ABSTRACT
A cultura de células, iniciada nos primórdios do século XIX, vem sendo cada vez mais adaptada à manutenção do metabolismo fundamental celular in vitro, permitindo o desenvolvimento da maioria das funções fisiológicas básicas inerentes a uma célula e ao conjunto delas (tecido). A permanência da informação genética trazida do organismo que lhe deu origem, faz com que as células em cultura correspondam a um modelo biológico ôvivoö para um número cada vez maior de testes laboratoriais aplicados aos campos da Medicina e da Biologia. O advento da Biologia Molecular e o esclarecimento do papel das células-tronco no desenvolvimento do organismo tornaram a cultura de células uma ferramenta fundamental na elucidação e solução de problemas, visando a melhoria da qualidade de vida dos indivíduos, como, também, o aprimoramento da manutenção de espécies animais e vegetais. Este trabalho tem o objetivo de mostrar a importância do uso das culturas de células, chamando a atenção para a biodiversidade intra e inter-específica, observada principalmente na região Amazônica, como também para a adaptabilidade das células in vitro.(AU)
Cell culture techniques, initiated in the early XIX century, evolved to increasingly being able to maintain the basic cell metabolism in vitro, allowing the development of most inherent basic cell and tissue physiological functions. The maintenance of the organism genetic information, makes cells culture to correspond to an ôaliveö biological model for a increasingly number of laboratorial tests, applied to the fields of Medicine and Biology. The advent of Molecular Biology and the clarification of the role of stem cells in the development of the organism, had promoted the use of cell culture as a basic tool for the elucidation solution of problems aiming at improving the quality of life of the individuals as well as, the maintenance of vegetables and animal species. The objective of this work is to show the importance of the use of cell cultures, calling attention to the biodiversity intra and inter-species observed in the Amazon region, as well as for the adaptability of the cells in vitro.(AU)
Subject(s)
Cell Culture Techniques , Tissue Culture TechniquesSubject(s)
Animals , Mice , Infant, Newborn , Alphavirus , Alphavirus , Primary Cell Culture , Cell Culture Techniques , Fluorescent Antibody Technique , Negative StainingABSTRACT
In skin lesions, the development of microbial infection affects the healing process, increasing morbidity and mortality rates in patients with severe burns, diabetic foot, and other types of skin injuries. Synoeca-MP is an antimicrobial peptide (AMP) that exhibits activity against several bacteria of clinical importance, but its cytotoxicity can represent a problem for its positioning as an effective antimicrobial compound. In contrast, the immunomodulatory peptide IDR-1018 presents low toxicity and a wide regenerative potential due to its ability to reduce apoptotic mRNA expression and promote skin cell proliferation. In the present study, we used human skin cells and a 3D skin equivalent models to analyze the potential of the IDR-1018 peptide to attenuate the cytotoxicity of synoeca-MP, as well as the influence of synoeca-MP/IDR-1018 combination on cell proliferation, regenerative processes, and wound repair. We found that the addition of IDR-1018 significantly improved the biological properties of synoeca-MP on skin cells without modifying its antibacterial activity against S. aureus. Likewise, in both melanocytes and keratinocytes, the treatment with synoeca-MP/IDR-1018 combination induces cell proliferation and migration, while in a 3D human skin equivalent model, it can accelerate wound reepithelization. Furthermore, treatment with this peptide combination generates an up-regulation in the expression of pro-regenerative genes in both monolayer cell cultures and in 3D skin equivalents. This data suggests that the synoeca-MP/IDR-1018 combination possesses a good profile of antimicrobial and pro-regenerative activity, opening the door to the development of new strategies for the treatment of skin lesions.
