Microencapsulation of human fibroblasts in a water-insoluble polyacrylate

Viable human diploid fibroblasts have been micro-encapsulated in EUDRAGIT RL, a commercially available water-insoluble polyacrylate, by an interfacial precipitation technique. Cells in medium and polymer solution (in diethyl phthalate) were coextruded and formed into droplets by a coaxial air stream. The droplets fell into a corn-oil/mineral-oil mixture to extract the solvent to precipitate the polymer around the cells. Capsules were ca. 500 mum in diameter depending on the air flowrate with a ca. 10-mum thick wall. When collagen (1 mg/mL) was added to the cell suspension prior to encapsulation and base-washed corn oil was used, cell growth occurred with one doubling achieved after five to six days as the collagen gel contracted inside the capsule. In the absence of collagen, cells spread on the inner wall of the capsule but did not grow, presumably because the surface charge on the capsule was inadequate. In similar fashion fibroblasts spread but did not grow on films of EUDRAGIT RL but did grow on blends of EUDRAGIT RL and EUDRAGIT E containing 10-30% of the latter more highly aminated polyacrylate. Although not suitable for anchorage-dependent cell growth by itself, EUDRAGIT RL has been suitable as a model polymer to demonstrate the feasibility of using water insoluble polyacrylates and organic solvents and nonsolvents for the micro-encapsulation of fibroblasts. Such microcapsules are of potential interest as a mode of large scale tissue culture for the production of novel therapeutic agents.     

New immobilization method of mammalian cells using alginate and polyacrylate

Mouse-mouse hybridoma cells were immobilized in polyacrylate-alginate gels. The immobilized hybridoma cells were cultured semi-continuously using a fluidized bed reactor, and allowed continuous antibody production without any gel destruction for one month. It has been proved that the polyacrylate-alginate gels were tolerant against physical stress. The composition of the gels suitable for cell growth and antibody production was given as follows; viscosity of alginate at 1% solution: 60–100 cP, alginate concentration: 0.8%, and polyacrylate concentration: 0.2%. In the semi-continuous culture using gels prepared under suitable conditions, the viable cell number was estimated as 2.5×10⁷ cells/ml-gel, and the antibody production rate was 2.2 mg/ml-gel/d, at maximum.     

Microencapsulation of microbial cells

The high level of biocatalysts such as microbial cells and enzymes plays an important role in increasing the productivity of a bioreactor. The beads entrapped with microbial cells are not strong enough for long-term use. The small void space of polymer matrix and the leakage of cells limit a final cell loading in the beads. The recent success of encapsulating microbial cells makes it possible to prepare dense biocatalyst composed of recombinant microbial cells. In addition to encapsulating microbial cells, immobilization of animal and plant cells in capsules is also briefly described.     

Interferon production by mammalian cells grown in a serum-free medium

Summary Interferon, produced by rabbit heart cells grown in a serum-free medium, failed to protect rabbit heart serum-free cells, but protected rabbit heart serum-containing-medium cells against vaccinia and vesicular stomatitis virus. Interferon produced in serum-free cells had a greater species specificity than that produced in serum-containing media. The difference in activity was shown to be due to lack of adsorption by serum-free-medium cells.     

Microencapsulation of yeast cells and their use as a biocatalyst in organic solvents

Stable, semipermeable polyamide microcapsules were prepared by interfacial polymerization from a mixture of 1,6-hexanediamine and poly(allylamine) crosslinked with di-acid chlorides and were used to encapsulate baker's yeast. The size and distribution of cells within the capsules were investigated by a combination of laser confocal, electron scanning, and transmission electron microscopy. The encapsulated cells were studied as a biocatalyst for the model reduction of 1-phenyl-1,2-propanedione to 2-hydroxy-1-phenyl-1-propanone in a number of organic solvents. The polymerization conditions were extensively investigated and were found to greatly influence the product yield. Microencapsulated yeast cells, prepared under optimized conditions, carried out the reduction more efficiently than free cells as well as those immobilized in alginate and kappa-carrageenan beads. The developed methodology should be broadly applicable to other biotransformations of interest. (c) 1996 John Wiley & Sons, Inc.     

Induction of a deoxycytidineless state in cultured mammalian cells by bromodeoxyuridine

Bromodeoxyuridine triphosphate is an allosteric inhibitor of ribonucleotide reductase, and bromodeoxyuridine can therefore kill cells by starving them for deoxycytidine nucleotides. The toxicity of bromodeoxyuridine for some cell lines is reduced many fold when deoxycytidine is also present. For example, wild type 3T6 cells can be grown serially in 1.5 × 10^?4 Molar bromodeoxyuridine and 2 × 10^?4 Molar deoxycytidine, remain healthy, and incorporate bromodeoxyuridine extensively into cellular DNA. Some of the numerous effects of this drug on the behavior of cells and viruses may be due to a deoxycytidineless state, rather than to the incorporation of the bromodeoxyuridine into DNA.     

