Polyacrylamide Microbeads Research Articles

Fabricating biocompatible polyacrylamide microbeads for cell-generated mechanical force quantification via photoinitiated polymerization

Mechanical forces have been known to influence or even determine embryonic development, cancer progression or even stem cell differentiation for more than a decade now. However, the specific physical and molecular mechanisms by which these forces, exerted on surface-adhesion receptors, are able to trigger gene expression and affect cellular function is far from being completely understood. Several studies have been published in the past few years to try and come up with a diverse set of strategies to quantify isotropic and anisotropic mechanical forces in living tissues.

Cell-like pressure sensors reveal increase of mechanical stress towards the core of multicellular spheroids under compression

The surrounding microenvironment limits tumour expansion, imposing a compressive stress on the tumour, but little is known how pressure propagates inside the tumour. Here we present non-destructive cell-like microsensors to locally quantify mechanical stress distribution in three-dimensional tissue. Our sensors are polyacrylamide microbeads of well-defined elasticity, size and surface coating to enable internalization within the cellular environment. By isotropically compressing multicellular spheroids (MCS), which are spherical aggregates of cells mimicking a tumour, we show that the pressure is transmitted in a non-trivial manner inside the MCS, with a pressure rise towards the core. This observed pressure profile is explained by the anisotropic arrangement of cells and our results suggest that such anisotropy alone is sufficient to explain the pressure rise inside MCS composed of a single cell type. Furthermore, such pressure distribution suggests a direct link between increased mechanical stress and previously observed lack of proliferation within the spheroids core.

Fabricating polyacrylamide microbeads by inverse emulsification to mimic the size and elasticity of living cells.

Inverse emulsification was used to fabricate polyacrylamide (PAAm) microbeads with size and elastic properties similar to typical, mammalian cells. These biomimicking microbeads could be fluorescently stained and functionalized with a collagen type-I coating, post-polymerization, for tracking bead locations and promoting cell recognition/binding, respectively. By occupying a previously unfilled range of sizes and mechanical properties, these microbeads may find unique use in both biomedical and materials applications.

Mice selected for acute inflammatory response display a higher lung inflammation induced by Tityus serrulatus scorpion venom

Mice selected for acute inflammatory response display a higher lung inflammation induced by Tityus serrulatus scorpion venom

Analysis of cytokine production by inflammatory mouse macrophages at the single-cell level: selective impairment of IL-12 induction in Leishmania-infected cells.

Intracellular staining for cytokines and parasites, combined with two-color flow cytometric analyses, were used to examine the frequencies of IL-12-, TNF-alpha- and IL-6-producing macrophages in response to Leishmania major infection and/or activation with IFN-gamma/lipopolysaccharide (LPS). Inflammatory macrophages were obtained from nonimmune granulomas, initiated by the injection of polyacrylamide microbeads (Bio-gel P-100) into subcutaneous pouches of different mouse strains. Infection of inflammatory macrophages in vitro using metacyclic promastigotes produced identical effects on cytokine responses regardless of whether cells from genetically resistant or susceptible mouse strains were used: IL-12 was not produced in response to infection itself, virtually every infected cell lost its ability to produce IL-12 in response to IFN-gamma/LPS, and the IL-6 response was partially inhibited, while the TNF-alpha response of infected cells was unimpaired. Low-multiplicity infection of inflammatory macrophages in vivo using either metacyclic promastigotes or tissue amastigotes also resulted in the complete and selective inhibition of IL-12 responses in infected cells. These data establish the physiologic relevance of prior observations regarding the selective impairment of IL-12 induction pathways in infected macrophages, and suggest a mechanisms for the delayed onset of cell-mediated control mechanisms that is typical of even self-limiting forms of leishmanial disease.

Effect of genetic modification of acute inflammatory responsiveness on tumorigenesis in the mouse.

