Electrophoretic cell separation by means of microspheres.

The electrophoretic mobility of fixed human erythrocytes immunologically labeled with poly(vinylpyridine) or poly(glutaraldehyde) microspheres was reduced by approximately 40%. This observation was utilized in preparative scale electrophoretic separations of fixed human and turkey erythrocytes, the mobilities of which under normal physiological conditions do not differ sufficiently to allow their separation by continuous flow electrophoresis. We suggest that resolution in the electrophoretic separation of cell subpopulations, currently limited by finite and often overlapping mobility distributions, may be significantly enhanced by immunospecific labeling of target populations using microspheres.     

PEGylation of microspheres generates a heterogeneous population of particles with differential surface characteristics and biological performance

Surface PEGylation of polystyrene microspheres with methoxy-poly(ethylene glycol)-5000 (mPEG-5000) generated a heterogeneous population of entities that differed in surface characteristics and in vitro biological performance (phagocytosis and complement activation). Surface heterogeneity was determined by hydrophobic interaction chromatography, measurements of particle electrophoretic mobility in a defined field and adlayer thickness of the projected mPEG chains. The particle population separation by hydrophobic interaction chromatography demonstrated a remarkable linear relationship between the particle zeta potential and phagocytosis by J774 A1 macrophage-like cells. Microsphere populations bearing a predominant surface of mPEG molecules as high-density mushroom-brush intermediate and/or brush configuration were most resistant to phagocytosis and activated the human complement system poorly. Conversely, those populations with predominant surface mPEGs in a mushroom regime were potent activators of the complement system and were prone to phagocytosis. Therefore, surface heterogeneity explains why a fraction of intravenously injected 'long-circulating' nanoparticles is cleared rapidly by macrophages of the reticuloendothelial system. Hydrophobic interaction chromatography can readily assess the extent of surface heterogeneity of PEGylated particulate drug delivery systems and pre-select particles with optimal retention times in the blood. These observations may also be relevant with respect to successful surface camouflaging of cells, drug depots and implantable devices.     

Macroporous gel particles as robust macroporous matrices for cell immobilization

A new design of robust matrices for cell immobilization is described. Macroporous gels (MGs) with immobilized microbial cells were prepared at subzero temperatures and were formed inside a plastic core (so-called, protective housing). Due to the protective housing the macroporous gel particles with immobilized cells can be used in well-stirred bioreactors. High retained activity of yeast (77-92%) and Escherichia coli (50-91%) cells immobilized in MGs after drying and storage in the dried state was due to the high structural stability and heterogeneous porous structure of the MGs.     

Aging of erythrocytes results in altered red cell surface properties in the rat,but not in the human Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have now found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, counter-current distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter.These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

On the kinetics of phase separation in aqueous two-phase systems

The effect of the tie-line location (phase volume ratio) on the kinetics of phase separation in batch PEG/salt aqueous two-phase systems (ATPS) has been investigated. PEG/sulphate systems with a stability ratio (sr) of 0.34 and 0.37 and relative tie-line lengths in the range 0.1 to 0.6 for a continuous top phase and in the range 0.03 to 0.15 for a continuous bottom phase were used in the batch studies. A continuous settler was designed with three different inlet geometries. Phase separation is much faster when the bottom phase is continuous and in this case the location on the tie-line and the presence or absence of Bacillus subtilis extract makes little difference. When the top phase is continuous the relative sizes of the phases (phase ratio, R, relative distance on tie-line, rd) has an important effect, the larger the top phase (larger R and rd) the slower the phase separation. The presence of Bacillus extract also makes the operation slower which is more marked at the largest values of R (and rd). At the largest volume ratios (R or rd) three different settling regions have been recognised, a region of coalescence, a region of drops moving to the interphase and a region where drops queue at the interphase to coalesce into the large phase. A modified correlation that takes into account the location on the tie-line and thus volume ratio (R) and relative distance (rd) has been proposed and successfully tested. The behavior of batch and continuous systems in the presence and absence of Bacillus subtilis extract in systems with continuous bottom phase was also studied. The settling velocity was lower in the continuous than in the batch systems, and in both cases the initial rate was lower in the presence of Bacillus extract.     

