Application of a multiple surface tissue culture propagator for the production of cell monolayers,virus, and biochemicals

A new concept of tissue culture equipment and procedures was developed for the mass-scale growth of several types of animal tissue cells in monolayers on multiple glass surfaces. Continuous, cell lines, primary and diploid cell strains were grown in this equipment. Cells studied include primary bovine kidney, human diploid WI-38, human foreskin, and mouse CCL1 cells. Photomicrographic comparisons of cells grown by these techniques indicate they are morphologically identical to tissue culture cells grown in glass bottles or tubes. The growth of the tissue culture cells in the propagator was monitored by carbohydrate Utilization and acid production. Large-scale production of viruses and biochemicals on cells grown in the multiple-plate tissue culture propagator was accomplished. Virus titers were equal to those obtained from conventional bottle or tube cultures for several strains of influenza, parainfluenza, and respiratory syneytial viruses. High-titred mouse interferon was also produced in this system. In addition to tissue culture cell production, Eaton agent, Mycoplasma pneumoniae was grown on the multiple glass surfaces on a mass scale.     

Susceptibility of cell lines of primate and nonprimate origin to certain human viruses

A comparative study was made of the susceptibility of 11 cell lines of human and animal origin, the WI-38 cell strain and fresh cultures of human thyroid, monkey kidney and hamster embryo tissues to certain human viruses. The animal cell lines were derived from monkey, rabbit, mouse, pig and calf tissues. The viruses used were strains of influenza A₂ and B viruses, parainfluenza viruses types 1, 2 and 3, RS virus, adenoviruses types 3, 4 and 21, poliovirus type 1 and Coxsackie A type 21 and Coxsackie B type 3 viruses. Cell lines derived from nonprimate tissues were generally less susceptible than cell cultures of human and simian origin. The combined use of fresh cultures of human thyroid and monkey kidney tissues and of a human cell line seems to provide a satisfactory indicator system for the viruses employed in this study.     

Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media

Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal‐derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum‐free, protein‐free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single‐cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD‐CHO? and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Optimization of vaccine production for animal health

Vaccines on the basis of mammalian cell cultures are of major importance for human and animal health. Therefore efforts are undertaken for the improved production of more effective vaccines. Of course, the main purpose of all these approaches is to save lives and improve the quality of life for human beings. However, there is also some remarkable effort in the food industry and the associated animal production, especially in the case of some Flaviviridal viruses (BVD), where>80% of all cattle herds are found to be infected. These viruses can cause tremendous economic losses of calfs and embryos (Ames, 1990). Because of these facts, there is a continuous endeavour for improving the manufacturing of therapeutics or preventing agents such as vaccines for the treatment of cattle. The competitive economic situation and the specific market demands still require effective and high yield production methods, especially in the case of one of the most widespread viral diseases in cattle like BVD (Ames, 1990).We have succeeded in establishing an improved method for the production of BVD on the basis of a continuous fermentation mode, that consist of modifications of the corresponding process and media improvements.     

Physiological studies of the Pediococcus pentosaceus biofilm

Pediococcus pentosaceus, a bacterium recently used in human and animal probiotics, was used in combination with supports made from polylactic acid composite soybean meal was used to study biofilm formation, and it was found that dense biofilms developed by Day 1. Proteomic comparison between planktonic and biofilm cultures of P. pentosaceus showed distinct expression patterns of intracellular and extracellular proteins. Type I glyceraldehyde-3-phosphate dehydrogenase was upregulated in biofilm cultures and mediated cell adhesion and encouraged biofilm production. GMP synthase, which regulates GMP synthesis and acts as an intracellular signal molecule to control cell mechanisms and has been exploited in the development of new therapeutic agents, was also upregulated in the biofilm mode of growth. The present work serves as a basis for future studies examining the complex network of systems that regulate lactic acid bacterial (LAB) biofilm formation and can serve as a framework for studies of production of therapeutic agents from LAB.     

