Growth of animal cells on microbeads. II. Estimation of biomass in situ and observation of cytopathic effects after infection of McCoy cells with Chlamydia trachomatis

The Channelyzer C256 together with the ZB Coulter Counter have been used to study in situ the growth kinetics of McCoy cells attached on Cytodex 3 microbeads. The number of cells per microbead varied linearly with the average size of the covered microbeads, showing that electronic particle sizing can be used to measure biomass or cell numbers. As the cells became confluent, an increase in cell volume with little accompanying increase in cell number was detected. The technique also enables the experimenter to follow in situ the development of cytopathic effects resulting from the infection by pathogens such as Chlamydia trachomatis serotype L1.     

X-ray Phase Contrast Imaging of Cell Isolation with Super-Paramagnetic Microbeads

Super-paramagnetic microbeads are widely used for cell isolation. Evaluation of the binding affinity of microbeads to cells using optical microscopy has been limited by its small scope. Here, magnetic property of microbeads was first investigated by using synchrotron radiation (SR) in-line x-ray phase contrast imaging (PCI). The cell line mouse LLC (Lewis lung carcinoma) was selected for cell adhesion studies. Targeted microbeads were prepared by attaching anti-VEGFR2 (vascular endothelial growth factor receptor-2) antibody to the shell of the microbeads. The bound microbeads were found to better adhere to LLC cells than unbound ones. PCI dynamically and clearly showed the magnetization and demagnetization of microbeads in PE-50 tube. The cells incubated with different types of microbeads were imaged by PCI, which provided clear and real-time visualization of the cell isolation. Therefore, PCI might be considered as a novel and efficient tool for further cell isolation studies.     

A novel cytomedical vehicle capable of protecting cells against complement

We have developed "Cytomedicine," which consists of functional cells entrapped in semipermeable polymer, and previously reported that APA microcapsules could protect the entrapped cells from injury by cellular immune system. However, microencapsulated cells were not protected from humoral immune system. Here, we developed a novel APA microcapsule, in which APA microbeads (APA(Ba) microbeads) were modified to contain a barium alginate hydrogel within their centers in an attempt to make it more difficult for antibody and complement to permeate the microcapsules. The permeability of APA(Ba) microbeads was clearly less than that of APA microcapsules, presumably due to the presence of barium alginate hydrogel. Cells encapsulated within APA(Ba) microbeads were protected against treatment with xenogeneic anti-serum. Furthermore, murine pancreatic beta-cells encapsulated in APA(Ba) microbeads remained viable and continued to secrete insulin in response to glucose. Therefore, APA(Ba) microbeads may be a useful carrier for developing anti-complement device for cytomedical therapy.     

16. Environmental specimen cryobanking

Density gradient centrifugation usually allows efficient separation of mononuclear cells from granulocytes using fresh human blood samples. However, we have found that with cryopreserved blood samples, density gradient centrifugation fails to separate granulocytes from mononuclear cells and have explored using immunomagnetic anti-CD15 microbeads as an alternate method to separate these cell populations. Using cryopreserved blood samples from 10 healthy donors we have shown that granulocytes express a significantly higher level of CD15 antigen than monocytes and lymphocytes, which allows for their efficient separation from mononuclear cells using anti-CD15 microbeads. This procedure is critical for purification of individual cell populations from cryopreserved leukocyte samples and could also potentially be applied to avoid granulocyte contamination of mononuclear cells isolated from stored blood and from patients with sepsis or thermal injury.     

Phagocytosis of Protein Coated Colloidal-Gold-Agarose-Gelatin Microbeads by Cultured Uterine Glandular Epithelial and Stromal Cells

A procedure for fixing and immunostaining whole cells from primary cultures of ovine and bovine uterine gland fragments was used to identify keratin in intermediate filaments of epithelial cells to distinguish them from stromal cells. Colloidal gold encapsulated aga-rose-gelatin microbeads were coated with different proteins and used to investigate uptake by epithelial and stromal cells in culture. Micro-beads were taken up by stromal cells and by epithelial cells on the outskirts of colonies. These cells formed ridges where they contacted and grew above stromal cells. Electron microscopy demonstrated that the microbeads had been internalized and appeared to be nontoxic. Individual cells could harbor more than 90 microbeads within their cytoplasm for at least seven to ten days with no apparent harm. Some cells with microbeads were seen to divide.     

An arabinogalactan protein(s) is a key component of a fraction that mediates local intercellular communication involved in tracheary element differentiation of zinnia mesophyll cells

Local intercellular communication is involved in tracheary element (TE) differentiation of zinnia (Zinnia elegans L.) mesophyll cells and mediated by a proteinous macromolecule, which was designated xylogen. To characterize and isolate xylogen, a bioassay system to monitor the activity of xylogen was developed, in which mesophyll cells were embedded in microbeads of agarose gel at a low (2.0-4.3x10(4) cells ml(-1)) or high density (8.0-9.0x10(4) cells ml(-1)) and microbeads of different cell densities were cultured together in a liquid medium to give a total density of 2.1-2.5x10(4) cells ml(-1). Without any additives, the frequency of TE differentiation was much smaller in the low-density microbeads than in the high-density microbeads. This low level of TE differentiation in the low-density microbeads was attributable to the shortage of xylogen. When cultures were supplemented with conditioned medium (CM) prepared from zinnia cell suspensions undergoing TE differentiation, the frequency of TE differentiation in the low-density microbeads increased remarkably, indicating the activity of xylogen in the CM. The xylogen activity in CM was sensitive to proteinase treatments. Xylogen was bound to galactose-specific lectins such as Ricinus communis agglutinin and peanut agglutinin, and precipitated by beta-glucosyl Yariv reagent. These results indicate that xylogen is a kind of arabinogalactan protein.     

