Animal Cell Culture Research Articles

What the COVID-19 crisis is telling humanity

The planet is in a global health emergency exacting enormous medical and economic tolls. It is imperative for us as a society and species to focus and reflect deeply upon what this and other related human health crises are telling us about our role in these increasingly frequent events and about what we can do to prevent them in the future. Cause: It is human behavior that is responsible for the vast majority of zoonotic diseases that jump the species barrier from animals to humans: (1) hunting, capture, and sale of wild animals for human consumption, particularly in live-animal markets; (2) massive overcrowding of animals for human consumption in stressful and unhygienic industrial “factory farm” environments; (3) large-scale close confinement of animals for human consumption, a major direct cause of mounting antibiotic resistance; (4) vast numbers of wildlife species threatened with extinction from habitat incursion and destruction. Action: Intensive confinement of animals in factory farm operations should be discontinued worldwide for the sake of animals, humans, and the environment, and we should rapidly evolve to eating other forms of protein that are safer for humans, including plant-based meat alternatives and meat produced by culturing animal cells. Additional investment in plant-based agriculture to grow crops to feed humans rather than livestock for human consumption will feed more people while utilizing far less land and water, allowing for the preservation of vital ecosystems for innumerable species. Rather than simply attempting to react to crises like COVID-19 after death and destruction are already upon us, we need to address the fundamental underlying causes and act now to prevent the numerous disasters that are literally waiting to happen.

Isolation of primary mice splenocytes for in vitro research

Abstract Animal cell culture is of great importance in research. We isolated mouse splenocytes from Swiss albino mice by using a mechanical method and cultured them in medium with fetal bovine serum, 1% penicillin-streptomycin and 1% amphotericin as an antibiotic and an antifungal, but without mitogen. The cells were confluent after 72 h, and their viability, measured by trypan blue assay, was 95%. The splenocytes were passaged four times and no fungal or bacterial contamination was observed during carrying out the experiment.

A novel method based on nonparametric regression with a Gaussian kernel algorithm identifies the critical components in CHO media and feed optimization.

As the composition of animal cell culture medium becomes more complex, the identification of key variables is important for simplifying and guiding the subsequent medium optimization. However, the traditional experimental design methods are impractical and limited in their ability to explore such large feature spaces. Therefore, in this work, we developed a NRGK (nonparametric regression with Gaussian kernel) method, which aimed to identify the critical components that affect product titres during the development of cell culture media. With this nonparametric model, we successfully identified the important components that were neglected by the conventional PLS (partial least squares regression) method. The superiority of the NRGK method was further verified by ANOVA (analysis of variance). Additionally, it was proven that the selection accuracy was increased with the NRGK method because of its ability to model both the nonlinear and linear relationships between the medium components and titres. The application of this NRGK method provides new perspectives for the more precise identification of the critical components that further enable the optimization of media in a shorter timeframe.

Molecular genetic techniques and oligonucleotides for mycoplasma identification – a review

Control of distribution of mycoplasmal infections in cattle herds is essential in the majority of countries world-wide. Various PCR procedures are available to detect mycoplasmas in cell cultures and bovine mycoplasma in different types of samples. We reviewed some common PCR techniques and specific primers targeted to different bacterial genetic regions of mycoplasma. Several researchers used the same PCR approach and Mycoplasma spp. as a target but their results could not be compared because different primer pairs were used. These methods and primers were first developed to identify mycoplasma species that contaminate animal cell cultures, and then were used by other researchers to differentiate mycoplasmas as a cow infecting agent. Our analysis of the specificity of these primer pairs to nucleotide sequences of five Mycoplasma spp. showed that oligonucleotides have less specificity to them. Numerous commercially available PCR kits are applicable to find mycoplasma contamination in cell cultures and fewer of them can be used in veterinary diagnostics. Although serological and culture techniques are still used, it is necessary to develop a new multiplex PCR technique with a more specific primer set especially in agrarian countries.

