A Novel Wick-Like Paper-Based Microfluidic Device for 3D Cell Culture and Anti-Cancer Drugs Screening

Paper is increasingly recognized as a portable substrate for cell culture, due to its low-cost, flexible, and special porous property, which provides a native cellular 3D microenvironment. Therefore, paper-based microfluidics has been developed for cell culture and biomedical analysis. However, the inability of continuous medium supply limits the wide application of paper devices for cell culture. Herein, a paper-based microfluidic device is developed with novel folded paper strips as wick-like structure, which is used for medium self-driven perfusion. The paper with patterns of hydrophilic channel, culture areas, and hydrophobic barrier could be easily fabricated through wax-printing. After printing, the hydrophilic paper strip at the periphery of the lower layer is then folded at 90° and extended into the medium container for continuous automatic supply of medium to the cell culture area. Tumor cells cultured in the paper device are tested for anti-cancer drug screening. Visualized cell viability and chemical sensitivity testing can be achieved by colorimetry combined with simple smartphone imaging, effectively reducing precision instrument dependence. The wick paper-based microfluidic device for cell culture endows the method the advantages of lower cost, ease-of-operation, miniaturization, and shows a great potential for large-scale cell culture, antibody drug production, and efficient screening.     

Promoting effect of rapeseed proteins and peptides on Sf9 insect cell growth

The Baculovirus Expression Vector System has become widely used for the production of recombinant proteins for research and diagnostics. Serum-free culture media able to support high cell densities have been developed for the large scale culture of insect cells. While serum elimination aims at avoiding the risks associated with the introduction of an ill defined component of bovine origin, additives such as protein hydrolysates from animal sources are still used. An alternative could be the supplementation of culture media with protein hydrolysates derived from plants. In this study, we describe the replacement of lactalbumin hydrolysate with a laboratory produced hydrolysate of rapeseed proteins. Its effect on Sf9 cell growth kinetics, substrate consumption and by-product formation in low-serum or serum-free medium was evaluated. Cells were unable to grow in the presence of a rapeseed protein hydrolysate generated by PTN 3.0 Special^® enzyme and containing only 24% of peptides under 1 kDa in size. On the other hand, serum-free medium supplementation with a rapeseed protein hydrolysate obtained with Orientase 90N^® enzyme had a strong growth promoting effect, leading to a 60% increase in maximal cell density without affecting cell metabolism. This significant positive effect could be explained by the higher degree of hydrolysis of this digest, with 74% of peptides under 1 kDa in size.     

Quantitation of interaction of lipids with polymer surfaces in cell culture

As cell culture medium development efforts have progressed towards leaner, serum-free, and chemically defined formulations, it has become increasingly important to ensure that the appropriate concentrations of all nutrients are maintained and delivered at point of use. In light of concurrent efforts to progress to disposable polymeric storage and culture platforms, the characterization and control of medium component interactions with container surfaces can be a key issue in ensuring consistent delivery of these medium formulations. These studies characterize the interactions of lipids with culture surfaces typically encountered in the bioprocess industry using model systems. The extent and kinetics of lipid association with polymeric surfaces were determined using radio-labeled linoleic acid and cholesterol. The effect of methyl-beta-cyclodextrin, a component commonly used to solubilize lipids in culture media, on association kinetics was also examined. In addition, loss of lipids across a sterilizing membrane filter was quantified. We find that there is potential for significant loss of hydrophobic components due to non-specific binding to surfaces at timescales relevant to a typical cell culture process. The extent of loss is dependent on the nature of the hydrophobic component as well as the type of surface. These studies highlight the potential of the extracellular environment to modify medium composition and also emphasize the importance of medium formulation strategies, including those used in the delivery of hydrophobic components. It is noted, however, that the level of loss is very dependent on the specific system including the composition of the culture medium used.     

