Synthesis and characterization of a novel thermoresponsive copolymer series and their application in cell and cell sheet regeneration

A series of poly-(N-isopropyl acrylamide)-based copolymers were developed with a view to biomedical applications, specifically cell cultivation and recovery. Ethylpyrrolidone methacrylate (EPM), the monomer of poly-(ethylpyrrolidone methacrylate) (pEPM), which is itself thermoresponsive, was copolymerized with N-isopropylacrylamide in varying ratios to create this novel thermoresponsive copolymer series. Characterization indicated a moderate increase of copolymer lower critical solution temperature with increasing EPM content. Films of the copolymers successfully hosted cells to monolayer. Cells detached from the copolymers upon temperature reduction with cell to cell junctions maintained, avoiding the damage which can be caused using conventional detachment techniques. These results indicate that these copolymers are highly cell compatible and may be useful for a range of biomedical applications.     

Thermosensitive N-isopropylacrylamide-N-propylacrylamide-vinyl pyrrolidone terpolymers: synthesis, characterization and preliminary application as embolic agents

In this article, thermosensitive N-isopropylacrylamide (NIPAAm)-N-propylacrylamide (NPAAm)-vinyl pyrrolidone (VP) terpolymers (PNINAVP) were prepared by varying feed ratios with free radical copolymerization method. The composition ratios and molecular weights of PNINAVP were examined by NMR and GPC. The thermo-responsive behaviors of copolymer solutions in the absence and with addition of Iohexol, a radiopaque agent, were investigated by differential scanning calorimetry (DSC) and rheometer. The sol-gel transition of the copolymer solutions occurred reversibly within 1 min in response to temperature. Incorporation of Iohexol increased the transition time and transition temperature of PNINAVP solutions; the rheological properties were also influenced. It was observed that at body temperature, PNINAVP and Iohexol could form an integrated bulky hydrogel presumably due to the hydrogen bonding between them, which was favorable for the clinical follow-up and reducing toxic side effects. In vitro embolic model experiment indicated that 5 wt% 16:16:1H PNINAVP solution containing Iohexol displayed a satisfactory embolization effect. This solution was injected into the rete mirabiles (RM) of six swines through a microcatheter. The angiographical results obtained immediately after the operation showed a complete occlusion of the RM, and no recanalization was observed at postoperative month 1. The histological examination demonstrated no acute inflammatory reaction inside the RM and surrounding tissue. This work could provide a beneficial guidance for designing a new temperature-sensitive polymer-based embolic agent.     

Synthesis and application of new microcarriers for animal cell culture. Part II: Application of polystyrene microcarriers

In this work (Part II) the application of new polystyrene based microcarriers in cell culture technology is demonstrated. Carriers with a variety of surface modifications were tested as a growth support for cell line BHK 21. The growth behavior of the cells and cell to surface attachment were compared to Cytodex 3 (Pharmacia), which was used as a reference carrier. To select carriers with growth supporting surfaces, broad screening in petri dish experiments was carried out. Candidates with the highest growth rates were investigated in spinner flask experiments in further detail. Polystyrene carrier with a surface modification like triethylamine, maltamine or N-methylglucosamine were able to support growth as good or better as the reference carrier Cytodex 3. Economies of ingredients and ease in laboratory handling could make amine-modified polystyrenes a competitive alternative to currently commercially available microcarrier types.     

Gallic acid loaded disulfide cross-linked biocompatible polymeric nanogels as controlled release system: synthesis,characterization, and antioxidant activity

In this article, a sustained release formulation of the antioxidant gallic acid (GA) is presented in the form of glutathione responsive disulfide cross-linked poly(ethylene glycol)-based nanogels synthesized via aqueous inverse miniemulsion using atom transfer radical polymerization. The particle size was found to be in the range from 227?±?51.78 to 573.3?±?207.2?nm at three drug loading levels achieved i.e. 6.6, 14.26, and 18.29 wt.% of the nanogels with loading efficiency in the range of 60–70%. The release profile of the GA studied at three drug loading levels suggested a controlled release and the nanogels were capable of scavenging radicals and retained the antioxidant activity. The GA-loaded nanogels were found to be biocompatible on human cervical cancer cell lines (HeLa). DCFH-DA (2,7-dichlorofluorescin diacetate) assay evidenced that the nanogels were capable of scavenging the reactive oxygen species in cellular environment.     