A Review on Therapeutic Potentials of Crocetin-A Carotenoid Derived from Saffron

Natural dyes recognized as carotenoids have found application in health care system due to their wide biological activities, high safety margins and lower cost. Crocetin, a carotenoid constituent of saffron has shown wide range of pharmacological applications due to its significant antioxidant properties. It is used as antifungal, antimicrobial and antiallergic agent. Crocetin has shown significant potential as an anti- tumor agent in animal models and cell culture systems. Current review has shown crocetin’s multispectrum pharmacological benefits for the treatment of various chronic diseases such as cancer, diabetes, parkinson’s and alzheimer’s disease. This review focus on various studies done on crocetin explaining its broa d spectrum pharmacological activities. In addition, this review will also focus on the pharmacokinetic studies of crocetin on human and animals. Crocetin is a highly potent molecule because of its strong antioxidant properties. In the near future, increasing its bioavailability using novel drug delivery methods having minimum side effects will get this promising natural molecule to the forefront of therapy for the treatment of various chronic human diseases like cancer, diabetes, parkinson’s and alzheimer’s disease.

Silk Protein as a Fetal Bovine Serum Substitute for Animal Cell Culture

Fetal Bovine Serum (FBS) is an essential substance added to animal cell culture medium. However, its composition is unclear causing problems such as development of an immune response when cultured cells are transplanted into the human body. In this study, silk sericin, silk fibroin, and hemolymph obtained from silkworms were added to the cell culture medium in order to determine if it can replace FBS. After establishment of the cell culture, cell proliferation and expression levels of cell growth-related genes were compared with those of control cells (cells cultured in the medium with 10% FBS). Results showed that the test group treated with silk fibroin extracted from a Korean silkworm variety, Kumokjam could replace 10% FBS. In addition, expression levels of cell growth related genes such as Fibronectin and TGF-β1 increased significantly in cells cultured using silk fibroin, depending on the concentration used in cell adhesion and cell proliferation [24]. To date, no studies have been conducted to find a replacement for FBS. Thus, this study was carried out to develop a substitute for FBS by using silkworm-derived alternatives such as silkworm hemolymph, silk sericin, and silk fibroin, which are cheap and have various physiological effects, cell promoting effects, and can be mass produced.

An Optical Biosensor for Continuous Glucose Monitoring in Animal Cell Cultures.

Biosensors for continuous glucose monitoring in bioreactors could provide a valuable tool for optimizing culture conditions in biotechnological applications. We have developed an optical biosensor for long-term continuous glucose monitoring and demonstrated a tight glucose level control during cell culture in disposable bioreactors. The in-line sensor is based on a commercially available oxygen sensor that is coated with cross-linked glucose oxidase (GOD). The dynamic range of the sensor was tuned by a hydrophilic perforated diffusion membrane with an optimized permeability for glucose and oxygen. The biosensor was thoroughly characterized by experimental data and numerical simulations, which enabled insights into the internal concentration profile of the deactivating by-product hydrogen peroxide. The simulations were carried out with a one-dimensional biosensor model and revealed that, in addition to the internal hydrogen peroxide concentration, the turnover rate of the enzyme GOD plays a crucial role for culture monitoring is an integral part of animal cell cultivation. For several culture parameters, in situ sensors exist; others are predominantly monitored off-line. One important cell culture parameter is glucose concentration. Despite many efforts, there is still a lack of in situ sensors for continuous glucose monitoring. Such biosensors could provide a valuable tool for optimizing culture conditions in biotechnological applications. In this contribution, the manufacture of a long-term stable optical glucose sensor is described which is used to demonstrate glucose level monitoring during cell culture in disposable bioreactors. The in situ sensor is based on a commercially available oxygen sensor that is coated with cross-linked glucose oxidase and a hydrophilic perforated diffusion membrane. Glucose was measured in shake flasks and wave bags with only minor drifts of the sensor sensitivity during batch and fed-batch fermentations.

The neurotoxicity of Nε-(carboxymethyl)lysine in food processing by a study based on animal and organotypic cell culture.