Isolation and characterization of an adherent, 8-azaguanine resistant variant of the Burkitt lymphoma cell line, Raji

A substrate adherent, fibroblast-like cell line (Raji-A) has been isolated from a suspension culture of an established Burkitt lymphoma cell line (Raji). Except for the altered morphology associated with substrate attachment, Raji-A is identical to Raji with respect to karyotype, isozyme composition, susceptibility to Epstein-Barr virus (EBV) infection and inducibility of latent EBV. In order to facilitate fusion experiments with Raji-A, drug resistant variants were induced by treating cells with ethylmethane sulfonate followed by selection in growth medium which contains 8-azaguanine. Three clones (AGRO, AGR3 and AGR6) were found to be resistant to 50 μg/ml of 8-azaguanine. They had only 10–15% as much hypoxanthineguanine phosphoribosyltransferase activity as wild type cells and a low plating efficiency of 1–3 × 10⁻⁶ in the selective medium, HAT. Potential uses of these variants for studying EBV-lymphoblastoid cell interactions as well as human genetics by cell hybridization are discussed.     

Growth and differentiation of epidermal cells from the rainbow trout established as explants and maintained in various media

Growth and a number of differentiated characteristics of cultured epidermal cells from the rainbow trout Oncorhynchus mykiss were compared using two commercially available serum–free media, a dermal substrate/serum free kit and a serum–containing medium which had been previously optimized for epidermal cell culture. Each medium supported short term growth over 15 days. Only the medium supplied for dermal substrate culture supported longer growth periods. This medium was supplied for use with a collagen/stromal substrate but gave good cultures even without the substrate. Differentiation, measured by examining mucous cells, cytokeratins, epidermal growth factor receptor, gap junction status and ultrastructure showed that serum–free media gave quantitatively and qualitatively superior expression and short term retention of differentiation over serum–containing medium. Epithelial cell growth with expression of differentiated characteristics can be maintained in primary culture in serum–free medium for at least as long as in serum–containing medium. This provides a useful technique for use when serum presence in medium is undesirable or proves toxic to the specialized cell type under investigation.     

Facile development of medium optimization for antibody production: implementation in spinner flask and hollow fiber reactor

Most bio-industrial mammalian cells are cultured in serum-free media to achieve advantages, such as batch consistency, suspended growth, and simplified purification. The successful development of a serum-free medium could contribute to a reduction in the experimental variation, enhance cell productivity, and facilitate biopharmaceuticals production using the cell culture process. Commercial serum-free media are also becoming more and more popular. However, the cell line secrets its own recombinant product and has special nutritional requirements. How can the composition of the proprietary medium be adjusted to support the specific cell’s metabolism and recombinant protein? This article uses statistical strategies to modify the commercial medium. A design of experiments is adopted to optimize the medium composition for the hybridoma cell in a serum-free condition. The supplements of peptone, ferric citrate, and trace elements were chosen to study their impact on hybridoma growth and antibody production using the response surface methodology. The stimulatory effect of the developed formulation on hybridoma growth was confirmed by the steepest ascent path. The optimal medium stimulated the hybridoma growth and antibody production in three diverse systems: a static plate, an agitated spinner flask, and a hollow fiber reactor. The cells in the developed serum-free medium had a better antibody production as compared to that in the commercial medium in the hollow fiber reactor. Our results demonstrated that the facile optimization for medium and antibody production was successfully accomplished in the hybridoma cells.     

A new method for the production of fine plant cell suspension cultures

Fine, almost single cell, suspensions were produced from both existing suspension cultures containing large cell clumps and from chopped callus pieces by immobilizing the cells in 4–5 mm diameter calcium alginate beads. The immobilized cells continued to divide inside the beads and at the bead surface, and after 2–3 weeks' culture, fine cell suspensions were formed as a result of loss of the surface cells into the medium. After removal of the cell suspensions by filtration, subsequent culture of the beads in fresh medium resulted in the further production of homogeneous cell suspensions after 1–2 weeks. In this way an almost continuous supply of fine cell suspensions could be obtained from cultures containing large clumps of cells. The cells produced by this method remained in this state for at least one culture period, although in some instances repeated subculture resulted in an increase in the size of cell groups. The technique has been successfully applied to the production of fine cell suspensions ofCatharanthus roseus, Nicotiana tabacum andDaucus carota.     

