Silk scaffolds connected with different naturally occurring biomaterials for prostate cancer cell cultivation in 3D

In the present work, different biopolymer blend scaffolds based on the silk protein fibroin from Bombyx mori (BM) were prepared via freeze‐drying method. The chemical, structural, and mechanical properties of the three dimensional (3D) porous silk fibroin (SF) composite scaffolds of gelatin, collagen, and chitosan as well as SF from Antheraea pernyi (AP) and the recombinant spider silk protein spidroin (SSP1) have been systematically investigated, followed by cell culture experiments with epithelial prostate cancer cells (LNCaP) up to 14 days. Compared to the pure SF scaffold of BM, the blend scaffolds differ in porous morphology, elasticity, swelling behavior, and biochemical composition. The new composite scaffold with SSP1 showed an increased swelling degree and soft tissue like elastic properties. Whereas, in vitro cultivation of LNCaP cells demonstrated an increased growth behavior and spheroid formation within chitosan blended scaffolds based on its remarkable porosity, which supports nutrient supply matrix. Results of this study suggest that silk fibroin matrices are sufficient and certain SF composite scaffolds even improve 3D cell cultivation for prostate cancer research compared to matrices based on pure biomaterials or synthetic polymers.     

Three-dimensional cell culture models for investigating human viruses

Three-dimensional (3D) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput experiments. Moreover, these models bridge the gap between traditional two-dimensional (2D) monolayer cultures and animal models. 3D culture systems have significantly advanced basic cell science and tissue engineering, especially in the fields of cell biology and physiology, stem cell research, regenerative medicine, cancer research, drug discovery, and gene and protein expression studies. In addition, 3D models can provide unique insight into bacteriology, virology, parasitology and host-pathogen interactions. This review summarizes and analyzes recent progress in human virological research with 3D cell culture models. We discuss viral growth, replication, proliferation, infection, virus-host interactions and antiviral drugs in 3D culture models.

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Fabrication techniques of biomimetic scaffolds in three‐dimensional cell culture: A review

In the last four decades, several researchers worldwide have routinely and meticulously exercised cell culture experiments in two‐dimensional (2D) platforms. Using traditionally existing 2D models, the therapeutic efficacy of drugs has been inappropriately validated due to the failure in generating the precise therapeutic response. Fortunately, a 3D model addresses the foregoing limitations by recapitulating the in vivo environment. In this context, one has to contemplate the design of an appropriate scaffold for favoring the organization of cell microenvironment. Instituting pertinent model on the platter will pave way for a precise mimicking of in vivo conditions. It is because animal cells in scaffolds oblige spontaneous formation of 3D colonies that molecularly, phenotypically, and histologically resemble the native environment. The 3D culture provides insight into the biochemical aspects of cell–cell communication, plasticity, cell division, cytoskeletal reorganization, signaling mechanisms, differentiation, and cell death. Focusing on these criteria, this paper discusses in detail, the diversification of polymeric scaffolds based on their available resources. The paper also reviews the well‐founded and latest techniques of scaffold fabrication, and their applications pertaining to tissue engineering, drug screening, and tumor model development.     

Evaluation of cellular adhesion and organization in different microporous polymeric scaffolds

The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug‐induced liver injury (DILI), and has led to a demand for robust, human cell‐based, in vitro assays for drug discovery. Microporous polymer‐based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high‐throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi‐like structures. Poly‐l‐ lysine and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 μm; B: 175 ± 6 μm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost‐effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer‐based scaffolds suitable for drug discovery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:505–514, 2018     

大规模动物细胞培养的问题及对策

大规模动物细胞培养在生物技术产业化进程中显示出强大的潜力。本文综述了大规模动物细胞培养过程中出现的问题及其解决办法 ,包括细胞培养环境、基因工程途径改建细胞系及过程监控等。对于这些进展的充分了解对优化细胞培养工艺、提高产品质量具有重要意义     

Three-dimensional multicellular cell culture for anti-melanoma drug screening: focus on tumor microenvironment