Nε-(carboxymethyl)lysine (CML) is a potentially noxious compound that is causing widespread concern due to its use in various food products. In this study, we investigated CML neurotoxicity via an in vivo experiment with mice, and an in vitro experiment using a 3D microvascular network model (with human brain vascular endothelial cell and human astrocyte) that simulated the blood-brain barrier. We found that CML could induce cell survival status variations, and histopathological changes to the brain. In addition, CML increased levels of oxidative stress, prompted the protein expression of the receptor for advanced glycation end-products (RAGE). CML up-regulated both the gene expression of RAGE, the activating protein-1 (AP-1), the inflammatory cytokines Interleukin-6 (IL-6), vascular cell adhesion molecule1 (VCAM-1), monocyte chemotactic protein1 (MCP-1). We, therefore, postulated that CML has the potential to deleteriously affect the nervous system through oxidative stress and that activation of the p38 MAPK-AP-1 signaling pathway might be implicated in this pathological process.

Progerin-Induced Transcriptional Changes in Huntington's Disease Human Pluripotent Stem Cell-Derived Neurons.

Huntington's disease (HD) is a neurodegenerative late-onset genetic disorder caused by CAG expansions in the coding region of the Huntingtin (HTT) gene, resulting in a poly-glutamine (polyQ) expanded HTT protein. Considerable efforts have been devoted for studying HD and other polyQ diseases using animal models and cell culture systems, but no treatment currently exists. Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer an elegant solution for modeling human diseases. However, as embryonic or rejuvenated cells, respectively, these pluripotent stem cells (PSCs) do not recapitulate the late-onset feature of the disease. Here, we applied a robust and rapid differentiation protocol to derive electrophysiologically active striatal GABAergic neurons from human wild-type (WT) and HD ESCs and iPSCs. RNA-seq analyses revealed that HD and WT PSC-derived neurons are highly similar in their gene expression patterns. Interestingly, ectopic expression of Progerin in both WT and HD neurons exacerbated the otherwise non-significant changes in gene expression between these cells, revealing IGF1 and genes involved in neurogenesis and nervous system development as consistently altered in the HD cells. This work provides a useful tool for modeling HD in human PSCs and reveals potential molecular targets altered in HD neurons.

Исследование цитотоксичности ультрамалых фосфониевых золотых наночастиц с использованием культур растительных и животных клеток

Биосовместимость ультрамалых коллоидных золотых наночастиц представляется важной проблемой, что обусловлено расширяющимся с каждым годом спектром биомедицинских и технических приложений данных наноматериалов. Согласно большинству исследований данный тип наночастиц является токсичным для живых организмов. Однако не вполне ясно, какой именно компонент коллоидной системы проявляет токсичность: связана ли она с собственно частицами или с дисперсионной средой. Также не ясен механизм токсического действия. Решается задача, связанная с идентификацией источника токсичности препарата ультрамалых фосфониевых золотых наночастиц, полученных методом Даффа, с использованием клеточных тест-систем: культур микроводоросли Dunaliella salina и культур животных клеток линий HeLa и Vero. Трижды отмытые от среды наночастицы не обладали токсичностью для животных клеток и были слаботоксичны для D. Salina. Обнаружено, что токсичность препарата ультрамалых фосфониевых золотых наночастиц обусловлена токсичностью дисперсионной среды; можно предположить, что основным источником токсичности дисперсионной среды являются комплексно-ионные формы золота.

Machine learning to predict developmental neurotoxicity with high-throughput data from 2D bio-engineered tissues.

There is a growing need for fast and accurate methods for testing developmental neurotoxicity across several chemical exposure sources. Current approaches, such as in vivo animal studies, and assays of animal and human primary cell cultures, suffer from challenges related to time, cost, and applicability to human physiology. Prior work has demonstrated success employing machine learning to predict developmental neurotoxicity using gene expression data collected from human 3D tissue models exposed to various compounds. The 3D model is biologically similar to developing neural structures, but its complexity necessitates extensive expertise and effort to employ. By instead focusing solely on constructing an assay of developmental neurotoxicity, we propose that a simpler 2D tissue model may prove sufficient. We thus compare the accuracy of predictive models trained on data from a 2D tissue model with those trained on data from a 3D tissue model, and find the 2D model to be substantially more accurate. Furthermore, we find the 2D model to be more robust under stringent gene set selection, whereas the 3D model suffers substantial accuracy degradation. While both approaches have advantages and disadvantages, we propose that our described 2D approach could be a valuable tool for decision makers when prioritizing neurotoxicity screening.