Effects of blocking integrin β1 and N-cadherin cellular interactions on mechanical properties of vascular smooth muscle cells

Experimental measurements of cellular mechanical properties have shown large variability in whole-cell mechanical properties between cells from a single population. This heterogeneity has been observed in many cell populations and with several measurement techniques but the sources are not yet fully understood. Cell mechanical properties are directly related to the composition and organization of the cytoskeleton, which is physically coupled to neighboring cells through adherens junctions and to underlying matrix through focal adhesion complexes. This high level of heterogeneity may be attributed to varying cellular interactions throughout the sample. We tested the effect of cell-cell and cell-matrix interactions on the mechanical properties of vascular smooth muscle cells (VSMCs) in culture by using antibodies to block N-cadherin and integrin β1 interactions. VSMCs were cultured on substrates of varying stiffness with and without tension. Under each of these conditions, cellular mechanical properties were characterized by performing atomic force microscopy (AFM) and cellular structure was analyzed through immunofluorescence imaging. As expected, VSMC mechanical properties were greatly affected by the underlying culture substrate and applied tension. Interestingly, the cell-to-cell variation in mechanical properties within each sample decreased significantly in the antibody conditions. Thus, the cells grown with blocking antibodies were more homogeneous in their mechanical properties on both glass and soft substrates. This suggests that diversified adhesion binding between cells and the ECM is responsible for a significant amount of mechanical heterogeneity that is observed in 2D cell culture studies.     

Structural alterations in fibronectin correlated with morphological changes in smooth muscle cells

Nontransformed cultures of vascular smooth muscle cells proliferate until they form a confluent sheet of cells. Subsequently, the cells become reorganized to form multicellular nodules that are loosely attached to the substrate. The formation of nodules is facilitated by the addition of medium conditioned by nodular cultures. Nodulation is inhibited by the addition of fibronectin. Fibronectins derived from monolayer culture conditioned medium or from plasma are maximally effective while fibronectin isolated from nodular cell conditioned medium is inactive. Analysis by NaDodSO4-polyacrylamide gel electrophoresis reveals that the nodular cell fibronectin has a molecular weight that is about 20-30 kd less than that of monolayer cell fibronectin. Further, nodular cell conditioned medium contains an activity that can convert both plasma fibronectin and monolayer cell fibronectin to the lower molecular weight correlated with the loss of biological activity.     

The extracellular topographical environment influences ovarian cancer cell behaviour

The importance of the biophysical cellular environment in cancer development has been increasingly recognised but so far has been only superficially studied. Here we investigated the effect of cell-like substrate topography on ovarian cancer cell behaviour and potential underlying signalling pathways. We observed changes in cell morphology in response to substrate topography, which implies modification of structure-function associations. Differences in focal adhesion signalling and Rho/ROCK activity suggested their involvement in the biomechanically-driven cellular responses. Cell-like topography was also shown to modulate the MAPK pathway and hence potentially regulate cell proliferation. The selective regulation of the cells by the mechanotransduction pathways that we noted has wide ranging implications for understanding cancer development. We established that the physical architecture of cell culture substrate is sufficient to influence cancer cell behaviour, independent of genetic composition or biochemical milieu.     

Acylceramides and lanosterol-lipid markers of terminal differentiation in cultured human keratinocytes: Modulating effect of retinoic acid

Summary Epidermal differentiation is accompanied by profound changes in the synthesis of a variety of intracellular proteins and intercellular lipids. In conventional, submerged culture keratinocytes have been shown to lose the ability to synthesize the protein markers of differentiation. They re-express them, however, when they are cultured in medium supplemented with delipidized [retinoic acid (RA)-depleted] serum or in air-exposed cultures using de-epidermized dermis (DED) as a substrate. Recent studies have revealed that acylceramides (AC) and lanosterol (LAN), which are present only in trace amounts in cultures of keratinocytes grown under submerged conditions on DED in medium supplemented with normal serum, become expressed in significant amounts when the culture is lifted to the air-liquid interface. Inasmuch as culture conditions may markedly affect the extent of keratinocyte differentiation, the present study aimed to investigate the effect of normal (RA-containing) or delipidized (RA-depleted) serum and of RA administration on lipid composition (especially of the AC and LAN contents) in cells cultured under submerged and air-exposed conditions. To test a possible effect of dermal substrate (used in the air-exposed model), the lipid composition of keratinocytes grown under submerged conditions on a plastic and on a dermal substrate (de-epidermized dermis, DED) has also been compared. The results revealed that under all culture conditions, RA deprivation of fetal bovine serum resulted in a marked increase of total ceramide content. Even under submerged conditions, the presence of both AC and LAN could be detected. In air-exposed culture, the content of these lipids was markedly increased. Addition of RA at 1 μM concentration to cultures grown in RA-depleted medium induced marked changes in lipid composition under all culture conditions tested. In cells grown under submerged conditions (both on plastic and on DED) AC and LAN were no longer present in detectable amounts. Also in air-exposed culture, a marked decrease in the content of these lipids was observed. These results suggest that liposoluble serum components, like RA, control the synthesis of lipids that are present in later stages of epidermal differentiation.     