Subject(s)
Antimicrobial Peptides , Staphylococcus aureus , Humans , Cell Culture Techniques , Cell ProliferationABSTRACT
Gastric organoids are biological models constructed in vitro using stem cell culture and 3D cell culture techniques, which are the latest research hotspots. The proliferation of stem cells in vitro is the key to gastric organoid models, making the cell subsets within the models more similar to in vivo tissues. Meanwhile, the 3D culture technology also provides a more suitable microenvironment for the cells. Therefore, the gastric organoid models can largely restore the growth condition of cells in terms of morphology and function in vivo. As the most classic organoid models, patient-derived organoids use the patient's own tissues for in vitro culture. This kind of model is responsive to the 'disease information' of a specific patient and has great effect on evaluating the strategies of individualized treatment. Herein, we review the current literature on the establishment of organoid cultures, and also explore organoid translational applications.
Subject(s)
Stomach Neoplasms , Humans , Stem Cells , Cell Culture Techniques , Models, Biological , Organoids , Tumor MicroenvironmentABSTRACT
Cutaneous squamous cell carcinoma (cSCC) is the second-most common type of non-melanoma skin cancer and is linked to long-term exposure to ultraviolet (UV) radiation from the sun. Rocuronium bromide (RocBr) is an FDA-approved drug that targets p53-related protein kinase (PRPK) that inhibits the development of UV-induced cSCC. This study aimed to investigate the physicochemical properties and in vitro behavior of RocBr. Techniques such as thermal analysis, electron microscopy, spectroscopy and in vitro assays were used to characterize RocBr. A topical oil/water emulsion lotion formulation of RocBr was successfully developed and evaluated. The in vitro permeation behavior of RocBr from its lotion formulation was quantified with Strat-M® synthetic biomimetic membrane and EpiDerm™ 3D human skin tissue. Significant membrane retention of RocBr drug was evident and more retention was obtained with the lotion formulation compared with the solution. This is the first systematic and comprehensive study to report these findings.
Subject(s)
Carcinoma, Squamous Cell , Skin Neoplasms , Humans , Rocuronium/pharmacology , Carcinoma, Squamous Cell/pathology , Skin Neoplasms/pathology , Skin/metabolism , Pharmaceutical Preparations/metabolism , Cell Culture TechniquesABSTRACT
Mutations in the GBA1 gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), cause Gaucher disease (GD) and are the most common genetic risk factor for Parkinson's disease (PD). Pharmacological chaperones (PCs) are being developed as an alternative treatment approach for GD and PD. To date, NCGC00241607 (NCGC607) is one of the most promising PCs. Using molecular docking and molecular dynamics simulation we identified and characterized six allosteric binding sites on the GCase surface suitable for PCs. Two sites were energetically more preferable for NCGC607 and located nearby to the active site of the enzyme. We evaluated the effects of NCGC607 treatment on GCase activity and protein levels, glycolipids concentration in cultured macrophages from GD (n = 9) and GBA-PD (n = 5) patients as well as in induced human pluripotent stem cells (iPSC)-derived dopaminergic (DA) neurons from GBA-PD patient. The results showed that NCGC607 treatment increased GCase activity (by 1.3-fold) and protein levels (by 1.5-fold), decreased glycolipids concentration (by 4.0-fold) in cultured macrophages derived from GD patients and also enhanced GCase activity (by 1.5-fold) in cultured macrophages derived from GBA-PD patients with N370S mutation (p < 0.05). In iPSC-derived DA neurons from GBA-PD patients with N370S mutation NCGC607 treatment increased GCase activity and protein levels by 1.1-fold and 1.7-fold (p < 0.05). Thus, our results showed that NCGC607 could bind to allosteric sites on the GCase surface and confirmed its efficacy on cultured macrophages from GD and GBA-PD patients as well as on iPSC-derived DA neurons from GBA-PD patients.