In situ cell differentiation monitoring of Catharanthus roseus suspension culture processes by NIR spectroscopy.

Plant suspension culture is attracting interest as a promising platform to produce biological medicines due to the absence of virus, prions or DNA related to mammals during the production process. However, the heterogenic plant cell proliferation nature is particularly challenging for establishing industrial processes based on innovative approaches currently used, particularly in the animal cell culture industry. In this context, while Process Analytical Technology (PAT) tools have been used to monitor classical parameters such as biomass dry weight, its use in cells heterogeneity has received limited attention. Therefore, the feasibility of in situ monitoring of cell differentiation in plant cell suspensions employing NIR spectroscopy and chemometrics was investigated. Off-line measurements of cell heterogeneity in term of cell differentiation and in-line NIR spectra captured in 3 L bioreactor cultures were employed to generate calibration models. Then models were tested to estimate the population distribution of parenchyma, collenchyma and sclerenchyma cells during Catharanthus roseus suspension cultures. Results have proven in situ NIR spectroscopy as a capable PAT tool to monitor differentiated cells accurately and in real-time. These results are the starting point to follow-up PAT systems so that plant cell culture heterogeneity may be better understood and controlled in biopharmaceutical plant cell cultures.

Towards in situ continuous feeding via controlled release of complete nutrients for fed-batch culture of animal cells

Small-scale culture of animal cells in suspension is of importance for many applications. At a small-scale, fed-batch is achieved either by manual bolus feeding or the use of liquid handling robots. In this study, we report an alternate application of a hydrogel for in situ continuous delivery of a nutrient feed comprising 18 amino acids, vitamins, antioxidants, and trace elements. We show that amino acid release is sustained for at least seven days. Importantly, release rates of individual amino acids can be independently modulated by changing their loading. We demonstrate the application of this hydrogel for complete in situ feeding of nutrients to a suspension adapted CHO cell line expressing IgG leading to 2.7-fold and 4-fold improvement in integral viable cell density (IVCD) and volumetric productivity respectively. This is similar to improvements obtained by bolus liquid feeding. Further, supplying glucose from the same hydrogel to eliminate manual feeding led to a 1.8-fold increase in IVCD accompanied by a 3-fold increase in volumetric productivity as compared to batch culture. In summary, this study provides a proof-of-concept that hydrogels can enable completely closed in situ feeding for mammalian cell culture requiring no external intervention. Such continuous in situ delivery can potentially enable closed culture systems maintaining nutrients at low levels mimicking physiological concentrations.

Protein trap: a new Swiss army knife for geneticists?

The protein trap is a powerful tool for genetic and biochemical studies of gene function in the animal kingdom. Although the original protein trap was developed for flies, it can be easily adapted to other multicellular organisms, both known models and ones with an unsequenced genome. The protein trap has been successfully applied to the fruit fly, crustaceans Parhyale hawaiensis, zebrafish, andinsect and animal cell cultures. This approach is based on the integration into genes of an artificial exon that carries DNA encoding a fluorescent marker, standardized immunoepitopes, an integrase docking site, and splice acceptor and donor sites. The protein trap for cell cultures additionally contains an antibiotic resistance gene, which facilitates the selection of trapped clones. Resulting chimeric tagged mRNAs can be interfered by dsRNA against GFP (iGFPi-in vivo GFP interference), or the chimeric proteins can be efficiently knocked down by deGradFP technology. Both RNA and protein knockdowns produce a strong loss of function phenotype in tagged cells. The fluorescent and protein affinity tags can be used for tagged protein localisation within the cell and for identifying their binding partners in their native complexes. Insertion into protein trap integrase docking sites allows the replacement of trap contents by any new constructs, including other markers, cell toxins, stop-codons, and binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS, that reliably reflect endogenous gene expression. A distinctive feature of the protein trap approach is that all manipulations with a gene or its product occur only in the endogenous locus, which cannot be achieved by any other method.