The derivation of clinical-grade human embryonic stem cell lines

The pluripotent nature of human embryonic stem cells (hESC) has attracted great interest in using them as a source of cells or tissue in cell therapy. However, in order to be used in regenerative medicine, the pluripotent hESC lines should be established and propagated according to good manufacturing practice quality requirements. The cultures should be animal substance free in order to exclude the risk of infections and immunogenity. They should also be genetically and epigenetically normal. The detailed molecular mechanisms of their pluripotency are still not defined. Using human feeder cells, a medium containing only human proteins, the mechanical isolation of the inner cell mass and mechanical passaging of hESC, is a safe option until a functional defined medium containing physiological concentrations of regulatory factors is available.     

Adaptation of the Se301 insect cell line to suspension culture. Effect of turbulence on growth and on production of nucleopolyhedrovius (SeMNPV)

As chemical pesticides are being banned as control agents for agricultural pests, the use of the highly specific, safe to non-target organisms baculoviruses has been proposed. These viruses can be produced either in vivo or in vitro. In vitro production requires appropriated host insect cell lines with the ability for growing as freely-suspended cells. In this work, the Spodoptera exigua Se301 cell line was used to produce the commercially available S. exigua nucleopolyhedrovirus (SeMNPV) in suspension. Se301 cells showed to be very sensitive to the hydrodynamic shear rates developed in bioreactors. A process of progressive adaptation to freely-suspended cultures using protective additives against shear stress and disaggregant was proposed. The best combinations were polyvinyl alcohol (PVA) or polyvinyl pyrrolidone (PVP) with the disaggregant dextran sulfate (DS). Both static and freely-suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Production of occluded baculovirus (OB) increased with the multiplicity of infection (MOI > 0.1).     

Production of oncolytic adenovirus and human mesenchymal stem cells in a single‐use,Vertical‐Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier‐based cell culture processes

Anchorage‐dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large‐scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage‐dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single‐use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical‐Wheel? technology was evaluated for its potential to support scalable cell culture process development. Two anchorage‐dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow‐derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical‐Wheel bioreactors (PBS‐VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS‐VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS‐VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA‐DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS‐VW, and scale‐up was successfully carried out for two different microcarrier‐based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical‐Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier‐based cell cultures of complex biopharmaceuticals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1600–1612, 2015     

Gamma irradiation of animal sera for inactivation of viruses and mollicutes – A review

Animal-derived materials such as animal sera represent a low, but finite, risk for introduction of an adventitious agent (virus or mollicute) into a biological bulk harvest during upstream manufacturing processes involving mammalian cell substrates. Viral and mollicute (Mycoplasma sp. and Acholeplasma sp.) contamination events have been relatively rare, but many of those that have been reported have been attributed to use of infected animal sera in growth media during cell expansion. The risk of introduction of viruses and mollicutes may be mitigated by elimination of the use of animal sera and implementation instead of chemically defined or serum- and animal-derived material-free cell culture media. When use of animal sera is unavoidable, however, mitigation of the risk of introducing an adventitious contaminant may involve treatment of the sera to inactivate potential contaminants. Gamma irradiation is one of the most widely employed methods for viral and mollicute inactivation in animal sera. In this article, we review the inactivation results reported for viral and mollicute inactivation in frozen serum. Studies performed to assess the impact of gamma irradiation on serum quality and performance are also discussed. The available data indicate that inactivation of mollicutes in serum is essentially complete at the gamma radiation doses normally employed (25–40 kGy), while the efficacy and kinetics for viral inactivation in serum by gamma irradiation appear to be dependent in part upon the size of the target virus.     

Viral metagenome analysis to guide human pathogen monitoring in environmental samples

Aims: The aim of this study was to develop and demonstrate an approach for describing the diversity of human pathogenic viruses in an environmentally isolated viral metagenome. Methods and Results: In silico bioinformatic experiments were used to select an optimum annotation strategy for discovering human viruses in virome data sets and applied to annotate a class B biosolid virome. Results from the in silico study indicated that <1% errors in virus identification could be achieved when nucleotide‐based search programs (BLASTn or tBLASTx), viral genome only databases and sequence reads >200 nt were considered. Within the 51 925 annotated sequences, 94 DNA and 19 RNA sequences were identified as human viruses. Virus diversity included environmentally transmitted agents such as parechovirus, coronavirus, adenovirus and aichi virus, as well as viruses associated with chronic human infections such as human herpes and hepatitis C viruses. Conclusions: This study provided a bioinformatic approach for identifying pathogens in a virome data set and demonstrated the human virus diversity in a relevant environmental sample. Significance and Impact of the Study: As the costs of next‐generation sequencing decrease, the pathogen diversity described by virus metagenomes will provide an unbiased guide for subsequent cell culture and quantitative pathogen analyses and ensures that highly enriched and relevant pathogens are not neglected in exposure and risk assessments.     