Monitoring hybridoma metabolism in continuous suspension culture at the intracellular level

A model mouse hybridoma cell line was grown in continuous culture experiments in a serum-free low-protein lipid-free medium. The steady-state responses of cell numbers, extra- and intracellular metabolite concentrations, substrate and (by) product consumption/production rates, and yield coefficients were investigated as a function of step changes in the glutamine concentration of the feed medium. In addition to the commonly performed analysis of metabolites in culture supernatants, we prepared perchloric acid extracts of cells and determined the amount and the composition of intracellular amino acids and organic acids. Significant differences were found with respect to intracellular metabolite pools for cells growing at nearly identical specific growth rates. To our knowledge this is the first time that data on the intracellular concentrations (pools) of amino acids and Krebs cycle intermediates are reported in the literature that were obtained under carefully defined culture conditions such as those attained in continuous culture experiments.     

Influence of organic nutrients and cocultures on the competitive behavior of 1,2-dichloroethane-degrading bacteria.

The effects of organic nutrients and cocultures on substrate removal by and competitive behavior of 1,2-dichloroethane-degrading bacteria were investigated. Xanthobacter autotrophicus GJ10 needed biotin for optimal growth on 1,2-dichloroethane. In continuous culture, dilution of biotin to a concentration below 0.2 nM resulted in washout. Growth could be restored by inoculation with the 2-chloroethanol utilizer Pseudomonas sp. strain GJ1, leading to a new steady state in which about 1% of the mixed culture consisted of cells of strain GJ1. This indicates that strain GJ1 excreted biotin or a precursor for its synthesis. Inoculation of the mixed culture with Ancylobacter aquaticus AD25 did not result in washout of strain GJ10, although strain AD25 has a 10-fold-lower Ks for growth on 1,2-dichloroethane. Strain AD25 did not become dominant because of the lack of vitamins, which are necessary for its optimal growth. The results indicate that medium composition and the presence of other species strongly influence the effect of substrate limitation on the composition of a bacterial population that degrades a xenobiotic compound in a continuous culture.     

Cell in situ zymography: an in vitro cytotechnology for localization of enzyme activity in cell culture

In situ zymography is a unique technique for detection and localization of enzyme?Csubstrate interactions majorly in histological sections. Substrate with quenched fluorogenic molecule is incorporated in gel over which tissue sections are mounted and then incubated in buffer. The enzymatic activity is observed in the form of fluorescent signal. With the advancements in the field of biological research, use of in vitro cell culture has become very popular and holds great significance in multiple fields including inflammation, cancer, stem cell biology and the still emerging 3-D cell cultures. The information on analysis of enzymatic activity in cell lines is inadequate presently. We propose a single-step methodology that is simple, sensitive, cost-effective, and functional to perform and study the ??in position?? activity of enzyme on substrate for in vitro cell cultures. Quantification of enzymatic activity to carry out comparative studies on cells has also been illustrated. This technique can be applied to a variety of enzyme classes including proteases, amylases, xylanases, and cellulases in cell cultures.     

Characterization of cultured epithelial cells using a novel technique not requiring enzymatic digestion for subculturing

Our laboratory had developed a methodology to expand epithelial cells in culture by growing keratinocyte monolayers, under large volumes of medium that produces large numbers of keratinocytes that leave the monolayer and move into suspension. The cells have been defined as epithelial Pop Up Keratinocytes or ePUKs cells and appear to be highly suitable for clinical applications. In this publication we extend the characterization of the cells with a detailed analysis of the capabilities of the monolayer of a single culture flask to produce, over time, ePUK cells. The cells were characterized using standard epithelial markers for proliferation and differentiation. Analysis of morphology of the monolayer formed and total number of cells produced is presented for a variety of human epithelial cell strains. These keratinocytes provide an additional controlled human cell system for investigation of the mechanisms regulating epithelia cell growth and differentiation and since they are produced in large numbers, they are highly suitable for use in epithelial cell banking.     

