Biocompatibility of cornea implants from polymeric materials and bio-artificial cornea in a model of human keratocytes cell culture

2018 ◽  
pp. 129-135
Author(s):  
Д.С. Островский ◽  
С.А. Борзенок ◽  
И.Н. Сабурина ◽  
Б.Э. Малюгин ◽  
И.А. Мушкова ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Growth ◽  
Comparison Group ◽  
Cultured Cells ◽  
Polymeric Materials ◽  
Biologically Active ◽  
Immunocytochemical Staining ◽  
Biological Compatibility ◽  
Study Results

Предложена модель in vitro изучения биологической совместимости и токсичности полимерных материалов на культурах клеток стромы роговицы человека - кератоцитах (КЦ). Цель исследования - изучение возможности использования выделенных культур кератоцитов донора-трупа человека для оценки биосовместимости полимерных материалов. Методика. Из роговицы трупного донорского глаза получали первичную культуру КЦ и пересевали до 4-го пассажа. Фенотип КЦ подтверждали методом иммуноцитохимического окрашивания с выявлением основных клеточных маркеров. КЦ культивировали в присутствии образцов полимерных материалов - четырех модификаций бисфенол-А-глицедилметакрилата (бис-ГМА) по 24 образца каждого (4 экспериментальные группы). В качестве группы сравнения 1 использовали образцы из полиметилметакрилата (ПММА) идентичной геометрии (24 образца), группа сравнения 2 - КЦ, культивированные по стандартной методике без полимерных образцов (24 образца). КЦ в каждой группе распределяли по 24 лункам культурального планшета, культивировали на протяжении 6 сут., ежедневно КЦ извлекали из четырех лунок в каждой группе и подсчитывали. Анализируя динамику клеточного роста и качественное состояние образцов полимеров, делали вывод о типе биологической совместимости исследуемых материалов. Результаты. Все кривые клеточного роста имели восходящую S-образную форму, количество клеток статистически значимо увеличивалось со 2-х по 4-е сут. и замедлялось к 6-м сут. Среди исследованных материалов бис-ГМА № 3 проявил наименьшую способность обеспечивать адгезию культивируемых клеток, бис-ГМА № 1 и № 2 - наибольшую. Результаты статистически значимы. Заключение. Проведенные исследования показали высокую информативность использования предложенной методики для определения биологической совместимости искусственных материалов. Исходя из полученных результатов, все исследуемые материалы были отнесены к числу биологически активных. Образцы исследуемых материалов в клеточной культуре статистически значимо влияют на уровень клеточной адгезии и степень пролиферации. An in vitro model was proposed for studying biocompatibility and toxicity of polymeric materials in cultures of human corneal stromal cells, keratocytes (KCs). The aim of the present research was to study a possibility of using cultures of isolated human KCs to assess biocompatibility of polymeric materials. Materials and methods. The primary KC culture was obtained from donor’s eye cornea and cultured to the 4th passage. The KC phenotype was confirmed by immunocytochemical staining, and the major cell markers were identified. KCs were cultured in the presence of four modifications of bisphenol-A-glycidyl methacrylate (bis-HMA) polymeric materials (24 replicate samples for each modification). Polymethylmethacrylate (PMMA) samples of identical geometry were used in the first comparison group (24 samples). In the second comparison group, KCs were cultured according to a standard procedure without polymer samples (24 wells). In each group, KCs were distributed to 24 wells of the culture plate and cultured for 6 days; cells were counted daily. Based on the dynamics of cell growth and qualitative condition of polymer samples, we made a conclusion about the type of biological compatibility of the materials under study. Results. All cell growth curves had an upward S shape; the number of cells increased statistically significantly from day 2 to day 4 (p <0.05) and slowed by day 6 (p>0.05). Among the studied materials, bis-HMA #3 showed the weakest ability (p <0.05) and bis-HMA #1 and #2 - the greatest ability (p<0.05) to provide adhesion of cultured cells. Conclusion. The study showed a high informative value of the proposed method for determining biological compatibility of artificial materials. All studied materials were classified as biologically active. Samples of the studied materials statistically significantly affected cell adhesion and proliferation in the cell culture.