Selective precipitation of ribonucleic acid from a mixture of total cellular nucleic acids extracted from cultured mammalian cells

A simple and reproducible method is described for precipitating RNA selectively from total mammalian-cell nucleic acids extracted by the phenol-sodium dodecyl sulphate procedure at pH8.0. Under specified conditions bulk RNA is precipitated almost quantitatively whereas bulk DNA remains in solution. Minor components of RNA (detected by pulse-labelling and chromatography on methylated albumin-kieselguhr) and rapidly labelled components of DNA containing single-stranded regions are also precipitated. The usefulness of the method is discussed in the context of isolating separately both RNA and DNA from cultured cells that are difficult to obtain in quantity.     

Encapsulation of viable cells within polyacrylate membranes

A new, semipermeable hydrogel membrane for encapsulating viable cells has been developed. The encapsulation was performed by consecutively introducing droplets of a suspension of hybridoma cells in a solution of a polyanionic acrylic copolymer into aqueous solutions of three polycationic polymers. As a result of interpolymeric ionic interactions and some chemical reactions, a polyelectrolyte complex membrane was formed at the interface between each droplet and the polycationic polymeric solutions. The hybridoma cells, used as a model system, were derived by fusing spleen cells from immunized BALB/c mice with the NS-1 murine plasmacytoma cell line. The cells divided and gradually filled the microcapsules over a period of 8 days. Prior to encapsulation of the hybridoma cells, the polyanions were tested for toxicity and inhibition of cell growth. A direct relationship was observed between hybridoma cell viability in the acrylic polyanion/RPMI-1640/10% (w/v) fetal calf serum (FCS) solutions and the kinematic viscosities of the solutions of the polyanions. Antihuman IgM was produced by the encapsulated hybridoma cells and immunoassay showed that the antibody concentration was 3 mug/ml of the total culture medium.     

Non-exponential growth by mammalian cells in culture

The concept of exponential growth by mammalian cells in culture is based upon the apparent linearity of semilogarithmic data plots. This method of graphical analysis is known to be an unreliable test of the exponential hypothesis. We have re-examined the question of growth exponentiality using the more sensitive method of Smith plots, in which specific growth rate is plotted against either time or density on transformed graphical coordinates which linearize the mathematical expression of the growth hypothesis being tested. With exponential growth, data points fall on a horizontal straight line when specific growth rate is plotted against time or density. Using both our own and literature data, we have performed Smith plot analyses on the growth of 125 different mammalian and avian cell lines. Of these, only eleven exhibited an exponential phase. The remaining cell lines all had non-exponential growth patterns. The most common of these consisted of an initial period of growth acceleration followed by a later phase of deceleratory growth. A smaller number of lines exhibited deceleratory kinetics at all times after plating. We conclude that mammalian cell growth in culture is predominantly non-exponential, and that the apparent exponentiality of semilogarithmic data plots is usually a methodological artifact.     

Interferon production by mammalian cells grown in a serum-free medium.

Interferon, produced by rabbit heart cells grown in a serum-free medium, failed to protect rabbit heart serum-free cells, but protected rabbit heart serum-containing-medium cells against vaccinia and vesicular stomatitis virus. Interferon produced in serum-free cells had a greater species specificity than that produced in serum-containing media. The difference in activity was shown to be due to lack of adsorption by serum-free-medium cells.     

Conditional gene silencing in mammalian cells mediated by a stress-inducible promoter

MicroRNAs (miRNAs) are endogenous non-coding small RNAs, which negatively regulate gene expression in a sequence-specific manner through the RNA interference (RNAi) pathway. Here we describe a new miRNA-based conditional RNAi expression system that relies on cellular stress-response mechanisms in mammalian cells. In our constructs, expression of miRNA mimics is tightly controlled by a heat shock-inducible promoter. This system is highly effective in silencing permanently or conditionally expressed luciferase. The stress inducible vectors also effectively deplete co-expressed pro-apoptotic protein CHOP with heat shock. Furthermore, we demonstrate cloning of a protein-coding sequence between the stress-inducible promoter and the miRNA expression cassette allows simultaneous silencing of a target gene and activation of synthesis of a protein of choice in response to stress stimulation. This new conditional gene silencing approach could be an invaluable tool for various areas of basic and applied research and for therapeutic intervention.     