Synthesis and characterization of glycolide,L-lactide,and PDMS-based terpolymers as a support for cell cultures

Poly(lactic acid)/poly(glycolic acid)/poly(dimethylsiloxane) (PLGA/TEGOMER) terpolymers have been synthesized by the ring-opening polymerization of L-lactide and glycolide with α,ω-amine-terminated poly(dimethylsiloxane) prepolymer, using stannous octoate as a catalyst. The resulting terpolymers were characterized by various analytical techniques including size exclusion chromatography, ¹H-nuclear magnetic resonance (¹H-NMR), Fourier transform infrared spectroscopy, and differential scanning calorimetry. The data showed that the terpolymers presented an amorphous structure. The glass transition temperature decreased with increasing TEGOMER unit content. For in vitro degradation studies, porous films were fabricated using a solvent-casting, particulate leaching technique. Degradation of the PLGA/TEGOMER terpolymer was studied in phosphate-buffered saline at pH 7.4 and 37°C. The degradation was followed by intrinsic viscosity, mass loss, and molecular weight measurements, and ¹H-NMR spectroscopy. The mass loss after 55 days was 76% for the PLGA/TEGOMER (71/24/5) sample. Cell growth experiments using Swiss 3T3 fibroblasts demonstrated that PLGA/TEGOMER terpolymer matrices allow the attachment and growth of cells.     

Mosaicism in amniotic fluid cell cultures: Classification and significance

The last decade has witnessed increasing application of human cytogenetic technology to prenatal chromosome analysis. However, unlike the rather uniform peripheral blood T-lymphocyte system which has provided most of our experience in human cytogenetics, long-term amniotic-fluid cell cultures display extreme cellular heterogeneity and disproportionate growth of certain cell types as a consequence of clonal amplification. When they enter cell culture, many of these cells are approching the terminal stages of their respective life spans and may have accumulated chromosomal aberrations. Concern about the possibility of true fetal mosaicism seems warranted chiefly in situations were multiple colonies display potentially viable aberrations. Clonal analysis, preferable of multiple clonal types, and attention to details of clonal morphology are likely to minimize diagnostic errors and undue apprehension resulting from mosaicism in amniotic-fluid cell cultures.     

Primary epithelial cell cultures derived from canine prostate: Isolation,culture, and characterization

Epithelial-cell-enriched primary cultures were established from canine prostate. Minced tissue was dissociated with 750 units/ml of collagenase in F12K tissue culture medium containing 1% fetal bovine serum. This treatment resulted in the gradual removal of stromal elements from the base of the epithelial cells. After 30 minutes of digestion, aggregates of epithelial cells free of stroma were dislodged from the minced pieces of prostate. These aggregates were washed and plated at high density in F12K plus 10% fetal bovine serum. After 12–16 hours in vitro the unattached cellular aggregates were removed from the culture dishes, washed, and reinoculated into new culture vessels containing fresh medium. After 48 hours in vitro, the aggregates had attached to the culture vessels and spread out to yield discrete patches of epithelial cells. By 120 hours in vitro the patches of cells had grown and coalesced to form a confluent monolayer of epithelial cells. Ultrastructural examination of these cultures indicated that adjacent cells were joined by desmosomes and tight junctions and had tonofilaments and microvilli, giving the cells an epithelial appearance. The cells contained rough endoplasmic reticulum, Golgi apparatus, and secretory granules similar to those of the epithelial cells in the intact organ. In addition, intracellular “blebs” containing acid phosphatase were observed in the monolayers and were found to increase in size and number with time in vitro. Differentiated function of the cultures was demonstrated by the presence of ornithine decarboxylase and acid phosphatase and the ability of the cultures to metabolize testosterone to primarily 5α-reduced metabolites.     

Identification and characterization of non-dividing cell populations in phase II cultures of human glial cells

It has been reported that cultures of normal cells in phase II contain a non-multiplying cell population, the size of which increases with passage number. In phase II cultures of normal human glial cells we have found two subpopulations of non-proliferating cells, one of which has a characteristic morphology, and differs from the actively dividing cells in a number of respects: (1) they are larger although of various sizes and are well spread over a very large substratum area: (2) they contain a great number of granules showing acid phosphatase activity, being heavy metal positive and displaying the characteristic natural fluorescence of lipofuscin pigment; and (3) they frequently contained a central somewhat irregular nucleus with various numbers of darkly staining nucleolar-like structures. Cytophotometric nuclear DNA measurements of the described "large" cell population show a decreased proportion of diploid cells as compared to their smaller sister cells. Moreover, with increasing passage number, the DNA values for large cells shift towards higher ploidy levels resulting in a scattered aneuploid pattern in the oldest passage. This "large" cell subpopulation consists of between 2% and 3% of all passages and becomes greatly decreased following subcultivation. The other subpopulation of non-dividing cells is generally morphologically similar to the dividers, increases in size with passage number and is the more important in the phase III phenomenon.     