Two distinct bidirectional selective breedings for quantitative traits were initiated from identical genetically heterogeneous mouse populations. The resulting lines are characterized by maximal or minimal acute inflammatory responsiveness (AIR): AIRmax and AIRmin lines, respectively, and by resistance or susceptibility to chemical skin tumorigenesis: Car-R and Car-S lines, respectively. The AIR response to s.c. injection of polyacrylamide microbeads, measured by cell content in the local exudate, was 10 times higher in AIRmax than in AIRmin mice. The response to selection was asymmetrical: the realized heritability was 0.26 in AIRmax and 0.008 in AIRmin, and resulted from the additive effect of 7-11 quantitative trait loci (QTL). Low responsiveness was globally dominant in F1 and 48% of F2 segregant variance was found to be due to genetic factors. These findings are the first demonstration of innate regulation of AIR by germ line genes. Susceptibility to skin tumorigenesis induced by a two-stage initiation (DMBA)-promotion (TPA) protocol was lower in AIRmax mice than in AIRmin mice, a 6-fold difference in tumor induction rate. Intense AIR was found to be associated with resistance, and low AIR with susceptibility to tumorigenesis, in F2 segregants chosen for extreme AIR phenotypes. At least some of the AIR QTLs therefore contain genes controlling tumorigenesis. Tumor phenotypes differed more in Car-R and Car-S than in AIRmax and AIRmin lines, indicating that QTLs unrelated to AIR, contribute to the host response to tumorigenesis. The extreme phenotypes/genotypes of the four selected lines and the known genetic constitution of their foundation population, offer new possibilities to discriminate the genes/mechanisms controlling two important traits: AIR and response to chemical tumorigenesis. Collaborative projects will be favorably considered. The description of tumor resistance genes in AIRmax and Car-R mice may be helpful for epidemiology and therapy of human cancer.

Genetics of nonspecific immunity: I. Bidirectional selective breeding of lines of mice endowed with maximal or minimal inflammatory responsiveness.

The genetic regulation of acute inflammatory reaction (AIR) was studied by the method of bidirectional selective breeding, used to produce a line of mice giving the maximal and a line of mice giving the minimal inflammatory reaction (AIR max and AIR min, respectively). The AIR was triggered by subcutaneous injection of a neutral substrate (suspension of polyacrylamide microbeads), and measured by the leukocyte and serum protein accumulation in the exudate. The two parameters are positively correlated and present a normal frequency distribution. The highly genetically heterogeneous foundation population was produced by the equipoised intercrossing of eight inbred strains of mice, and selective breeding carried out by assortative matings of extreme phenotypes. The response to selection in 11 consecutive generations was highly asymmetrical: a marked AIR increase in the AIR max and no change in the AIR min line occurred. The mean value of realized heritability in the AIR max line was 0.26 and 0.18 for cell and protein concentrations, respectively. The response to selection must have resulted from the interaction of seven to nine independent gene loci endowed with additive effects. The lack of response to selection of the AIR min line is discussed. The large inter-line difference opens new possibilities for studying the biochemistry and molecular genetics of inflammation, and also for investigating the beneficial or detrimental effect of inflammatory responses.

Modification of host cell membrane lipid composition by the intra-erythrocytic human malaria parasite Plasmodium falciparum

The phospholipid and fatty acid compositions of the host infected erythrocyte plasma membrane (IEPM) have been determined for erythrocytes infected with the human malaria parasite Plasmodium falciparum. IEPM were prepared by selective lysis of the host erythrocyte (but not of the parasite membranes) with 0.1% saponin, followed by differential centrifugation. The purity of the IEPM was determined by measuring the membrane-specific enzyme markers acetylcholinesterase, glutamate dehydrogenase and lactate dehydrogenase, and by immunoelectron microscopy using monoclonal antibodies specific for human erythrocyte glycophorin A (4E7) and for a 195 kDa parasite membrane glycoprotein (Pf6 3B10.1). Both approaches demonstrated that the host erythrocyte plasma membrane preparation was free from contamination by parasite membranes. During intra-erythrocytic development of the parasite, the phospholipid composition of the erythrocyte membrane was strikingly altered. IEPM contained more phosphatidylcholine (38.7% versus 31.7%) and phosphatidylinositol (2.1% versus 0.8%) and less sphingomyelin (14.6% versus 28.0%) than normal uninfected erythrocytes. Similar alterations in phospholipid composition were determined for erythrocyte membranes of parasitized cells isolated by an alternative method utilizing polycationic polyacrylamide microbeads (Affigel 731). The total fatty acid compositions of the major phospholipids in IEPM were determined by g.l.c. The percentage of polyunsaturated fatty acids in normal erythrocyte phospholipids (39.4%) was much higher than in phospholipids from purified parasites (23.3%) or IEPM (24.0%). The unsaturation index of phospholipids in IEPM was considerably lower than in uninfected erythrocytes (107.5 versus 161.0) and was very similar to that in purified parasites (107.5 versus 98.5). Large increases in palmitic acid (C16:0) (from 21.88% to 31.21%) and in oleic acid (C18:1) (from 14.64% to 24.60%), and major decreases in arachidonic acid (C20:4) (from 17.36% to 7.85%) and in docosahexaenoic acid (C22:6) (from 4.34% to 1.8%) occurred as a result of infection. The fatty acid profiles of individual phospholipid classes from IEPM resembled in many instances the fatty acid profiles of parasite phospholipids rather than those of uninfected erythrocytes. Analysis of IEPM from P. falciparum-infected erythrocytes (trophozoite stage) revealed that, during intra-erythrocytic maturation of the parasite, the host erythrocyte phospholipid composition was markedly refashioned. These alterations were not dependent on the method used to isolate the IEPM, with similar results obtained using either a saponin-lysis method or binding to Affigel beads. Since mature erythrocytes have negligible lipid synthesis and metabolism, these alterations must occur as a result of parasite-directed metabolism of erythrocyte lipids and/or trafficking of lipids between the parasite and erythrocyte membranes.