Developments in three-dimensional cell culture technology aimed at improving the accuracy of in vitro analyses

2010 ◽  
Vol 38 (4) ◽  
pp. 1072-1075 ◽  
Author(s):  
Daniel J. Maltman ◽  
Stefan A. Przyborski
Keyword(s):  
Cell Culture ◽  
Cell Growth ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Proliferation And Differentiation ◽  
In Vitro Systems

Drug discovery programmes require accurate in vitro systems for drug screening and testing. Traditional cell culture makes use of 2D (two-dimensional) surfaces for ex vivo cell growth. In such environments, cells are forced to adopt unnatural characteristics, including aberrant flattened morphologies. Therefore there is a strong demand for new cell culture platforms which allow cells to grow and respond to their environment in a more realistic manner. The development of 3D (three-dimensional) alternative substrates for in vitro cell growth has received much attention, and it is widely acknowledged that 3D cell growth is likely to more accurately reflect the in vivo tissue environments from which cultured cells are derived. 3D cell growth techniques promise numerous advantages over 2D culture, including enhanced proliferation and differentiation of stem cells. The present review focuses on the development of scaffold technologies for 3D cell culture.

Use of Reduced Temperature Cell Pausing to Enhance Flexibility of Cell-Based Assays

2009 ◽  
Vol 14 (6) ◽  
pp. 716-722 ◽  
Author(s):  
Helen Wise ◽  
Paul William Abel ◽  
Darren Cawkill
Keyword(s):  
Cell Culture ◽  
Cell Growth ◽  
Protein Function ◽  
Normal Cell ◽  
Simple Technique ◽  
Cultured Cells ◽  
Experimental Process ◽  
G Protein Coupled ◽  
Reduced Temperature

Construction and supply of cell-based reagents for in vitro plate-based screens are often highlighted as a bottleneck within drug discovery. Recent years have seen the successful application of both cryopreservation and automation to increase the capacity and flexibility of cell provision. However, routine cell culture remains a fixed experimental process that requires cells to be prepared and used at specific times. We have investigated the potential of reduced temperature incubation to be used as a simple methodology for stopping and starting cell growth and introduce further flexibility into cell provision. Our results show that incubation of CHOK1, HEK293, and 1321N1 cells at 23 °C arrested growth while maintaining cell viability. Recovery of these paused cells at 37 °C resulted in resumption of normal cell growth and target protein function. Experiments demonstrated that paused cells, expressing either a recombinant G-protein-coupled receptor or an ion channel, performed comparably with the equivalent continuously cultured cells in a 384-well cell-based assay. This simple technique offers the potential to introduce flexibility into cell culture experiments and processes that were previously considered to be fixed. ( Journal of Biomolecular Screening 2009:716-722)

IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S27-S40 ◽  
Author(s):  
T. Kobayashi ◽  
T. Kigawa ◽  
M. Mizuno ◽  
T. Watanabe
Keyword(s):  
Cell Culture ◽  
Organ Culture ◽  
Cultured Cells ◽  
Cell Sheet ◽  
Short Term ◽  
Hypothalamic Extract ◽  
Numerous Cell ◽  
Single Cell Culture ◽  
Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

Change in mutation frequency at a TP53 hotspot during culture of ENU-mutagenised human lymphoblastoid cells

Mutagenesis ◽  
2019 ◽  
Author(s):  
Masahiko Watanabe ◽  
Masae Toudou ◽  
Taeko Uchida ◽  
Misato Yoshikawa ◽  
Hiroaki Aso ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Growth ◽  
Mutation Frequency ◽  
Cultured Cells ◽  
Tp53 Gene ◽  
Tumour Suppressor Genes ◽  
Restriction Sites ◽  
Mutation Spectra ◽  
Risk Of Cancer ◽  
Assay Conditions

Abstract Mutations in oncogenes or tumour suppressor genes cause increases in cell growth capacity. In some cases, fully malignant cancer cells develop after additional mutations occur in initially mutated cells. In such instances, the risk of cancer would increase in response to growth of these initially mutated cells. To ascertain whether such a situation might occur in cultured cells, three independent cultures of human lymphoblastoid GM00130 cells were treated with N-ethyl-N-nitrosourea to induce mutations, and the cells were maintained for 12 weeks. Mutant frequencies and spectra of the cells at the MspI and HaeIII restriction sites located at codons 247–250 of the TP53 gene were examined. Mutant frequencies at both sites in the gene exhibited a declining trend during cell culture and reached background levels after 12 weeks; this was also supported by mutation spectra findings. These results indicate that the mutations detected under our assay conditions are disadvantageous to cell growth.