Generation of selective microenvironment at the cell periphery through bulk-phase hydrophilic polymers

In order to understand the previously demonstrated effect of poly(ethylene glycol) on the stimulation of lymphocyte responses to syngeneic tumor cells (Ben-Sasson, S.A. and Henkart, P.A. (1977) J. Immunol. 119, 227–231), we have investigated the effects of addition of poly(ethylene glycol) to the medium in a number of cellular systems. The binding of trimeric IgG to tumor-lymphocyte Fc receptors was greatly enhanced by poly(ethylene glycol); a substantial increase in binding of trimeric IgG to non-Fc-receptor-bearing tumor cells was also observed. Similarly, the binding of labeled bovine serum albumin to lymphocyte surfaces was increased by poly(ethylene glycol), implying that nonspecific binding of proteins to cells was generally enhanced. The dose-response curve of concanavalin A mitogenesis was shifted to the right, as would be expected from a local increase in concanavalin A concentration. Antibody binding to erythrocytes as detected by complement lysis was similarly increased. It was found that in aqueous two-phase mixtures created by poly(ethylene glycol) and dextran, erythrocytes partition into the dextran phase through exclusion into dextran-rich microdroplets. It is proposed that addition of poly(ethylene glycol) to cell culture media creates a similar separate phase around the cell surface in which the local concentration of proteins is greater than that in the bulk medium. This concept explains many of the diverse effects of addition of poly(ethylene glycol) to the medium. It also can partially explain the requirement for serum to observe the poly(ethylene glycol) effect on the lymphocyte response to syngeneic tumor cells.     

High gradient magnetic separation of rosette-forming cells

High Gradient Magnetic Separation (HGMS) is a rapid and straightforward technique that has previously been proven effective in extracting erythrocytes from a flowing cell suspension if the red cell hemoglobin is in a paramagnetic state. In this work it was applied to the enrichment of the small population (<2%) of splenocytes from an immune mouse that bound sheep red cells to form rosettes. Samples flowed through the HGMS column in a strong magnetic field where rosettes and free sheep cells were selectively retained. These were subsequently eluted by simply removing the magnetic field. The process required 20–30 min per mouse spleen. Rosettes in the initial sample and in the fractions that passed through, or were retained by, the column were enumerated under the microscope. Under the conditions used here, the retained and eluted cells typically showed a 20–50-fold increase in the frequency of rosetted cells, and the cells that passed through the magnet showed 90–100% depletion of rosettes. The recovery of intact rosettes and the overall cell recovery were generally both in the range of 80–90%.     

Blood progenitor cell separation from clinical leukapheresis product by magnetic nanoparticle binding and magnetophoresis

Positive selection of CD34+ blood progenitor cells from circulation has been reported to improve patient recovery in applications of autologous transplantation. Current magnetic separation methods rely on cell capture and release on solid supports rather than sorting from flowing suspensions, which limits the range of therapeutic applications and the process scale up. We tested CD34+ cell immunomagnetic labeling and isolation from fresh leukocyte fraction of peripheral blood (leukapheresis) using the continuous quadrupole magnetic flow sorter (QMS), consisting of a flow channel (SHOT, Greenville, IN) and a quadrupole magnet with a maximum field intensity (B(o)) of 1.42 T and a mean force field strength (S(m)) of 1.45 x 10(8) TA/m(2). Both the sample magnetophoretic mobility (m) and the inlet and outlet flow patterns highly affect the QMS performance. Seven commercial progenitor cell labeling reagent combinations were quantitatively evaluated by measuring magnetophoretic mobility of a high CD34 expression cell line, KG-1a, using the cell tracking velocimeter (CTV). The CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) showed the strongest labeling of KG-1a cells and was selected for progenitor cell enrichment from 11 fresh and 11 cryopreserved clinical leukapheresis samples derived from different donors. The CD34+ cells were isolated with a purity of 60-96%, a recovery of 18-60%, an enrichment rate of 12-169, and a throughput of (1.7-9.3) x 10(4) cells/s. The results also showed a highly regular dependence of the QMS performance on the flow conditions that agreed with the theoretical predictions based on the CD34+ cell magnetophoretic mobility.     

Development of 3D hydrogel culture systems with on-demand cell separation

Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3D co-culture methods lack the ability to effectively separate two cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3D hydrogel co-culture system that allows us to culture different cell types for up to 7 days and subsequently separate and isolate the different cell populations using enzyme-sensitive glues. Separable 3D co-culture laminates were prepared by laminating PEG-based hydrogels with enzyme-degradable hydrogel adhesives. Encapsulated cell populations exhibited good segregation with well-defined interfaces. Furthermore, constructs can be separated on-demand upon addition of the appropriate enzyme, while cell viability remains high throughout the culture period, even after laminate separation. This platform offers great potential for a variety of basic cell signaling studies as the incorporation of an enzyme-sensitive adhesive interface allows the on-demand separation of individual cell populations for immediate analysis or further culture to examine persistence of co-culture effects and paracrine signaling on cell populations. See accompanying commentary by Danielle R. Bogdanowicz and Helen H. Lu DOI: 10.1002/biot.201300054     

Poly(ADP-ribose) signaling in cell death

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.     