Inflammation and anti‐tumor resistance. V. Production of a cytostatic factor following cooperation of elicited polymorphonuclear leukocytes and macrophages

When Lewis tumor cells (3LL) included in a gel of polyacrylamide microbeads are injected into mice and recovered 1 day later, incorporation of 125I-UdR is strongly reduced. In contrast, no reduction is observed in irradiated mice. The cytostatic effect is non-existent in 6 hr-old granuloma (granulocytes 90%, macrophages 10%), but is maximal in 48 hr-old granuloma (granulocytes 50%, macrophages 50%). When 6 hr-old granuloma cells (which are not cytostatic) are incubated with bone-marrow-derived macrophages (BMs) (which are slightly cytostatic), considerable cytostasis against 3LL is observed, such an effect being strongly reduced if the 2 cell populations are separated. This suggests that close contact of live polymorphs with macrophages is necessary for full expression of cytostasis. No increase in cytostasis of bone-marrow macrophages is observed when BMs are incubated with a cell-free extract of freeze-thawed 6-hr-old granuloma cells. Cell contact is not necessary between granuloma cells and 3LL since the cytostatic activity is found in the supernatant of incubated phagocytic cells. Cytostasis is also observed with B16, P815, J774 cells and normal mouse bone-marrow cells. An active cytostatic fraction has been purified from the supernatant of 48-hr-old granuloma cells following molecular filtration and HPLC. The cytostatic factor has a low molecular weight (400-500 daltons), is not a peptide and presents maximum absorption at 267 nm.

Chemiluminescence monitoring of phagocyte oxidative metabolism in mice bearing polyacrylamide induced granulomas

A technical protocol was recently described by Fauve et al. (J. Immunol. Methods 1983, 64, 345) for inducing subcutaneous granuloma with polyacrylamide microbeads. The present study using this technique demonstrates that the capacity of host phagocytes to generate reactive oxygen species can be easily monitored by chemiluminescence, both locally in granuloma infiltrating cells and sites remote from the inflammatory reactions, i.e., within microamounts of whole blood and in spleen cells. We observed that both resting and stimulated (zymosan or phorbol-myristate acetate) production by C57BL/6 mouse phagocytes are significantly higher in granulomas induced with high porosity polyacrylamide beads (P300) than in those induced with beads of low polyacrylamide porosity (P4). Since this selective modulation of phagocyte oxidative metabolism is also detectable within microamounts of whole blood and in spleen cells, it could serve as a model for investigating the role od reactive oxygen species in the inflammatory reaction.

A solid-phase radioimmunoassay of serum dehydroepiandrosterone sulphate using a monoclonal antibody

A monoclonal antibody directed against dehydroepiandrosterone, but with high affinity for dehydroepiandrosterone sulphate (DHA-S), has been used to develop a solid phase radioimmunoassay for measuring serum DHA-S. The antibody was eovalently linked to polyacrylamide microbeads with no change in binding characteristics. The procedure requires only the chromatography of serum on anion-exchange cellulose before assaying the equivalent of 0.25 μl serum. The method is precise, accurate and specific and can detect 19.5pg of DHA-S. Serum DHA-S levels measured by this method were in good agreement with those found in a validated radioimmunoassay method involving hydrolysis. The method is quick and one operator could assay 50 blood specimens per day. DHA-S levels in serum from 50 men and 86 women were in agreement with those in the literature. With the availability of theoretically limitless quantities of consistently high quality monoclonal antibodies the advantages of developing solid phase radioimmunoassays for steroids is discussed.