Microencapsulation of dopamine neurons derived from human induced pluripotent stem cells

Background

Dopamine neurons derived from induced pluripotent stem cells have been widely studied for the treatment of Parkinson's disease. However, various difficulties remain to be overcome, such as tumor formation, fragility of dopamine neurons, difficulty in handling large numbers of dopamine neurons, and immune reactions. In this study, human induced pluripotent stem cell-derived precursors of dopamine neurons were encapsulated in agarose microbeads. Dopamine neurons in microbeads could be handled without specific protocols, because the microbeads protected the fragile dopamine neurons from mechanical stress.

Methods

hiPS cells were seeded on a Matrigel-coated dish and cultured to induce differentiation into a dopamine neuronal linage. On day 18 of culture, cells were collected from the culture dishes and seeded into U-bottom 96-well plates to induce cell aggregate formation. After 5 days, cell aggregates were collected from the plates and microencapsulated in agarose microbeads. The microencapsulated aggregates were cultured for an additional 45 days to induce maturation of dopamine neurons.

Results

Approximately 60% of all cells differentiated into tyrosine hydroxylase-positive neurons in agarose microbeads. The cells released dopamine for more than 40 days. In addition, microbeads containing cells could be cryopreserved.

Conclusion

hiPS cells were successfully differentiated into dopamine neurons in agarose microbeads.

General significance

Agarose microencapsulation provides a good supporting environment for the preparation and storage of dopamine neurons.     

Synthesis and utilization of E. coli‐encapsulated PEG‐based microdroplet using a microfluidic chip for biological application

We report herein an effective strategy for encapsulating Escherichia coli in polyethylene glycol diacrylate (PEGDA) microdroplets using a microfluidic device and chemical polymerization. PEGDA was employed as a reactant due to the biocompatibility, high porosity, and hydrophilic property. The uniform size and shape of microdroplets are obtained in a single‐step process using microfluidic device. The size of microdroplets can be controlled through the changing continuous flow rate. The combination of microdroplet generation and chemical polymerization techniques provide unique environment to produce non‐toxic ways of fabricating microorganism‐encapsulated hydrogel microbeads. Due to these unique properties of micro‐sized hydrogel microbeads, the encapsulated E. coli can maintain viability inside of microbeads and green fluorescent protein (GFP) and red fluorescent protein (RFP) genes are efficiently expressed inside of microbeads after isopropyl‐β‐D ‐thiogalactopyranoside induction, suggesting that there is no low‐molecular weight substrate transfer limitation inside of microbeads. Furthermore, non‐toxic, gentle, and outstanding biocompatibility of microbeads, the encapsulated E. coli can be used in various applications including biotransformation, biosensing, bioremediation, and engineering of artificial cells. Biotechnol. Bioeng. 2010;107:747–751. © 2010 Wiley Periodicals, Inc.     

Osteogenic differentiation of encapsulated rat mesenchymal stem cells inside a rotating microgravity bioreactor: in vitro and in vivo evaluation

The objective of this study is to evaluate the in vitro and in vivo osteogenic potential of rat bone marrow mesenchymal stem cells (BM-MSCs) using chitosan/hydroxyapatite (C/HAp) microbeads as encapsulation matrix under osteoinductive medium and dynamic culture conditions. The degradation characteristics of C/HAp microbeads were evaluated under in vitro and in vivo conditions for 180 days. BM-MSCs were encapsulated in C/HAp microbeads with >?85% viability, and were cultured in a slow turning lateral vessel-type rotating bioreactor simulating microgravity conditions for 28 days, under the effect of osteogenic inducers. MTT assay showed that the metabolic activity of encapsulated cells was preserved >?80% after a week. In vitro experiments confirmed that the encapsulated BM-MSCs differentiated into osteoblastic cells, formed bone-like tissue under osteogenic microgravity bioreactor conditions. Preliminary in vivo study indicated C/HAp microbeads containing BM-MSCs were able to repair the surgically-created small bone defects in the rat femur. BM-MSCs-C/HAp composite microbeads may have potential for modular bone regeneration.     