AbstractThe development of new treatments for malignant melanoma, which has the worst prognosis among skin neoplasms, remains a challenge. The tumor microenvironment aids tumor cells to grow and resist to chemotherapeutic treatment. One way to mimic and study the tumor microenvironment is by using three-dimensional (3D) co-culture models (spheroids). In this study, a melanoma heterospheroid model composed of cancer cells, fibroblasts, and macrophages was produced by liquid-overlay technique using the agarose gel. The size, growth, viability, morphology, cancer stem-like cells population and inflammatory profile of tumor heterospheroids and monospheroids were analyzed to evaluate the influence of stromal cells on these parameters. Furthermore, dacarbazine cytotoxicity was evaluated using spheroids and two-dimensional (2D) melanoma model. After finishing the experiments, it was observed the M2 macrophages induced an anti-inflammatory microenvironment in heterospheroids; fibroblasts cells support the formation of the extracellular matrix, and a higher percentage of melanoma CD271 was observed in this model. Additionally, melanoma spheroids responded differently to the dacarbazine than the 2D melanoma culture as a result of their cellular heterogeneity and 3D structure. The 3D model was shown to be a fast and reliable tool for drug screening, which can mimic the in vivo tumor microenvironment regarding interactions and complexity.Graphic abstract      

Step-fortifications of nutrients in mammalian cell culture

A series of high-density media for mammalian cell culture were developed by step-fortifications of most nutrient components in RPMI-1640 medium. Each medium constituting the series was constructed to meet in vitro cell growth limitations. Four different cell lines were cultivated in the media series, and their growth characteristics were observed. Maximum cell densities varied in the range of 0.4 to 1.3 x 10(7) cells/mL, depending on cell lines. Cell growth responses to each of the media series were analyzed in terms of cell density and cell mass. Step increases of cell mass in the range of 1.3 to 3.7 g/L were observed according to the step-fortifications of nutrients. Also, the characteristics of each cell line were compared in terms of metabolic yields and specific productions of lactic acid and ammonium ion. The effect of step-fortifications of nutrients on the production of monoclonal antibody was also examined. Apparent differences in metabolic characteristics among cell lines were observed. Experimental results suggested that the different cell sizes and metabolic characteristics of each cell line resulted in cell-line-specific responses to the step-fortifications. The significant influence of nutritional fortifications on high-density culture of mammalian cells was evaluated. (c) 1993 John Wiley & Sons, Inc.     

Three-dimensional culture models to study drug resistance in breast cancer

Despite recent advances in breast cancer treatment, drug resistance frequently presents as a challenge, contributing to a higher risk of relapse and decreased overall survival rate. It is now generally recognized that the extracellular matrix and cellular heterogeneity of the tumor microenvironment influences the cancer cells' ultimate fate. Therefore, strategies employed to examine mechanisms of drug resistance must take microenvironmental influences, as well as genetic mutations, into account. This review discusses three-dimensional (3D) in vitro model systems which incorporate microenvironmental influences to study mechanisms of drug resistance in breast cancer. These bioengineered models include spheroid-based models, biomaterial-based models such as polymeric scaffolds and hydrogels, and microfluidic chip-based models. The advantages of these model systems over traditionally studied two-dimensional tissue culture polystyrene are examined. Additionally, the applicability of such 3D models for studying the impact of tumor microenvironment signals on drug response and/or resistance is discussed. Finally, the potential of such models for use in the development of strategies to combat drug resistance and determine the most promising treatment regimen is explored.     

Three-dimensional perfused cell culture

Compelling evidence suggests the limitation and shortcomings of the current and well established cell culture method using multi-well plates, flasks and Petri dishes. These are particularly important when cell functions are sensitive to the local microenvironment, cell–cell and cell–extracellular matrix interactions. There is a clear need for advanced cell culture systems which mimic in vivo and more physiological conditions. This review summarises and analyses recent progress in three dimensional (3D) cell culture with perfusion as the next generation cell culture tools, while excluding engineered tissue culture where three dimensional scaffold has to be used for structural support and perfusion for overcoming mass transfer control. Apart from research activities in academic community, product development in industry is also included in this review.     