Inflammation et résistance antibactérienne: III.—Influence d'une réaction inflammatoire induite par l'injection de gels de polyacrylamide sur la résistance des souris à l'infection par Listeria monocytogenes

Summary Inflammation was induced in the dorsal area of pathogen-free mice following subcutaneous injection of polyacrylamide microbeads (Biogel) of varying particle size (200–400 mesh) and pore size (exclusion limit ranging from a molecular weight of 2,000 to 300,000). Six days after injection of the microbeads, mice were infected with 105 Listeria monocytogenes. Only animals injected with “Biogel P2, P4, P6” (exclusion limit 2,000, 4,000 and 6,000 respectively) survived this lethal inoculum of Listeria. A study of the kinetics of this increased resistance revealed that homogenates of granulomas, induced eight days before injection into mice, significantly decreased the number of listeria in the spleen of treated mice. Such increased resistance was not found in cell homogenates, but only in the exudate of 8-day old granulomas. Fractionation studies of the inflammatory exudates with ammonium sulfate showed that immunostimulating substance were present in the supernatant following precipitation with 80% saturated (NH4)2SO4.

Polyacrylamide microbeads, a sustained release drug delivery system

Using tetracycline-HCl and theophylline as model drugs, polyacrylamide microbeads were prepared by w/o emulsion polymerization technique. Furthermore, drug-loaded polymer microbeads were prepared in the presence of different concentrations of gelatin, which were further cross-linked for different time intervals. The prepared microbeads were characterized through particle size analysis, electron microscopy, and in vitro dissolution. Relatively spherical free-flowing populations of microbeads were obtained. The presence of gelatin during the polymerization processes led to larger particles proportional to its concentration in the aqueous phase. The dried microbeads showed smooth pored or fissured surfaces. In aqueous media, they attained equilibrium hydration within 10 min, with 73.5% v/v water uptake forming spongy spheres. The products showed slower dissolution rates with higher gelatin concentrations and extended cross-linking.

Polyacrylamide microbeads with entrapped protein A from S. aureus for cell separation.

A technique was developed for the identification and separation of cells according to their surface antigenic markers, based on immobilization of protein A by entrapment in polyacrylamide microbeads. This method is further development of the previous techniques with SpA-containing S. aureus bacteria, but has the advantage of having less unspecific binding to the cells. We show that this technique might be a useful tool for the separation of cells, exemplified by separation of cells on Petri dishes coated with a monolayer of SpA-microbeads. The viability of separated cells was unaffected and adherent cells were easily removed by trypsin.

Immobilization of proteins by entrapment in polyacrylamide microbeads

The immobilization of protein A and IgG by entrapment in polyacrylamide microbeads with a diameter of 1 μm was studied with the aim of optimizing the yield of immobilized protein. The microbeads were prepared by a modified emulsion polymerization technique. The small size of the beads resulted in an increase in immobilized protein available for interaction at the bead surface when compared with beads of larger diameter (100 μm). The increase was approximately proportional to the increase of the total bead surface. The increase in total bead surface was, however, accompanied by a slight decrease in the degree of immobilization. Optimal immobilization of protein A and IgG was obtained at monomer compositions of T-C 10-5 and 20-5, respectively (for definitions of T and C, see below). The fraction of immobilized protein available for interaction at the bead surface was almost constant over a wide range of protein concentrations tested. The yield of immobilized protein is generally rather low. The immobilization of IgG could be increased, by the presence of urea or by increasing the apparent molecular weight of IgG by addition of soluble protein A, thus forming soluble IgG protein A complexes. A large fraction of the non-immobilized protein (20–90%) could be recovered from the washings by affinity chromatography. The entrapment seemed to be more related to the molecular weight than to the hydrodynamic properties of the protein. The density of the microbeads was determined in Percoll and was found to be dependent on the total monomer concentration (T). At high concentrations of cross-linker (C) the apparent density was increased, owing to diffusion of the density medium into the large pores of the gel.