CAP, a new human suspension cell line for influenza virus production

Forced by major drawbacks of egg-based influenza virus production, several studies focused on the establishment and optimization of cell-based production systems. Among numerous possible host cell lines from duck, monkey, canine, chicken, mouse, and human origin, only a few will meet regulatory requirements, accomplish industrial standards, and result in high virus titers. From primary virus isolation up to large-scale manufacturing of human vaccines, however, the most logical choice seems to be the use of human cell lines. For this reason, we evaluated the recently established CAP cell line derived from human amniocytes for its potential in influenza virus production in suspension culture in small scale shaker flask and stirred tank bioreactor experiments. Different human and animal influenza viruses could be adapted to produce hemagglutination (HA) titers of at least 2.0 log₁₀ HA units/100 μL without further process optimization. Adjusting trypsin activity as well as infection conditions (multiplicity of infection, infection medium) resulted in HA titers of up to 3.2 log₁₀ HA units/100 μL and maximum cell-specific virus productivities of 6,400 virions/cell (for human influenza A/PR/8/34 as a reference). Surface membrane expression of sialyloligosaccharides as well as HA N-glycosylation patterns were characterized. Overall, experimental results clearly demonstrate the potential of CAP cells for achieving high virus yields for different influenza strains and the option to introduce a highly attractive fully characterized human cell line compliant with regulatory and industrial requirements as an alternative for influenza virus vaccine production.     

The new medium MDSS2N, free of any animal protein supports cell growth and production of various viruses

The development of media free of serum and animal or human proteins is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination. In order to reduce the risk of contamination, we have modified the serum free medium MDSS2, a very efficient serum free medium for the production of various biologicals including experimental vaccines using different cell lines (Merten et al., 1994), by replacing the animal derived products by plant extracts. The new serum and animal protein free medium (MDSS2N) can be efficiently used for biomass production of various cell lines. These cells grow equally well or better in this new serum-free medium than in the old formulation (MDSS2): • BHK-21/BRS cells, adapted to MDSS2N, showed an overall specific growth rate of 0.0197 h-1 (μ_max = 0.0510±0.0058 h-1), whereas those cultivated in MDSS2 grew with an average specific growth rate of 0.0179 h-1 (μ_max = 0.0305±0.0177 h-1). • Vero cells grew with an average specific growth rate of 0.0159 h-1 and 0.0153 h-1 in MDSS2 and MDSS2N, respectively. Very similar growth rates were obtained in microcarrier cultures in stirred tank reactors: the specific growth rates were 0.0161 h-1 and 0.0166 h-1 for MDSS2 and MDSS2N cultures, respectively. • For MDCK cells, when cultured on microcarriers in bioreactors, a higher average specific growth rate was observed in MDSS2N than in MDSS2; values of 0.0248 h-1 and 0.0168 h-1, respectively, were obtained. The capacity of MDSS2N to support the production of different viruses was equally evaluated and it could be established that for certain viruses there are no or insignificant differences between MDSS2N and MDSS2 (influenza and polio virus), whereas, the production of rabies virus is somewhat reduced in MDSS2N when compared to MDSS2. The use of MDSS2N for cell culture and the production of various viruses is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Foreign protein production using plant cell and organ cultures: Advantages and limitations

Plants and plant tissue cultures are used as host systems for expression of foreign proteins including antibodies, vaccines and other therapeutic agents. Recombinant or stably transformed plants and plant cell cultures have been applied for foreign protein production for about 20 years. Because the product concentration achieved exerts a major influence on process economics, considerable efforts have been made by commercial and academic research groups to improve foreign protein expression levels. However, post-synthesis product losses due to protease activity within plant tissues and/or extracellular protein adsorption in plant cell cultures can negate the benefits of molecular or genetic enhancement of protein expression. Transient expression of foreign proteins using plant viral vectors is also a practical approach for producing foreign proteins in plants. Adaptation of this technology is required to allow infection and propagation of engineered viruses in plant tissue cultures for transient protein expression in vitro.     