Synthesis and characterization of multifunctional poly(glycidyl methacrylate) microspheres and their use in cell separation

Multifunctional poly(glycidyl methacrylate) (PGMA) microspheres containing magnetic, fluorescent, and cancer cell-specific moieties were prepared in four steps: (i) preparation of parent PGMA microspheres by dispersion polymerization and their reaction with ethylenediamine to obtain amino groups, (ii) precipitation of iron ions (Fe²⁺ and Fe³⁺) to form Fe₃O₄ nanoparticles within the microspheres, (iii) consecutive reactions of folic acid with the amino groups on PGMA, and (iv) incorporation of fluorescein isothiocyanate into the microspheres. The microspheres were superparamagnetic, highly monodispersive, intensively fluorescent, and capable of recognizing and binding cancer cells that overexpress folic acid receptors. It was demonstrated that with these microspheres, HeLa cells could be captured from their suspension and easily moved in the direction of the externally applied magnetic field.     

Dye-incorporated poly(EGDMA-HEMA) microspheres as specific sorbents for aluminum removal

Aluminum [Al(III)] adsorption onto dye-incorporated poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) [poly(EGDMA-HEMA)] microspheres was investigated. Poly(EGDMA-HEMA) microspheres, in the size range of 150–200 μm, were produced by a modified suspension polymerization of EGDMA and HEMA. The reactive dyes (i.e., Congo Red, Cibacron Blue F3GA and Alkali Blue 6B) were covalently incorporated to the microspheres. The maximum dye load was 14.5 μmol Congo Red/g, 16.5 μmol Cibacron Blue F3GA/g and 23.7 μmol Alkali Blue 6B/g polymer. The maximum Al(III) adsorption on the dye microspheres from aqueous solutions containing different amounts of Al(III) ions were 27.9 mg/g, 17.3 mg/g and 12.2 mg/g polymer for the Congo Red, Cibacron Blue F3GA and Alkali Blue 6B, respectively. The maximum Al(III) adsorption was observed at pH 7.0 in all cases. Non-specific Al(III) adsorption was about 0.84 mg/g polymer under the same conditions. High desorption ratios (95%) were achieved in all cases by using 0.1 M HNO₃. It was possible to reuse these dye-incorporated poly(EGDMA-HEMA) microspheres without significant losses in the Al(III) adsorption capacities.     

Electrophoretic separation and characterization of pea protoplasts

A carrier-free electrophoresis system was developed to separate protoplasts according to their zeta-potential and to study their behaviour in subsequent cell culture. Apex protoplasts from young cotyledon-free pea embryos ( Pisum sativum L. cv. Belman), including primordia, were analysed with respect to their electrophoretic mobility, viability, cell size, cell division capacity, chlorophyll content, and expression of a 50 kDa protein involved in early somatic embryogenesis. Protoplasts fractionated on the basis of their electrophoretic mobility were viable and able to divide and form microcallus in cell culture. Cell culture procedures were modified in order to handle low numbers of protoplasts (50–2000 per fraction). Electrophoretic separation revealed distinct classes of protoplasts, differing in all parameters tested except cell size. Therefore, a connection between zeta potential and other physiological parameters can be proposed.     

Structural analysis of the cell walls regenerated by carrot protoplasts

A procedure was developed to isolate protoplasts rapidly from carrot (Daucus carota L. cv. Danvers) cells in liquid culture. High purity of cell-wall-degrading enzymes and ease of isolation each contributed to maintenance of viability and initiation of regeneration of the cell wall by a great majority of the protoplasts. We used this system to re-evaluate the chemical structure and physical properties of the incipient cell wall. Contrary to other reports, callose, a (1 3)-d-glucan whose synthesis is associated with wounding, was not a component of the incipient wall of carrot protoplasts. Intentional wounding by rapid shaking or treatment with dimethyl sulfoxide initiated synthesis of callose, detected both by Aniline blue and Cellufluor fluorescence of dying cells and by an increase in (1 3)-linked glucan quantified in methylation analyses. Linkage analyses by gas-liquid chromatography of partially methylated alditol-acetate derivatives of polysaccharides of the incipient wall of protoplasts and various fractions of the cell walls of parent cells showed that protoplasts quickly initiated synthesis of the same pectic and hemicellulosic polymers as normal cells, but acid-resistant cellulose was formed slowly. Complete formation of the wall required 3 d in culture, and at least 5 d were required before the wall could withstand turgor. Pectic substances synthesized by protoplasts were less anionic than those of parent cells, and became more highly charged during wall regeneration. We propose that de-esterification of the carboxyl groups of pectin uronic-acid units permits formation of a gel that envelops the protoplast, and the rigid cellulose-hemicellulose frame-work forms along with this gel matrix.Abbreviations DEAE Diethylaminoethyl - DMSO dimethyl sulfoxide - ECP extracellular polymers - EDTA ethylenediaminetetraacetic acid - HGA nomogalacturonan - RG rhamnogalacturonan - Tes N-tris(hydroxymethyl)methyl-2-amino-ethanesufonic acid - TFA trifluoroacetic acid Journal paper No. 11,776 of the Purdue University Agriculture Experiment Station     