Radiosensitization of mammalian cells in vitro by nitroacridines

The nitroacridine nitracrine (1-NC) is a DNA intercalator and a hypoxia-selective, electron-affinic radiosensitizer. Sensitization of Chinese hamster fibroblast cultures at 0 degrees C by the nitro positional isomers of 1-NC has now been compared to help establish structure-activity relationships. The des-nitro analog (E(1) at pH 7 = -899 mV) did not sensitize, suggesting that an electron-affinic chromophore is required. All the nitroacridines (E(1) range -376 to -257 mV) sensitized hypoxic cells with a maximum sensitizer enhancement ratio of about 1.7, but with a 200-fold range in potency. When mean intracellular drug concentrations were compared, 2-, 3-, and 4-NC had potencies which were similar, independent of E(1), and no greater than predicted for non-DNA binding nitroheterocycles. Sensitization by these three isomers occurred at intracellular concentrations likely to saturate the potential intercalation sites on DNA. A large fraction of the radical sites sensitized by O2 are apparently inaccessible to these drugs. It is suggested that sensitization results from electron transfer from migrating transient charge carriers of low reduction potential to immobile bound intercalators. An additional sensitizing mechanism may be available to 1-NC, which was 20 times more potent, a potency not accounted for by E(1), cell uptake, or DNA binding affinity. The dissociation kinetics of the DNA-drug complex was faster for 1-NC than for the other isomers. The higher potency of 1-NC may reflect a short mean residence time (less than 1 ms) in its intercalation site, allowing significant mobility on the DNA within the lifetime of relatively stable radiation-induced target radicals.     

Inactivation of a common OGG1 variant by TNF-alpha in mammalian cells

Reactive oxygen species threaten genomic integrity by inducing oxidative DNA damage. One common form of oxidative DNA damage is the mutagenic lesion 8-oxoguanine (8-oxodG). One driver of oxidative stress that can induce 8-oxodG is inflammation, which can be initiated by the cytokine tumor necrosis factor alpha (TNF-α). Oxidative DNA damage is primarily repaired by the base excision repair pathway, initiated by glycosylases targeting specific DNA lesions. 8-oxodG is excised by 8-oxoguanine glycosylase 1 (OGG1). A common Ogg1 allelic variant is S326C-Ogg1, prevalent in Asian and Caucasian populations. S326C-Ogg1 is associated with various forms of cancer, and is inactivated by oxidation. However, whether oxidative stress caused by inflammatory cytokines compromises OGG1 variant repair activity remains unknown. We addressed whether TNF-α causes oxidative stress that both induces DNA damage and inactivates S326C-OGG1 via cysteine 326 oxidation. In mouse embryonic fibroblasts, we found that S326C-OGG1 was inactivated only after exposure to H₂O₂ or TNF-α. Treatment with the antioxidant N-acetylcysteine prior to oxidative stress rescued S326C-OGG1 activity, demonstrated by in vitro and cellular repair assays. In contrast, S326C-OGG1 activity was unaffected by potassium bromate, which induces oxidative DNA damage without causing oxidative stress, and presumably cysteine oxidation. This study reveals that Cys326 is vulnerable to oxidation that inactivates S326C-OGG1. Physiologically relevant levels of TNF-α simultaneously induce 8-oxodG and inactivate S326C-OGG1. These results suggest a mechanism that could contribute to increased risk of cancer among S326C-Ogg1 homozygous individuals.     

Photosensitization of mammalian cells by psoralens and porphyrins

Because of the ability of photosensitizers to induce specific photochemical reactions in vivo, leading to cell injury and death, many such molecules have been considered as therapeutic agents. Among them two classes of sensitizers, i.e. furocoumarins (psoralens) and porphyrins, are currently used for the photochemotherapy of various skin diseases and malignant lesions. Different types of cell responses can result according to the intracellular localization of the photosensitizer and to the nature of the photochemistry induced by the chromophore which absorbs photons. In this review, the cytological aspects of photosensitization by psoralens and porphyrins will be discussed.     

Microencapsulation of yeast cells in the calcium alginate membrane

Summary Cells of Saccharomyces cerevisiae (ATCC 24858) were encapsulated in the calcium alginate membrane and cultured. Swelling of the capsule was prevented by adding 0.2 g CaCl₂ to 1 L growth medium. The dry cell concentration based on the inner volume of the capsule reached 309 g/L, which was much higher than could be obtained by cell entrapment. All the cells remained inside the capsule during the cultivation. The flux of CO₂ through the capsule membrane increased approximately twice by adding a nonionic surfactant to the CaCl₂ solution during the step of capsule formation.