A New In Vitro Model for Predicting the Toxicity of Cosmetic Products

1992 ◽  
Vol 20 (1) ◽  
pp. 138-143
Author(s):  
Maria Carrara ◽  
Lorenzo Cima ◽  
Roberto Cerini ◽  
Maurizio Dalle Carbonare
Keyword(s):  
Cell Culture ◽  
Cultured Cells ◽  
Neutral Red ◽  
Total Protein Content ◽  
Cosmetic Products ◽  
Cell Culture Insert ◽  
A Cell ◽  
Vitro Model ◽  
Cosmetic Emulsions

A method has been developed whereby cosmetic products which are not soluble in water or in alcohol can be brought into contact with cell cultures by being placed in a cell culture insert, which is then placed in the cell culture well. Preliminary experiments were carried out with L929 cells, and cytotoxicity was evaluated by measuring neutral red uptake and the total protein content of treated cultured cells. Encouraging results were obtained in comparisons of three cosmetic emulsions and of one emulsion containing a range of concentrations of two preservatives, Kathon CG and Bronopol.

scholarly journals Mechanical Strain-Enabled Reconstitution of Dynamic Environment in Organ-on-a-Chip Platforms: A Review

Micromachines ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 765
Author(s):  
Qianbin Zhao ◽  
Tim Cole ◽  
Yuxin Zhang ◽  
Shi-Yang Tang
Keyword(s):  
Cell Culture ◽  
Shear Stress ◽  
Cultured Cells ◽  
Mechanical Strain ◽  
3D Cell Culture ◽  
Small Scale ◽  
Mechanical Stimuli ◽  
Human Organ ◽  
Organ On A Chip

Organ-on-a-chip (OOC) uses the microfluidic 3D cell culture principle to reproduce organ- or tissue-level functionality at a small scale instead of replicating the entire human organ. This provides an alternative to animal models for drug development and environmental toxicology screening. In addition to the biomimetic 3D microarchitecture and cell–cell interactions, it has been demonstrated that mechanical stimuli such as shear stress and mechanical strain significantly influence cell behavior and their response to pharmaceuticals. Microfluidics is capable of precisely manipulating the fluid of a microenvironment within a 3D cell culture platform. As a result, many OOC prototypes leverage microfluidic technology to reproduce the mechanically dynamic microenvironment on-chip and achieve enhanced in vitro functional organ models. Unlike shear stress that can be readily generated and precisely controlled using commercial pumping systems, dynamic systems for generating proper levels of mechanical strains are more complicated, and often require miniaturization and specialized designs. As such, this review proposes to summarize innovative microfluidic OOC platforms utilizing mechanical actuators that induce deflection of cultured cells/tissues for replicating the dynamic microenvironment of human organs.

scholarly journals Oxygen and Glucose Levels in Cell Culture Media Determine Resveratrol’s Effects on Growth, Hydrogen Peroxide Production, and Mitochondrial Dynamics

Antioxidants ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 157 ◽  
Author(s):  
Joao Fonseca ◽  
Fereshteh Moradi ◽  
Andrew Valente ◽  
Jeffrey Stuart
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Culture ◽  
Cell Growth ◽  
Cultured Cells ◽  
Culture Media ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Network ◽  
Hydrogen Peroxide Production ◽  
Network Characteristics ◽  
Glucose Levels

Resveratrol is a plant-derived polyphenol that has been widely studied for its putative health promoting effects. Many of those studies have been conducted in cell culture, in supra-physiological levels of oxygen and glucose. Resveratrol interacts with reactive oxygen species (ROS) as an antioxidant or pro-oxidant. Resveratrol affects the expression and activities of ROS-producing enzymes and organelles. It is therefore important to consider how cell culture conditions might determine the effects of resveratrol on cultured cells. We determined the effects of resveratrol on cell growth, hydrogen peroxide production, and mitochondrial network characteristics in C2C12 mouse myoblasts and PC3 human prostate cancer cells under conditions of physiological (5%) and supra-physiological (18%) oxygen, and normo- (5 mM) and hyper-glycemia (25 mM). Interestingly, most effects of resveratrol on the parameters measured here were dependent upon prevailing oxygen and glucose levels during the experiment. Many of the effects of resveratrol on cell growth, hydrogen peroxide production, and mitochondrial network characteristics that were seen in 25 mM glucose and/or 18% oxygen were absent under the physiologically relevant conditions of 5 mM glucose with 5% oxygen. These findings emphasize the importance of using physiologically meaningful starting conditions for cell-culture experiments with resveratrol and indeed any manipulation affecting ROS metabolism and mitochondria.

scholarly journals Alkaloids Production and Cell Growth of Cinchona ledgeriana Moens: Effects of Fungal Filtrate and Methyl Jasmonate Elicitors

2021 ◽  
Vol 6 (1) ◽  
pp. 31-40
Author(s):  
Yustiny Andaliza Hasibuan ◽  
Diah Ratnadewi ◽  
Zainal Alim Mas’ud
Keyword(s):  
Cell Culture ◽  
Cell Growth ◽  
Secondary Metabolites ◽  
Methyl Jasmonate ◽  
Woody Plant ◽  
Cultured Cells ◽  
Cinchona Alkaloids ◽  
Cell Biomass ◽  
Cinchona Ledgeriana ◽  
Culture Technology