Mycoplasma infection of cell cultures: Isolation and detection

Summary Mycoplasma contamination of cell cultures was first reported in 1956 and continues today to be a major concern to the investigator. Although awareness has increased over the years, mycoplasma infection of cell cultures remains a common, troublesome problem that affects many cell functions and can markedly influence proper interpretation of test results. Up-dated, sensitive mycoplasma test procedures are described. These include isolation of mycoplasmas by agar and large volume broth media procedures, and detection in an indicator cell culture system with a DNA-binding fluorochrome stain. Although prevention is the first line of defense, occasionally the considerable effort required to eliminate the contaminant from an irreplaceable cell line can be justified.     

A densitometer for the evaluation of cell growth in primary cultures: Construction and operation

Summary This report describes the design, construction and operation of a photovoltic cell densitometer. The densitometer has proven to be very useful in evaluating the effect of various culture medium supplements on the growth of primary cultures.     

Development and characterization of primary cell cultures from the hematopoietic tissues of the Dublin Bay prawn, Nephrops norvegicus

Improved maintenance in vitro of the hematopoietic tissue of the Dublin Bay prawn Nephrops norvegicus (L.) resulted by using 10% (v/v) 2× Leibovitz's medium prepared in seawater (salinity = 25), and supplemented with 10% (v/v) heat inactivated fetal bovine serum plus 5% (v/v) Nephrops serum or 5% (v/v) Nephrops muscle extract, and 0.06 g/l of L-proline and 1 g/l of glucose. Pronase at 100 g/ml improved tissue dissociation and subsequent spreading of hematopoietic cell cultures. The addition of epithelial growth factor (EGF), based fibroblast growth factor (bFGF) or insulin growth factor 1 (IGF-I) did not enhance cell growth. Cell culture contained several types of maturing hemocytes, in the size range of 6–24 µm diameter. Acid phosphatase, -naphthyl butyrate esterase, -naphthyl acetate esterase, naphthyl AS-D chloroacetate esterase activity and phenoloxidase activity was demonstrated, but not so alkaline phosphatase or peroxidase. Small PAS (= Periodic acid Schiff) positive granules, unsaturated lipids and phospholipids were observed. Cultures remained functional for over two weeks. Mitosis was noticed occasionally; however, cell proliferation was not recorded by use of nuclear proliferation markers.     

Solvent-free atom transfer radical polymerization for the preparation of poly(poly(ethyleneglycol) monomethacrylate)-grafted Fe3O4 nanoparticles: synthesis, characterization and cellular uptake

Poly(poly(ethyleneglycol) monomethacrylate) (P(PEGMA))-grafted magnetic nanoparticles (MNPs) were successfully prepared via a solvent-free atom transfer radical polymerization (ATRP) method. The macroinitiators were immobilized on the surface of 6.4±0.8 nm Fe₃O₄ nanoparticles via effective ligand exchange of oleic acid with 3-chloropropionic acid (CPA), which rendered the nanoparticles soluble in the PEGMA monomer. The so-obtained P(PEGMA)-grafted MNPs have a uniform hydrodynamic particle size of 36.0±1.2 nm. The successful grafting of P(PEGMA) on the MNP surface was ascertained from FTIR and XPS analyses. The uptake of the MNPs by macrophage cells is reduced by two-orders of magnitude to <2 pg Fe/cell after surface grafting with P(PEGMA). Furthermore, the morphology and viability of the macrophage cells cultured in a medium containing 0.2 mg/mL of P(PEGMA)-grafted MNPs were found similar to those of cells cultured without nanoparticles, indicating an absence of significant cytotoxicity effects. T₂-weighted magnetic resonance imaging (MRI) of P(PEGMA)-grafted MNPs showed that the magnetic resonance signal is enhanced significantly with increasing nanoparticle concentration in water. The R₁ and R₂ values per millimole Fe, and R₂/R₁ value of the P(PEGMA)-grafted MNPs were calculated to be 8.8 mm⁻¹ s⁻¹, 140 mm⁻¹ s⁻¹, and 16, respectively. These results indicate that the P(PEGMA)-grafted MNPs have great potential for application in MRI of specific biotargets.     