Extracellular Vesicles-Connecting Kingdoms.

It is known that extracellular vesicles (EVs) are shed from cells of almost every type of cell or organism, showing their ubiquity in all empires of life. EVs are defined as naturally released particles from cells, delimited by a lipid bilayer, and cannot replicate. These nano- to micrometer scaled spheres shuttle a set of bioactive molecules. EVs are of great interest as vehicles for drug targeting and in fundamental biological research, but in vitro culture of animal cells usually achieves only small yields. The exploration of other biological kingdoms promises comprehensive knowledge on EVs broadening the opportunities for basic understanding and therapeutic use. Thus, plants might be sustainable biofactories producing nontoxic and highly specific nanovectors, whereas bacterial and fungal EVs are promising vaccines for the prevention of infectious diseases. Importantly, EVs from different eukaryotic and prokaryotic kingdoms are involved in many processes including host-pathogen interactions, spreading of resistances, and plant diseases. More extensive knowledge of inter-species and interkingdom regulation could provide advantages for preventing and treating pests and pathogens. In this review, we present a comprehensive overview of EVs derived from eukaryota and prokaryota and we discuss how better understanding of their intercommunication role provides opportunities for both fundamental and applied biology.

Novel scale-up strategy based on three-dimensional shear space for animal cell culture

Animal cell cultivations are widely used for producing antibodies, vaccines, and recombinant protein drugs. However, the extreme sensitivity of animal cells to hydrodynamic stress often hinders its scale-up in large-scale stirred tank bioreactors. This study introduced a new scale-up strategy based on three-dimensional (3D) shear space for large-scale animal cell culture. First, the shear environments of bioreactors ranging from lab-scale (7.5 and 42 L) to industrial-scale (30, 90, 350, and 1000 L) were quantitatively analyzed through computational fluid dynamics (CFD) methods and successfully validated by particle image velocimetry (PIV) experiments. Moreover, the quantitative relationships between shear parameters (including shear rates in the impeller and tank zone, overall average shear rate, and maximum shear rate) and impeller tip velocity were established. In addition, a correlation analysis between shear-related parameters and viable cell densities in Spodoptera frugiperda Sf9 cultivations indicated that shear rates in the impeller and tank zone, and the overall average shear rate were the three key shear parameters required for scale-up. Further, an optimized 3D operation space for shear rate was established according to the three key shear parameters obtained under preferable operation conditions in lab-scale bioreactors. Based on the results, agitation rates in large-scale bioreactors were determined using the proposed correlation. Ultimately, we achieved successful scale-up of Spodoptera frugiperda Sf9 in industrial bioreactors with volumes up to 1000 L using this strategy. Thus, this study introduces a highly efficient and economical scale-up strategy for shear-sensitive cells.

The Use of Human Induced Pluripotent Stem Cells for Testing Neuroprotective Activity of Pharmacological Compounds.

Development of therapeutic preparations involves several steps, starting with the synthesis of chemical compounds and testing them in different models for selecting the most effective and safest ones to clinical trials and introduction into medical practice. Cultured animal cells (both primary and transformed) are commonly used as models for compound screening. However, cell models display a number of disadvantages, including insufficient standardization (primary cells) and disruption of cell genotypes (transformed cells). Generation of human induced pluripotent stem cells (IPSCs) offers new possibilities for the development of high-throughput test systems for screening potential therapeutic preparations with different activity spectra. Due to the capacity to differentiate into all cell types of an adult organism, IPSCs are a unique model that allows examining the activity and potential toxicity of tested compounds during the entire differentiation process in vitro. In this work, we demonstrated the efficiency of IPSCs and their neuronal derivatives for selecting substances with the neuroprotective activity using two classes of compounds - melanocortin family peptides and endocannabinoids. None of the tested compounds displayed cyto- or embryotoxicity. Both melanocortin peptides and endocannabinoids exerted neuroprotective effect in the neuronal precursors and IPSC-derived neurons subjected to hydrogen peroxide. The endocannabinoid N-docosahexaenoyl dopamine exhibited the highest neuroprotective effect (~70%) in the differentiated cultures enriched with dopaminergic neurons; the effect of melanocortin Semax was ~40%. The possibility of using other IPSC derivatives for selecting compounds with the neuroprotective activity is discussed.