Large scale purification and further characterization of a rat liver cell conditioned medium multiplication stimulating activity

The application of roller-bottle cell culture techniques and a relatively simple purification scheme has led to the isolation of milligram quantities of a polypeptide cell multiplication stimulating activity (MSA) from Buffalo rat liver cell conditioned medium. We have characterized the apparently homogeneous MSA with respect to its biological activity, its N-terminal amino acid residue, and its amino acid composition, and have tested the MSA for growth-promoting activity in a number of cell types.     

Isolation and culture of the terminally differentiated adult mammalian ventricular cardiac muscle cell

Summary We have carried out systematic studies to optimize and standardize methodology to isolate and culture the adult rat ventricular cardiac muscle cell. Four hearts were perfused simultaneously with a calcium-free medium containing collagenase. The ventricular tissue was then minced and further digested to liberate individual cells. Approximately 16 million rod-shaped muscle cells were obtained. The plating efficiency has been greatly improved by culturing the cells in a conditioned medium prepared from a rabbit corneal cell line. This medium also contained added fetal bovine serum, essential and nonessential amino acids, vitamins, insulin, transferrin, and 25 trace minerals. The culture flasks were precoated with rat-tail collagen. Fibroblast contamination was virtually eliminated by including cytosine arabinoside in the medium during the first 7 d of culture. After this time the cells could be cultured in the absence of serum in a chemically defined medium composed of MEM, vitamins, nonessential amino acids, and trace minerals. They continued to contract spontaneously and do well in this medium for at least 3 d thereafter. This improved methodology resulted in a reproducible culture system with improved plating efficiency. It provided a new and unique system to study the structure and function of the adult mammalian ventricular cardiac muscle cell. This investigation was supported by Grant HL 25873 from the National Institutes of Health, Bethesda, MD.     

Melanoma growth in non-chemically modified translucid bacterial nanocellulose hollow and compartimentalized spheres

BackgroundBacterial nanocellulose (BNC) has been used as cell support in numerous tissue engineering studies. Its use can be explained based on the fact its structure allows the creation of a required microenvironment for an ideal material, which supports 3D cell culture. Its structure and interconnected pores lead to animal cells adhesion and proliferation, also allowing oxygen and nutrients transportation.MethodsWe developed a new methodology to produce spherical platforms synthesized by Komagataebacter hansenii (ATCC 23769) under dynamic culture conditions in minimal medium. The chemical composition and physical properties of the platforms were evaluated. Then, human melanoma cells (SK-MEL-28) were encapsulated into the platforms and evaluated by metabolic activity, morphology and their ability on adhering to the Hollow Translucid BNC Spheres (BNC-TS-H) and Compartmentalized Translucid BNC Spheres (BNC-TS-C) up to 3 days.ResultsBNC-TS-H and BNC-TS-C platforms were produced as translucid spheroid platforms with distinct microenvironment under dynamic fermentation. The chemical and physical characterizations confirmed the platforms composition as BNC. The produced internal microenvironments in spherical platforms are relevant to determine tumor cell fate. In the first 12 h of culture, cells could adhere to nanocellulose microfibers assuming their typical tumorous phenotype in 72 h of culture.ConclusionThe dynamic fermentation in minimal medium produced distinct microstructured platforms of BNC-TS-H and BNC-TS-C. The platforms microstructure resulted in microenvironments that enabled distinct cell-cell and cell-matrix interactions. This behavior suggests several applications in tissue engineering.General significanceThe method produced translucid BNC sphere platforms with distinct microenvironments for 3D cell culture.     

THE RELATIONSHIP BETWEEN CELL-SUBSTRATE ADHESIVENESS AND CELL GROWTH: A STUDY ON CHONDROCYTES CULTURED IN VITRO WITH CONDITIONED MEDIUM

Sparsely cultured chondrocytes from the sternae of 14-day chick embryos grew vigorously with a liquid medium using Falcon tissue culture dishes, when the conditioned medium (CM) prepared from the high-density cultures of neural retinae was used as a growth medium. In CM most cells adhered to the culture substrate, while in the fresh medium, in which cell growth was very slow, many viable cells remained unattached to the substrate. Enhancement of cell growth by CM was much less marked in cultures of chondrocytes suspended in agarose. The main effect of CM in enhancing cell growth is thus considered to be related to the increase of cell adhesiveness to the substrate. The main active factor in CM is non-dialyzable and heat-stable.