An efficient and simple method of non-enzymatic detachment of monolayer cultures into single cell suspension

Summary Activating the Na⁺/H⁺ antiporter causes a distinctive and rapid endocytosis which internalizes the plasma membrane leading to cell retraction and detachment as rounded cells with much reduced surface area. Antiport activation is by a combination of two individually effective motivations, viz. a) steep [Na⁺] and [H⁺] gradients across the plasma membrane and b) allosteric activation by second messengers initiated with simple inorganic sulphate. The dissociated cells are as viable as those released by trypsinization. Thus it provides an effective enzyme-free alternative to trypsin digestion in cell dissociation.     

Silicone derivatives for contact lenses: Functionalization,chemical characterization,and cell compatibility assessment

Epoxy ring-opening functionalization of polymers at random sites along chains with various chemical groups has been demonstrated. The reaction is performed in an aqueous solution under mild conditions in order to minimize degradation of the macromolecular chains. Silicone lenses made of copolymers with epoxy side chains were functionalized with 4-hydroxybutyric acid, sodium salt. The carboxylated silicone derivatives were characterized by ESCA and radiotracers. A mean value of 30% reaction yield was concluded, based upon data from both methods; nevertheless, the latter can be improved up to 50% or more if the conditions of preparation of the epoxydized silicone lenses are optimized. Derivatized silicones were coated in the wells of culture plates to evaluate the cell compatibility of these new polymers with a fibroblast cell line (McCoy's). No cellular toxicity was observed.     

Cytokines, thyroid autoantibody synthesis and thyroid cell survival in culture

In autoimmune thyroid disease lymphoid cells infiltrating the thyroid gland occur in conspicuous aggregates or as a diffusely distributed population invading the thyroid follicles. Consequently cytokines secreted by activated T cells or macrophages could influence neighbouring thyroid cells as well as other lymphocytes. We have investigated this possibility using recombinant cytokines. Thyroid cell survival was assessed in terms of mitochondrial dehydrogenase activity in monolayers exposed to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1 (IL-1 alpha and beta) and interleukin-2 (IL-2) in the presence or absence of thyroid-stimulating hormone (TSH). Neither TNF-alpha nor IL-2 affected thyroid cell survival, IFN-gamma was usually inhibitory and IL-1 alpha slightly enhanced cell survival in some experiments. However, the effects were small and variable and were not enhanced by potentially synergistic combinations of cytokines, longer periods of exposure, or different culture conditions. In contrast, IFN-gamma, IL-2 and TNF-alpha inhibited the ability of thyroid lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis to synthesize autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg). Comparison of lymphoid populations isolated by digestion and/or mechanical disaggregation indicated that a population of activated B cells, plasma cells and T cells, intimately associated with thyroid cells since they could only be extracted by digestion, was influenced by cytokines. Our studies suggest that in addition to its well-recognized ability to induce MHC class II antigens on thyroid cells, IFN-gamma may inhibit thyroid cell proliferation and TNF-alpha, IFN-gamma and IL-2 may down-regulate thyroid autoantibody synthesis.     

Use of thermosensitive polymer material on the basis of N-isopropylacrylamide and N-tert-butylacrylamide copolymer in cell technologies

We developed thermosensitive polymer substrates on the basis of N-isopropylacrylamide and N-tert-butylacrylamide co-polymer and studied their interaction with cultured substrate-dependent mammalian cells. It was shown that these polymers promote cell adhesion and proliferation at a level comparable to polystyrene treated for cell culturing and provide effective cell detachment after lowering culturing temperature below a critical level determined by phase transition temperature in aqueous solutions of polymers. A dependence of phase transition temperature on the ratio between N-isopropylacrylamide and N-tert-butylacrylamide was demonstrated. Differences in the dynamics of cell detachment from the surface of polymer substrates with various proportions between the components were shown. __________ Translated from Kletochnye Tekhnologii v Biologii i Medicine, No. 4, pp. 231–234, December, 2006