The cytotoxic effect of fumonisin B<Subscript>1</Subscript> and ochratoxin A on human and pig lymphocytes using the Methyl Thiazol Tetrazolium (MTT) assay

Lymphocytes cell obtained from healthy human donors and pigs were exposed to fumonisin B₁ (FB₁) and ochratoxin A (OTA), which have been found to be immunosuppressive, carcinogenic and mutagenic, to ascertain their single and combined cytotoxic effects with time and to assess the suitability of animal lymphocytes as test agents in comparison to human cells. The main objectives of this work were to assess the use of animal lymphocytes, particularly pig lymphocytes, for their use in the Methyl Thiazol Tetrazolium (MTT) cytotoxicity test, making them more accessible to animal research-based institutes in comparison to human lymphocytes previously used, and to study the cytotoxic and synergism or antagonistic effects of FB1 and OTA. The MTT assay, which measures cell viability and proliferation based on reduction of MTT to a blue dye, also used the addition of phytohaemagglutinin (PHA) to stimulate the blood cells. The results showed a progressive decrease in lymphocytes viability with time of exposure to the toxins. It was also noted that FB1, as compared to OTA, had a lower cytotoxicity on both human and pig lymphocytes cells. In addition, when the two mycotoxins were combined, a synergistic decrease of cell viability in both human and pig lymphocytes was observed, with pig lymphocytes showing a greater sensitivity. This study has shown that the MTT assay can be used for the determination of cytotoxicity of mycotoxins using animal, and in particular pig, lymphocytes, which eliminates the use of human donors and other cell cultures.     

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.     

Recent developments in the use of transgenic plants for the production of human therapeutics and biopharmaceuticals

In recent years there has been a dramatic increase in the application of plant biotechnology for the production of a variety of commercially valuable simple and complex biological molecules (biologics) for use in human and animal healthcare. Transgenic whole plants and plant cell culture systems have been developed that have the capacity to economically produce large-scale quantities of antibodies and antibody fragments, antigens and/or vaccine epitopes, metabolic enzymes, hormones, (neuro)peptides and a variety of biologically active complexes and secondary metabolites for direct use as therapeutic agents or diagnostic tools in the medical healthcare industry. As the products of genetically modified plants make their way from concept to commercialization the associated risks and acceptance by the public has been become a focal point. In this paper, we summarize the recent advances made in the use of transgenic plants and plant cell cultures as biological factories for the production of human therapeutics and biopharmaceuticals and discuss the long-term potential of `molecular farming' as a low-cost, efficient method for the production of biological materials with demonstrated utility to the pharmaceutical industry or medical community.     

Validation of the safety of MDCK cells as a substrate for the production of a cell-derived influenza vaccine

Cell culture-based production methods may assist in meeting increasing demand for seasonal influenza vaccines and developing production flexibility required for addressing influenza pandemics. MDCK-33016PF cells are used in propagation of a cell-based seasonal influenza vaccine (Optaflu^®); but, like most continuous cell lines, can grow in immunocompromised mice to produce tumors. It is, therefore, essential that no residual cells remain within the vaccine, that cell lysates or DNA are not oncogenic, and that the cell substrate does not contain oncogenic viruses or oncogenic DNA. Multiple, redundant processes ensure the safety of influenza vaccines produced in MDCK-33016PF cells. The probability of a residual cell being present in a dose of vaccine is approximately 1 in 10³⁴. Residual MDCK-DNA is ≤10 ng per dose and the ß-propiolactone used to inactivate influenza virus results in reduction of detectable DNA to less than 200 base pairs (bp). Degenerate PCR and specific PCR confirm exclusion of oncogenic viruses. The manufacturing process has been validated for its capacity to remove and inactivate viruses. We conclude that the theoretical risks arising from manufacturing seasonal influenza vaccine using MDCK-33016PF cells are reduced to levels that are effectively zero by the multiple, orthogonal processes used during production.     