Cinchona alkaloids are known as antimalaria and anti-arrhythmic. Due to the long waiting time to harvest, cell culture technology is a challenge. This study aimed to determine the effects of elicitors, filtrate of two strains of endophytic fungi and methyl jasmonate (MeJA), in cell suspension culture of Cinchona ledgeriana on quinine and quinidine production. The cells were cultured for seven weeks in woody plant (WP) media treated with either of those elicitors in various concentrations. The cells growth was observed and the alkaloids were analyzed by HPLC. Cells treated with MeJA failed to grow that led to the cell biomass insufficiency for alkaloids determination.  It indicates that the cells are quite sensitive to even low concentration of MeJA that hampered the growth. Cells treated with the filtrate of Diaporthe sp. M13-Millipore filtered (S2M) gave the least cell biomass but presented the highest content of both alkaloids. Diaporthe sp. strain M-13 is stronger as elicitor than M-23 for this plant species. Filtrate of non-virulent fungi can elevate the biosynthesis of alkaloids. This reconfirms that cultured cells are capable to produce secondary metabolites and the productivity can be increased by using an appropriate elicitor.  

scholarly journals Which cytochrome P450 metabolizes phenazepam? Step by step in silico, in vitro, and in vivo studies

2018 ◽  
Vol 33 (2) ◽  
pp. 65-73 ◽  
Author(s):  
Dmitriy V. Ivashchenko ◽  
Anastasia V. Rudik ◽  
Andrey A. Poloznikov ◽  
Sergey V. Nikulin ◽  
Valeriy V. Smirnov ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cytochrome P450 ◽  
In Silico ◽  
In Vivo Studies ◽  
Cyp3a Activity ◽  
Cell Culture Study ◽  
Study Results ◽  
Three Stages

Abstract Background: Phenazepam (bromdihydrochlorphenylbenzodiazepine) is the original Russian benzodiazepine tranquilizer belonging to 1,4-benzodiazepines. There is still limited knowledge about phenazepam’s metabolic liver pathways and other pharmacokinetic features. Methods: To determine phenazepam’s metabolic pathways, the study was divided into three stages: in silico modeling, in vitro experiment (cell culture study), and in vivo confirmation. In silico modeling was performed on the specialized software PASS and GUSAR to evaluate phenazepam molecule affinity to different cytochromes. The in vitro study was performed using a hepatocytes’ cell culture, cultivated in a microbioreactor to produce cytochrome P450 isoenzymes. The culture medium contained specific cytochrome P450 isoforms inhibitors and substrates (for CYP2C9, CYP3A4, CYP2C19, and CYP2B6) to determine the cytochrome that was responsible for phenazepam’s metabolism. We also measured CYP3A activity using the 6-betahydroxycortisol/cortisol ratio in patients. Results: According to in silico and in vitro analysis results, the most probable metabolizer of phenazepam is CYP3A4. By the in vivo study results, CYP3A activity decreased sufficiently (from 3.8 [95% CI: 2.94–4.65] to 2.79 [95% CI: 2.02–3.55], p=0.017) between the start and finish of treatment in patients who were prescribed just phenazepam. Conclusions: Experimental in silico and in vivo studies confirmed that the original Russian benzodiazepine phenazepam was the substrate of CYP3A4 isoenzyme.

scholarly journals Eicosanoid content in fetal calf serum accounts for reproducibility challenges in cell culture

2020 ◽  
Author(s):  
Laura Niederstaetter ◽  
Benjamin Neuditschko ◽  
Julia Brunmair ◽  
Lukas Janker ◽  
Andrea Bileck ◽  
...  
Keyword(s):  
Cell Culture ◽  
High Resolution ◽  
Fetal Calf Serum ◽  
Cultured Cells ◽  
High Resolution Mass Spectrometry ◽  
Calf Serum ◽  
Protein Composition ◽  
Efficient Uptake ◽  
U937 Macrophage

AbstractReproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch-wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 macrophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spectrometry was used to identify FCS constituents and to explore their effects on cultured cells evaluating secreted cytokines, eicosanoids and other inflammatory mediators. Remarkably, the FCS eicosanoid composition showed more batch-dependent variations than the protein composition. Efficient uptake of fatty acids from medium by U937 macrophages and inflammation-induced release thereof was evidenced using C13-labelled arachidonic acid, highlighting rapid lipid metabolism. For functional testing, FCS batch-dependent nanomolar concentration differences of two selected eicosanoids, 5-HETE and 15-HETE, were balanced out by spiking in. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPARγ activation, independently corroborated by HETE-induced formation of peroxisomes observed by high-resolution microscopy. In conclusion, the present data demonstrate that FCS-contained eicosanoids, subject to substantial batch-wise variation, may modulate cellular effector functions in cell culture experiments.

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