Subject(s)
Gaucher Disease , Parkinson Disease , Humans , Parkinson Disease/drug therapy , Parkinson Disease/genetics , Glucosylceramidase/genetics , Glucosylceramidase/metabolism , Molecular Docking Simulation , Gaucher Disease/drug therapy , Gaucher Disease/genetics , Cell Culture Techniques , Binding Sites , Glycolipids , MutationABSTRACT
Since the sequence of hepatitis E virus (HEV) was determined from a patient with enterically transmitted non-A, non-B hepatitis in 1989, similar sequences have been isolated from many different animals, including pigs, wild boars, deer, rabbits, bats, rats, chicken, and trout. All of these sequences have the same genomic organization, which contains open reading frames (ORFs) 1, 2, and 3, although their genomic sequences are variable. Some have proposed that they be classified as new family, Hepeviridae, which would be further divided into different genera and species according to their sequence variability. The size of these virus particles generally ranged from 27 to 34 nm. However, HEV virions produced in cell culture differ in structure from the viruses found in feces. Those from cell culture have a lipid envelope and either lack or have a little ORF3, whereas the viruses isolated from feces lack a lipid envelope but have ORF3 on their surfaces. Surprisingly, most of the secreted ORF2 proteins from both these sources are not associated with HEV RNA.
Subject(s)
Deer , Hepatitis C , Hepatitis E virus , Animals , Rabbits , Rats , Swine , Hepatitis E virus/genetics , Cell Culture Techniques , Chickens , LipidsABSTRACT
Cell culture is an important research method in virology. Although many attempts have been conducted to culture HEV in cells, only a few cell culture systems were considered to be efficient enough for usage. Concentration of virus stocks, host cells, and medium components affects the culture efficiency and the genetic mutations during HEV passage were found to be associated with the increased virulence in cell culture. As an alternative method for traditional cell culture, the infectious cDNA clones were constructed. The viral thermal stability, factors that impact the host range, post-translation of viral proteins, and function of different viral proteins were studied using the infectious cDNA clones. HEV cell culture studies on progeny virus showed that the viruses secreted from host cells have an envelope and its formation was associated with pORF3. This result explained the phenomenon that virus could infect host cells in the presence of anti-HEV antibodies.
Subject(s)
Antibodies , Cell Culture Techniques , DNA, Complementary , Biological Transport , Clone CellsABSTRACT
Hepatitis E virus (HEV) infects over 20 million people worldwide per year, leading to 30,000-40,000 deaths. In most cases HEV infection in a self-limited, acute illness. However, chronic infections could occur in immunocompromised individuals. Due to scarcity of robust cell culture models in vitro and genetic tractable animal models in vivo, the details of HEV life cycle, as well as its interaction with host cells still remain elusive, which dampens antivirals discovery. In this chapter, we present an update in the HEV infectious cycle steps: entry, genome replication/subgenomic RNA transcription, assembly, and release. Moreover, we discussed the future prospective on HEV research and illustrates important questions urgently to be addressed.
Subject(s)
Hepatitis E virus , Animals , Hepatitis E virus/genetics , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Cell Culture Techniques , Immunocompromised Host , Life Cycle StagesABSTRACT
Sphingosine-1-phosphate (S1P) is a potent sphingolipid metabolite that regulates a wide range of biological functions such as cell proliferation, cell apoptosis and angiogenesis. Its cellular level is elevated in breast cancer, which, in turn, would promote cancer cell proliferation, survival, growth and metastasis. However, the cellular concentration of S1P is normally in the low nanomolar range, and our previous studies showed that S1P selectively induced apoptosis of breast cancer cells at high concentrations (high nanomolar to low micromolar). Thus, local administration of high-concentration S1P alone or in combination of chemotherapy agents could be used to treat breast cancer. The breast mainly consists of mammary gland and connective tissue stroma (adipose), which are dynamically interacting each other. Thus, in the current study, we evaluated how normal adipocyte-conditioned cell culture media (AD-CM) and cancer-associated adipocyte-conditioned cell culture media (CAA-CM) would affect high-concentration S1P treatment of triple-negative breast cancer (TNBC) cells. Both AD-CM and CAA-CM may suppress the anti-proliferative effect and reduce nuclear alteration/apoptosis caused by high-concentration S1P. This implicates that adipose tissue is likely to be detrimental to local high-concentration S1P treatment of TNBC. Because the interstitial concentration of S1P is about 10 times higher than its cellular level, we undertook a secretome analysis to understand how S1P would affect the secreted protein profile of differentiated SGBS adipocytes. At 100 nM S1P treatment, we identified 36 upregulated and 21 downregulated secretome genes. Most of these genes are involved in multiple biological processes. Further studies are warranted to identify the most important secretome targets of S1P in adipocytes and illustrate the mechanism on how these target proteins affect S1P treatment of TNBC.