Theoretical analysis of insulin-dependent glucose uptake heterogeneity in 3D bioreactor cell culture

Three-dimensional (3D) cell cultures in bioreactors are becoming relevant as models for biological and physiological in vitro studies. In such systems, mathematical models can assist the experiment design that links the macroscopic properties to single-cell responses. We investigated the relationship between biochemical stimuli and cell response within a 3D cell culture in scaffold with heterogeneous porosity. Specifically, we studied the effect of insulin on the local glucose metabolism as a function of 3D pore size distribution. The multiscale mathematical model combines the mass transport within a 3D scaffold and a signaling pathways model. It considers the scaffold heterogeneity, and it describes spatiotemporal concentration of metabolites, biochemical stimuli, and cell density. The signaling model was integrated into this model, linking the local insulin concentration at cell membrane to the glucose uptake rate through glucose transporter type 4 (GLUT4) translocation from the cytosol to the cell membrane. The integrated model determines the cell response heterogeneities in a single channel, hence the biological response distribution in a 3D system. It also provides macroscopic outcomes to evaluate the feasibility of an experimental measurement of the system response. From our analysis, it became apparent that the flow rate is the most important operative variable, and that an optimum value ensures a fast and detectable cell response. This model on insulin-dependent glucose consumption rate offers insight into the cell metabolism physiology, which is a fundamental requirement for the study metabolic disorder such as Type 2 diabetes mellitus, in which the physiological insulin-dependent glucose metabolism is impaired.     

Tryptophan oxidation catabolite,N‐formylkynurenine,in photo degraded cell culture medium results in reduced cell culture performance

Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC‐MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N‐formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:74–82, 2016     

A new bacterial cellulose substrate for mammalian cell culture

A new substrate for mammalian cell culture was developed using a cellulose membrane produced byAcetobacter aceti. Modification of the ionic charge of the membrane and adsorption of collagen to it promoted cellular adhesion to the membrane surface. The growth of eight kinds of cells on the membrane, was comparable to that achieved in plastic Petri dishes. The membrane was tested for use in the production of recombinant Erythroid Differentiation Factor (EDF)/activin A using genetically engineered Chinese hamster ovary cells. Both the viability of the cells and production of EDF/activin A were maintained for about 1 month, while cultures on plastic dishes lasted only 12 days. It was considered that the mechanism of improved cell viability was related to the ultrastructure of the cellulose membrane.     

红豆杉细胞培养中紫杉醇高产细胞株的筛选及其稳定性分析

对中国红豆杉〔Taxus chinensis(Pilger)Rehd.〕、云南红豆杉(T. yunnanensis Cheng et L.K.Fu)和东北红豆杉(T. cuspidata Sieb.et Zucc.)不同部位的愈伤组织进行培养及分析比较。结果表明,中国红豆杉愈伤组织生长较快,其叶片诱导的愈伤组织紫杉醇含量较高,且紫杉醇含量与培养物的外观特征有明显相关性,颜色浅、块状或颗粒较明显的细胞团紫杉醇含量较高。运用细胞看护培养技术,从中国红豆杉愈伤组织中筛选出生长速率达0.52g.L-1.d-1、紫杉醇含量超过0.01%的细胞优株,经20次继代培养,其生长和紫杉醇含量均较稳定。     

Enhanced cell viability via strain stimulus and fluid flow in magnetically actuated scaffolds

A novel magnetically actuated scaffold was used to explore the effects of strain stimulus on the proliferation and spatial distribution of smooth muscle cells and improve cell viability in the scaffold interior by pumping nutrients throughout the structure. Magnetically actuable scaffolds were fabricated in a tube shape by winding electrospun sheets of a biodegradable polymer modified with magnetic Fe₂O₃ nanoparticles. Prior to rolling, the sheets were seeded with smooth muscle cells and wound into tubes with diameter 5.2 mm and wall thickness 0.2 mm. The tubular scaffolds were actuated by a magnetic field to induce a cyclic crimping deformation, which applies strain stimulus to the cells and pumps nutrient fluid through the porous tube walls. Comparison with non‐actuated controls shows that magnetic actuation increases the total cell count throughout the scaffold after 14 days of incubation. Furthermore, whereas cell density as a function of position through the tube wall thickness showed a minimum in the mid‐interior in the controls after 14 days due to cell starvation, the actuated scaffolds displayed a maximum cell density. Comparison of cell distributions with the expected spatial variations in strain amplitude and nutrient flux implies that both strain stimulus and nutrient pumping are significant factors in cell proliferation. Biotechnol. Bioeng. 2013; 110: 936–946. © 2012 Wiley Periodicals, Inc.     