Muscle tissue engineering in fibrous gelatin: implications for meat analogs

Bioprocessing applications that derive meat products from animal cell cultures require food-safe culture substrates that support volumetric expansion and maturation of adherent muscle cells. Here we demonstrate scalable production of microfibrous gelatin that supports cultured adherent muscle cells derived from cow and rabbit. As gelatin is a natural component of meat, resulting from collagen denaturation during processing and cooking, our extruded gelatin microfibers recapitulated structural and biochemical features of natural muscle tissues. Using immersion rotary jet spinning, a dry-jet wet-spinning process, we produced gelatin fibers at high rates (~ 100 g/h, dry weight) and, depending on process conditions, we tuned fiber diameters between ~ 1.3 ± 0.1 μm (mean ± SEM) and 8.7 ± 1.4 μm (mean ± SEM), which are comparable to natural collagen fibers. To inhibit fiber degradation during cell culture, we crosslinked them either chemically or by co-spinning gelatin with a microbial crosslinking enzyme. To produce meat analogs, we cultured bovine aortic smooth muscle cells and rabbit skeletal muscle myoblasts in gelatin fiber scaffolds, then used immunohistochemical staining to verify that both cell types attached to gelatin fibers and proliferated in scaffold volumes. Short-length gelatin fibers promoted cell aggregation, whereas long fibers promoted aligned muscle tissue formation. Histology, scanning electron microscopy, and mechanical testing demonstrated that cultured muscle lacked the mature contractile architecture observed in natural muscle but recapitulated some of the structural and mechanical features measured in meat products.

A conceptual air-lift reactor design for large scale animal cell cultivation in the context of in vitro meat production

Cell-based meat, or cultured meat is at the early commercialization stage, but thorough considerations of the scaling-up challenges are scarce and the physiological data of animal muscle cells required by reliable process design are severely lacking. To make cultured meat an economically viable source of animal proteins, we propose a rational approach towards the design of a large scale air-lift reactor for cultured meat manufacturing. Computational fluid dynamics (CFD) modeling is used as a primary tool to provide an a priori estimation of the mass transfer and mixing performance of the reactor and the effects of reactor internals are investigated. The mass transfer coefficient of 36 1/h, energy dissipation rate of 46 W/m3 and a mixing time of 103 s are all within acceptable ranges typical to animal cells. It is estimated that a single reactor of 300 m3 can supply the meat demand of a population of 75,000.

Interest of locally weighted regression to overcome nonlinear effects during in situ NIR monitoring of CHO cell culture parameters and antibody glycosylation.

Animal cell culture processes have become the standard platform to produce therapeutic proteins such as recombinant monoclonal antibodies (mAb). Since the mAb quality could be subject to significant changes depending on manufacturing process conditions, real time monitoring and control systems are required to ensure mAb specifications mainly glycosylation and patient safety. Up to now, real time monitoring glycosylation of proteins has received scarce attention. In this article, the use of near infrared (NIR) to monitor mAb glycosylation has been reported for the first time. Whereas monitoring models are mainly constructed using linear partial least squares regressions (PLSR), evidences presented in this study indicate nonlinearity relationship between in situ captured spectra and compound concentrations, compromising the PLSR performances. A novel and simple approach was proposed to fit nonlinearity using the locally weighted regression (LWR). The LWR models were found to be more appropriate for handling information contained in spectra so that real time monitoring of cultures were accurately performed. Moreover, for the first time, the LWR calibration models allowed mAb glycosylation to be monitored, in a real time manner, by using in situ NIR spectroscopy. These results represent a further step toward developing active-control feedback of animal cell processes, particularly for ensuring properties of biologics.