Subject(s)
Triple Negative Breast Neoplasms , Humans , Adipocytes , Lysophospholipids , Cell Culture Techniques , Culture Media, ConditionedABSTRACT
Polycomb-group (PcG) of proteins are evolutionarily conserved transcription factors necessary for the regulation of gene expression during the development and the safeguard of cell identity in adulthood. In the nucleus, they form aggregates whose positioning and dimension are fundamental for their function. We present an algorithm, and its MATLAB implementation, based on mathematical methods to detect and analyze PcG proteins in fluorescence cell image z-stacks. Our algorithm provides a method to measure the number, the size, and the relative positioning of the PcG bodies in the nucleus for a better understanding of their spatial distribution, and thus of their role for a correct genome conformation and function.
Subject(s)
Cell Nucleus , Imaging, Three-Dimensional , Polycomb-Group Proteins , Cell Nucleus/metabolism , Cell Culture Techniques , Microscopy, Fluorescence/methodsABSTRACT
As many consider organ on a chip for better in vitro models, it is timely to extract quantitative data from the literature to compare responses of cells under flow in chips to corresponding static incubations. Of 2828 screened articles, 464 articles described flow for cell culture and 146 contained correct controls and quantified data. Analysis of 1718 ratios between biomarkers measured in cells under flow and static cultures showed that the in all cell types, many biomarkers were unregulated by flow and only some specific biomarkers responded strongly to flow. Biomarkers in cells from the blood vessels walls, the intestine, tumours, pancreatic island, and the liver reacted most strongly to flow. Only 26 biomarkers were analysed in at least two different articles for a given cell type. Of these, the CYP3A4 activity in CaCo2 cells and PXR mRNA levels in hepatocytes were induced more than two-fold by flow. Furthermore, the reproducibility between articles was low as 52 of 95 articles did not show the same response to flow for a given biomarker. Flow showed overall very little improvements in 2D cultures but a slight improvement in 3D cultures suggesting that high density cell culture may benefit from flow. In conclusion, the gains of perfusion are relatively modest, larger gains are linked to specific biomarkers in certain cell types.
Subject(s)
Cell Culture Techniques , Microphysiological Systems , Humans , Caco-2 Cells , Reproducibility of Results , BiomarkersABSTRACT
The creation of mutant mice has been invaluable for advancing biomedical science, but is too time- and resource-intensive for investigating the full range of mutations and polymorphisms. Cell culture models are therefore an invaluable complement to mouse models, especially for cell-autonomous pathways like the circadian clock. In this study, we quantitatively assessed the use of CRISPR to create cell models in mouse embryonic fibroblasts (MEFs) as compared to mouse models. We generated two point mutations in the clock genes Per1 and Per2 in mice and in MEFs using the same sgRNAs and repair templates for HDR and quantified the frequency of the mutations by digital PCR. The frequency was about an order of magnitude higher in mouse zygotes compared to that in MEFs. However, the mutation frequency in MEFs was still high enough for clonal isolation by simple screening of a few dozen individual cells. The Per mutant cells that we generated provide important new insights into the role of the PAS domain in regulating PER phosphorylation, a key aspect of the circadian clock mechanism. Quantification of the mutation frequency in bulk MEF populations provides a valuable basis for optimizing CRISPR protocols and time/resource planning for generating cell models for further studies.