Novel cell culture device enabling three-dimensional cell growth and improved cell function

A better understanding of cell biology and cell-cell interactions requires three-dimensional (3-D) culture systems that more closely represent the natural structure and function of tissues in vivo. Here, we present a novel device that provides an environment for routine 3-D cell growth in vitro. We have developed a thin membrane of polystyrene scaffold with a well defined and uniform porous architecture and have adapted this material for cell culture applications. We have exemplified the application of this technology by growing HepG2 liver cells on 2- and 3-D substrates. The performance of HepG2 cells grown on scaffolds was significantly enhanced compared to functional activity of cells grown on 2-D plastic. The incorporation of thin membranes of porous polystyrene to create a novel device has been successfully demonstrated as a new 3-D cell growth technology for routine use in cell culture.     

A quantitative analysis of shear effects on cell suspension and cell culture of perilla frutescens in bioreactors

The short-time effects of shear on suspended cells of Perilla frutescens were quantitatively analyzed by exposing the cells to a well-defined flow field in a rotating drum reactor. It was found that both shear rate and shearing time significantly affected cell viability. The quantitative effects of shear on cell growth and the production of anthocyanin, a secondary metabolite, by the cell cultures were further investigated in a series of batch cultivations using a 5-L plant cell bioreactor with a marine impeller. The results indicated that there was an optimum range of shear rate; i.e., an average shear rate of 20 to 30 s(-1) or an impeller tip speed of 5 to 8 dm/s, which maximized all the values of the following parameters: the specific growth rate, the maximum cell concentration, the (specific) production and productivity of anthocyanin, and the cell and anthocyanin yields. (c) 1994 John Wiley & Sons, Inc.     

Estimation of disruption of animal cells by laminar shear stress

Using mechanical cell properties measured by micromanipulation, and a model of cell distortion in laminar flow fields, a method has been developed for predicting disruption of animal cells by laminar shear stresses. Predictions of the model were compared with measured losses of cell number and viability of TB/C3 murine hybridomas sheared in a cone and plate viscometer at shear rates up to 3950 s(-1), and shear stresses up to 600 Nm(-2), achieved by enhancement of viscosity with dextran. In all cases, the experimental, results and predictions were within 30%. Such excellent agreement suggests it might be possible to use micromanipulation measurements of animal cell mechanical properties to predict cell damage in more complex flow fields, such as those in bioreactors. (c) 1992 John Wiley & Sons, Inc.     

Induction of mammalian cell death by simple shear and extensional flows

In this work we investigated whether the type of shear flow, to which cells are exposed, influences the initiation of cell death. It is shown that mammalian cells, indeed, distinguish between discrete types of flow and respond differently. Two flow devices were employed to impose accurate hydrodynamic flow fields: uniform steady simple shear flow and oscillating extensional flow. To distinguish between necrotic and apoptotic cell death, fluorescence activated cell sorting and the release of DNA in the culture supernatant was used. Results show that Chinese Hamster Ovaries and Human Embryonic Kidney cells will enter the apoptotic pathway when subjected to low levels of hydrodynamic stress (around 2.0 Pa) in oscillating, extensional flow. In contrast, necrotic death prevails when the cells are exposed to hydrodynamic stresses around 1.0 Pa in simple shear flow or around 500 Pa in extensional flow. These threshold values at which cells enter the respective death pathway should be avoided when culturing cells for recombinant protein production to enhance culture longevity and productivity. Biotechnol. Bioeng. 2009; 104: 360–370 © 2009 Wiley Periodicals, Inc.