines. For continuous cell lines, these issues revolve around the provision of nutrients and the removal of waste products. In cultures of primary cells, the nutrients may have other roles and directly influence the physiological performance of the culture. For instance, recently it has been shown that proline and oxygen levels play an important role in hepatocyte cultures.

In most cases, oxygen delivery is likely to be an important consideration. Too much oxygen will be inhibitory or toxic, whereas too little may alter metabolism. Some tissues, such as liver, kidney, and brain, have high oxygen requirements, whereas others require less. Controlling oxygen at the desired concentration levels, at the desired uniformity, and at the fluxes needed at high cell densities is likely to prove challenging. Further, the oxygen and nutritional requirements may vary among cell types in a reconstituted tissue that is comprised of multiple cell types. These requirements further complicate nutrient delivery. Thus, defining, designing, and controlling the biochemical characteristics of the microenvironment may prove difficult, especially given the constraints imposed by diffusion and any requirements for a particular microgeometry.

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BET mechanisms in cancer

Elizabeth Henderson , Panagis Filippakopoulos , in Histone Modifications in Therapy, 2020

5.11 Ovarian cancer

Ovarian cancer is the seventh most common cancer in women worldwide. 145 With the majority of women relapsing following platinum-based regimens, the median overall survival is 1 year, indicating the need for alternative therapeutic strategies. 146 Yet there are indications that BET BRD inhibitors may represent a possible therapeutic strategy; BRD4 expression has been found to correlate with disease progression and its amplification was found to inversely correlate with disease prognosis. 109, 111 Early studies found that the response of ovarian malignant models to BET BRD inhibition is inconsistent, with variable reduction in growth inhibition, apoptosis induction, and nonuniform cell cycle arrest; half of the tested ovarian cancer cell lines showed a modest decrease in the G2 population. 103, 107 However, two BET BRD inhibitors, (+)-JQ1 and GS-626510, led to reduced cell viability and colony formation in continuous cell lines with c MYC amplification. 110, 111 Findings in vivo using cMYC- or BRD4-amplified xenografts, whether originating from ovarian cancer primary cell line or chemotherapy-resistant patient-derived cells, were also promising, with reduced tumor growth and increased murine survival in both xenograft models. 108, 109, 111 This suggests cMYC amplification may stratify patients in terms of predicted response to BET BRD inhibitors.

In terms of the molecular response, some continuous cell lines demonstrated dependence on BRD4 for viability; investigations showed that one or both of FOSL and MYC, two BRD4 target genes, were downregulated across ovarian cell lines following (+)-JQ1 treatment. 103, 107 This finding was puzzling, however, owing to the inconsistent cellular phenotypes following (+)-JQ1 treatment, despite the finding that many ovarian cell lines displayed downregulation of MYC mRNA and protein levels. Beyond MYC, there is evidence that the transcription factor FOXM1 may act as a driver of ovarian cancer. Upon treatment with (+)-JQ1, FOXM1 mRNA and protein expression decreased; in vitro depletion of the factor mimicked the effects of BET BRD inhibitor, while overexpression of FOXM1 within a tumor xenografts rescued cells from the antiproliferative effects of (+)-JQ1. 109 While the studies presented here testify the importance of MYC, these studies point to other transcription factors as determinants of cellular response to treatment with BET BRD inhibitors.

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