Subject(s)
CRISPR-Cas Systems , Circadian Clocks , Animals , Mice , Fibroblasts/metabolism , Circadian Clocks/genetics , Cell Culture Techniques , Transcription Factors/metabolism , Disease Models, Animal , Circadian Rhythm/geneticsABSTRACT
Despite significant advances in investigative and therapeutic methodologies for cancer, 2D cell culture remains an essential and evolving competency in this fast-paced industry. From basic monolayer cultures and functional assays to more recent and ever-advancing cell-based cancer interventions, 2D cell culture plays a crucial role in cancer diagnosis, prognosis, and treatment. Research and development in this field call for a great deal of optimization, while the heterogenous nature of cancer itself demands personalized precision for its intervention. In this way, 2D cell culture is ideal, providing a highly adaptive and responsive platform, where skills can be honed and techniques modified. Furthermore, it is arguably the most efficient, economical, and sustainable methodology available to researchers and clinicians alike.In this chapter, we discuss the history of cell culture and the varying types of cell and cell lines used today, the techniques used to characterize and authenticate them, the applications of 2D cell culture in cancer diagnosis and prognosis, and more recent developments in the area of cell-based cancer interventions and vaccines.
Subject(s)
Cell Culture Techniques , Neoplasms , Humans , Cell Culture Techniques/methods , Cell Line , Neoplasms/diagnosisABSTRACT
Three-dimensional (3D) tumor spheroids and tumoroids are among the most exploited cell culture methods in the lung cancer field, finding applications in the investigation of tumor growth and proliferation, invasion, and drug screening. However, 3D tumor spheroids and tumoroids cannot fully mimic the architecture of the human lung adenocarcinoma tissue and, in particular, the direct contact of the lung adenocarcinoma cells with the air, as they lack polarity. Our method allows to overcome this limitation by enabling to grow tumoroids of lung adenocarcinoma cells and healthy lung fibroblasts at the Air-Liquid Interface (ALI). This ensures straightforward access to both the apical and basal surface of the cancer cell culture, with several advantages in drug screening applications.
Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Humans , Lung Neoplasms/metabolism , Cell Culture Techniques/methods , Cell Line, Tumor , Spheroids, Cellular/metabolismABSTRACT
Renewable and scalable human liver tissue platforms are a powerful tool to study organ physiology and model diseases, such as cancer. Stem cell-derived models provide an alternative to cell lines, which can display limited relevance to primary cells and tissue. Historically, two-dimensional (2D) cultures have been used to model liver biology as they are easy to scale and deploy. However, 2D liver models lack functional diversity and phenotypic stability in long-term culture. To address those issues, protocols for generating the three-dimensional (3D) tissue aggregates have been developed. Here, we describe a methodology to generate 3D liver spheres from pluripotent stem cells. Liver spheres are composed of three key liver cell types (hepatic progenitor cells, endothelial cells, and hepatic stellate cells) and have been used to study human cancer cell metastasis.
Subject(s)
Neoplasms , Pluripotent Stem Cells , Humans , Endothelial Cells , Cell Culture Techniques/methods , Liver , Hepatocytes/metabolism , Cell Differentiation , Neoplasms/metabolismABSTRACT
Magnetic hyperthermia is an innovative thermal therapy for the treatment of solid malignancies. This treatment approach utilizes magnetic nanoparticles that are stimulated by alternating magnetic fields to induce temperature elevations in tumor tissue, resulting in cell death. Magnetic hyperthermia is clinically approved for treating glioblastoma in Europe and is undergoing clinical evaluation for prostate cancer in the United States. Numerous studies have also demonstrated efficacy in other cancers, however, and its potential utility extends far beyond its current clinical indications. Despite this great promise, assessing the initial efficacy of magnetic hyperthermia in vitro is a complicated endeavor, with multiple hurdles worth considering, such as accurate thermal monitoring, accounting for nanoparticle interference, and a myriad of treatment controls that make robust experimental planning essential to evaluate treatment outcome. Presented here is an optimized magnetic hyperthermia treatment protocol to test the primary mechanism of cell death in vitro. This protocol can be applied to any cell line and ensures accurate temperature measurements, minimal nanoparticle interference, and controls for multiple factors that can influence experimental outcome.