50 Years of poly(ADP-ribosyl)ation

The seminal paper published in 1963 by Chambon, Weil and Mandel reporting a new NAD-dependent protein modification now known as poly(ADP-ribosyl)ation (PARylation) marked the launch of a new era in both protein research and cell biology. In the coming decades, the identity, biochemical characteristics and regulation of enzymes responsible for the synthesis and degradation of protein-bound poly(ADP-ribose) have been discovered and the surprisingly multifarious biological roles of PARylation have not ceased to amaze cell and molecular biologists ever since. The review series on PARylation following this preface is comprised of ten papers written by great experts of the field and aims to provide practicing physicians and basic scientists with the state-of-the-art on the “writers, readers and erasers” of poly(ADP-ribose), some recent paradigm shifts of the field and its translational potential.     

Monoclonal antibodies to adhesive cell coat glycoproteins secreted by zoospores of the green alga Enteromorpha

Zoospores of Enteromorpha compressa (L.) Grev. secrete an adhesive cell coat which is involved in their attachment to various substrata. Two monoclonal antibodies (mAbs), designated Ent 1 and Ent 6, were raised against settled zoospores displaying secreted adhesive. Both antibodies labelled specifically the anterior region of the cell containing putative adhesive vesicles. During settlement the antigens recognised by both mAbs were secreted but whereas Ent 6 recognised a fibrillar material released within a few minutes of settlement, Ent 1 recognised components which were associated predominantly with the developing cell wall at later time points. Both mAbs also labelled a Golgi-rich region of settled spores, suggesting that these antigens are also synthesised after settlement. Both mAbs labelled the cell walls of vegetative tissue. Competitive enzyme-linked immunosorbent assay indicated that the two antibodies recognise separate, but overlapping epitopes. In spore settlement assays the Ent 6 immunoglobulin strongly reduced initial adhesion at low concentration whereas the inhibitory effects of Ent 1 occurred at later time points. On analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) both MAbs recognised a major buffer- and SDS-soluble, polydisperse 110-kDa antigen. The 110-kDa component was present in extracts of zoospores and sporulating tissue, but absent, in soluble form, from vegetative tissue. Deglycosylation of zoospore extract with anhydrous HF and peptide N-glycosidase digestion, showed that the major 110-kDa antigen is an N-linked glycan, and that the epitope is borne by the protein component. Time-course experiments showed that the Ent 6 antigen became progressively insoluble after zoospore attachment. Taken together, the data indicate that the two antibodies recognise separate but closely related antigens which have distinctive roles in adhesion and cell wall development. Received: 8 February 1999 / Accepted: 26 July 1999     

Analysis of the cell kinetics during planarian regeneration by means of SAMBA 200 cell image processor

Summary The relationships between cell kinetics and nuclear transformations in regeneration were investigated in the planarianPolycelis nigra by means of image analysis. A SAMBA 200 cell image processor was used to compute densitometric, textural and morphological parameters on Feulgen-stained nuclei in the blastema and near the cut 2–96 h after decapitation. On the basis of these parameters, the phase of the cell cycle (G₁–G₀, S, G₂ and M) was identified and the variations in the percentage of cells in the various phases as well as the blastema cell number were computed against time after decapitation. It was demonstrated that the transection is followed by the sequential wasting of the M, G₂, S and G₁–G₀ compartments. The depletion of a compartment was interpreted as being responsible for the subsequent recovery observed in the next one. The results show that cell proliferation at the section level is not sufficient to account for the increase of the blastema cell number during the first 48 h of regeneration, since the doubling time is about 12 h while the average cycle time is 48 h. It is thus suggested that G₁–G₀ cells migrate toward the section level, at least during the first 2 days of regeneration. Analysis of the nuclear profiles demonstrated that there are two different classes of G₁–G₀ cells: one corresponding to mature cells with a lot of condensed chromatin distributed in clumps within the nucleus, the other to immature cells with chromatin regularly distributed according to a rather homogeneous pattern. About one G₁–G₀ cell out of five is immature at the section level before decapitation while four cells out of five are immature as early as 8 h after the cut. This early inversion of the ratio between mature and immature cells argues in favour of an immigration of immature G₁–G₀ cells to the young blastema, where they are expected to accomplish only one cell cycle, and thus gives rise to mature cells.