Review: Production, characterization, and testing of banked mammalian cell substrates used to produce biological products

Summary A critical component in controlling the production of biological products derived from human and animal cell lines in the characterization and testing of banked cell substrates. The objective is to confirm the identity, purity, and suitability of these cells for manufacturing use. Quality concerns for biological products derived from cell lines originate from the presence of cellular and adventitious contaminants. Well-characterized cell banks not only permit a consistent source of production cells throughout the life of a product but also decrease the likelihood of contamination by other cell lines and adventitious agents. An important part of qualifying a cell line is choosing the appropriate testing for the presence of adventitious contaminants. The qualification of cell banks includes tests for cell identity and endogenous and adventitious microbial contaminants (bacteria, fungi, mycoplasmas, and viruses). For cells producing recombinant deoxyribonucleic acid-derived products, analysis of the expression construct at the nucleic acid level (genetic stability) is also a primary concern. The strategy for designing a safety-testing program for banked cells should be based on sound scientific principles and current regulatory guidance.     

Production of retroviral vectors for gene therapy with the human packaging cell line FLYRD18.

The development of gene therapy is hampered by the difficulty of producing large stocks of retroviral vectors at high titer. This study aimed to improve culture conditions and to intensify the production of retroviruses by FLYRD18, a packaging cell line derived from the HT1080 human fibrosarcoma line. Batch virus production proved to be feasible in unsupplemented basal medium and provided significantly higher titers and productivities than medium supplemented with 10% serum. For longer-term production, however, AIM-V complete serum-free medium and basal medium supplemented with 2% serum gave superior results. Serum supplementation should nevertheless be optimized to take into account the presence of inhibitors of viral production. In monolayer cultures with 0.2 mL/cm(2), the cell concentration was increased up to 2 x 10(6) cells/mL without loss of cell productivity. A semicontinuous production process, which enables the collection of larger amounts of viruses from the same culture, has also been successfully used. Suspension culture processes were prevented by the anchorage dependency of the FLYRD18 cell line. Microcarrier cultures were able to produce viruses but will require further investigation and optimization for their performance to become competitive with monolayer cultures. In the course of this study, more than a 10-fold increase of titer has been achieved.     

乳酸脱氢酶基因调控对HEK-293细胞代谢和腺病毒生产的影响

减少乳酸积累一直是哺乳动物细胞生物技术产业的一个目标。体外培养动物细胞时,乳酸积累主要是2种代谢途径作用的综合结果:一方面,葡萄糖在乳酸脱氢酶A(lactate dehydrogenase A,LDHA)的作用下生成乳酸;另一方面,乳酸可通过乳酸脱氢酶B(LDHB)或乳酸脱氢酶C(LDHC)氧化为丙酮酸重新进入三羧酸循环。本研究综合评估了乳酸代谢关键基因调控对人胚胎肾细胞(human embryonic kidney 293 cells,HEK-293)细胞生长、代谢和人腺病毒(human adenovirus,HAdV)生产的影响,有效提高了HEK-293细胞的HAdV生产能力,并为哺乳动物细胞的乳酸代谢工程调控提供了理论基础。通过改造乳酸代谢关键调控基因(敲除ldha基因以及过表达ldhb和ldhc基因),有效改善了HEK-293细胞的物质和能量代谢效率,显著提高了HAdV的生产。与对照细胞相比,3个基因改造均能促进细胞生长,降低乳酸和氨的积累,明显增强细胞的物质和能量代谢效率,显著提高了HEK-293细胞的HAdV生产能力。ldhc基因过表达对HEK-293细胞的生长、代谢和HAdV生产调控最显著,最大细胞密度提高了约38.7%,乳酸对葡萄糖得率和氨对谷氨酰胺得率分别下降了33.8%和63.3%,HAdV滴度提高了至少16倍。此外,相比于对照细胞株,改造细胞株的腺苷三磷酸(adenosine triphosphate,ATP)生成速率、ATP/O_(2)比率、ATP与腺苷二磷酸(adenosine diphosphate,ADP)的比值以及还原型辅酶Ⅰ(nicotinamide adenine dinucleotide,NADH)含量均有不同程度的提高,能量代谢效率明显改善。