Subject(s)
Glioblastoma , Hyperthermia, Induced , Magnetite Nanoparticles , Male , Humans , Hyperthermia, Induced/methods , Glioblastoma/therapy , Magnetic Fields , Cell Death , Cell Culture Techniques , Cell Line, Tumor , Magnetite Nanoparticles/therapeutic useABSTRACT
Air-liquid interface (ALI) cell cultures are considered a valid tool for the replacement of animals in biomedical research. By mimicking crucial features of the human in vivo epithelial barriers (e.g., lung, intestine, and skin), ALI cell cultures enable proper structural architectures and differentiated functions of normal and diseased tissue barriers. Thereby, ALI models realistically resemble tissue conditions and provide in vivo-like responses. Since their implementation, they are routinely used in several applications, from toxicity testing to cancer research, receiving an appreciable level of acceptance (in some cases a regulatory acceptance) as attractive testing alternatives to animals. In this chapter, an overview of the ALI cell cultures will be presented together with their application in cancer cell culture, highlighting the potential advantages and disadvantages of the model.
Subject(s)
Epithelial Cells , Neoplasms , Animals , Humans , Cell Culture Techniques , Lung , Cell Differentiation , SkinABSTRACT
In vitro cell culture is one of the most widely used tools used today for increasing our understanding of various things such as protein production, mechanisms of drug action, tissue engineering, and overall cellular biology. For the past decades, however, cancer researchers have relied heavily on conventional two-dimensional (2D) monolayer culture techniques to test a variety of aspects of cancer research ranging from the cytotoxic effects of antitumor drugs to the toxicity of diagnostic dyes and contact tracers. However, many promising cancer therapies have either weak or no efficacy in real-life conditions, therefore delaying or stopping altogether their translating to the clinic. This is, in part, due to the reductionist 2D cultures used to test these materials, which lack appropriate cell-cell contacts, have altered signaling, do not represent the natural tumor microenvironment, and have different drug responses, due to their reduced malignant phenotype when compared to real in vivo tumors. With the most recent advances, cancer research has moved into 3D biological investigation. Three-dimensional (3D) cultures of cancer cells not only recapitulate the in vivo environment better than their 2D counterparts, but they have, in recent years, emerged as a relatively low-cost and scientifically accurate methodology for studying cancer. In this chapter, we highlight the importance of 3D culture, specifically 3D spheroid culture, reviewing some key methodologies for forming 3D spheroids, discussing the experimental tools that can be used in conjunction with 3D spheroids and finally their applications in cancer research.
Subject(s)
Antineoplastic Agents , Neoplasms , Humans , Spheroids, Cellular/pathology , Cell Culture Techniques/methods , Neoplasms/pathology , Antineoplastic Agents/pharmacology , Tumor MicroenvironmentABSTRACT
Cancer cell spheroids have been shown to mimic in vivo tumour microenvironment and are therefore suitable for in vitro drug screening. Microfluidic technology can provide conveniences for spheroid assays such as high-throughput, simplifying manual operation and saving reagent. Here, we propose a concentration gradient generator based on microfluidic technology for cell spheroid culture and assay. The chip consists of upper microchannels and lower microwells. After partitioning HepG2 suspension into the microwells with concave and non-adhesive bottoms, spheroids can spontaneously form. By controlling the fluid replacement and flow in microchannels, the doxorubicin solution is diluted automatically into a series of concentration gradients, which spanning more than one order of magnitude. And then the effect of doxorubicin on spheroids is measured in situ by fluorescent staining. This chip provides a very promising approach to achieve the high-throughput and standardized anti-cancer drug screening in future.