Magnetophoretic mobilities correlate to antibody binding capacities

METHODS: A methodology and a mathematical theory have been developed, which allow quantitation of the expression levels of cellular surface antigens using immunomagnetic labels and cell tracking velocimetry (CTV) technology. RESULTS: Quantum Simply Cellular (QSC) microbeads were immunomagnetically labeled with anti-CD2 fluorescein isothiocyanate (FITC) antibodies and anti-FITC MACS paramagnetic nanoparticles. Magnetophoretic mobility has been defined as the magnetically induced velocity of the labeled cell or microbead divided by the magnetophoretic driving force, proportional to the magnetic energy density gradient. DISCUSSION: Using computer imaging and processing technology, the mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads in a nearly constant magnetic energy gradient. A calibration curve correlating the measured magnetophoretic mobility of the immunomagnetically labeled microbeads to their antibody binding capacities (ABC) has been obtained. CONCLUSION: The results, in agreement with theory, indicate a linear relationship between magnetophoretic mobility and ABC for microbeads with less than 30,000 ABC. The mathematical relationships and QSC standardization curve obtained allow determination of the number of surface antigens on similarly immunomagnetically labeled cells.     

Combination effects of complement regulatory proteins and anti-complement polymer

We previously reported the development of a "cytomedicine" that consists of cells trapped in alginate-poly-L-lysine-alginate (APA) microcapsules and agarose microbeads. The functional cells that are entrapped in semipermeable polymer are completely isolated from cellular immune system. However, the ability of cytomedicine to isolate cells from the humoral immune system, which plays an essential role in xenograft rejection, is low. Therefore, the goal of the present study was to develop a novel cytomedicine that could protect the entrapped cells from injury of the complement system. We investigated the applicability of the complement regulatory protein (CRP), Crry, to cytomedicine. Crry-transfected cells entrapped within agarose microbeads resisted injury by complement to a degree, while entrapment of Crry transfected cells within agarose microbeads containing polyvinyl sulfate (PVS), a novel cytomedical device with anti-complement activity, clearly protected against complement attack. These data indicate that the combination of a CRP and a cytomedical device with anti-complement activity is a superior device for cytomedical therapy.     

Cell-density-dependent lysis and sporulation of Myxococcus xanthus in agarose microbeads.

Vegetative cells of Myxococcus xanthus were immobilized in 25-microns-diameter agarose microbeads and incubated in either growth medium or sporulation buffer. In growth medium, the cells multiplied, glided to the periphery, and then filled the beads. In sporulation buffer, up to 90% of the cells lysed and ca. 50% of the surviving cells formed resistant spores. A strong correlation between sporulation and cell lysis was observed; both phenomena were cell density dependent. Sporulation proficiency was a function of the average number of cells within the bead at the time that sporulation conditions were imposed. A minimum of ca. 4 cells per microbead was necessary for efficient lysis and sporulation to proceed. Increasing this number accelerated the lysis and sporulation process. No lysis occurred when an average of 0.4 cell was entrapped per bead. Entrapping an average of 1.7 cells per bead resulted in 46% lysis and 3% sporulation of survivors, whereas entrapping an average of 4.2 cells per bead yielded 82% lysis and 44% sporulation of the surviving cells. Sporulation and lysis also depended upon the cell density in the culture as a whole. The existence of these two independent cell density parameters (cells per bead and cells per milliliter) suggests that at least two separate cell density signals play a role in controlling sporulation in M. xanthus.     

使用旋转式生物反应器体外扩增人表皮细胞

目的：使用Cytodex-3微载体和高截面纵横比的旋转式生物反应器容器作为培养系统大规模扩增人表皮细胞（hECs）。方法：使用中性蛋白酶和胰蛋白酶．EDTA两步骤法从人皮肤中分离出人表皮细胞,使用DIL标记细胞后结合微载体后在旋转式生物反应器（RCCS）中培养,细胞贴附微载体的生长状态使用倒置显微镜,扫描电镜观测。并且分析细胞群体倍增时间来比较微重力培养与平面培养的体外增殖能力差异。结果：在旋转式生物反应器的微重力培养体系中,人表皮细胞能快速贴附到微载体表面,在培养过程中达到很大的细胞密度,并且表现出很强的增殖能力和细胞活性。结论：使用旋转式生物反应器和微载体悬浮培养人表皮细胞,是大量制备皮肤组织工程种子细胞的一种有效方法。     

Microcultivation of anaerobic bacteria single cells entrapped in alginate microbeads

Alginate microbeads, produced by emulsion/internal gelation, were studied for the entrapment and microcultivation of microbial cells with biotechnological potential. An anaerobic consortium which was selected for its capacity to degrade complex carbohydrates, and a pure culture of cellulose degrading bacteria were used for entrapment studies. Optimization of conditions for the formation of spherical alginate microbeads in sizes between 20 and 80 μm were examined. The best conditions were achieved by combining rapeseed methyl ester as oil phase and stirring at 100 rpm using a rotation impeller. Calcium alginate microbeads produced under these conditions were shown to present morphological stability, with large pores in the internal matrix that favours microcolony development. Finally, single cells were observed inside the beads after the entrapment procedure and microcolony formation was confirmed after cultivation in cellobiose.     

Automation of functional assays by flow injection fluorescence microscopy.

Bead-injection spectroscopy is a novel technique that uses immobilized eukaryotic cells on microbeads as a renewable biosensor for fluorescence microscopy. The use of a flow injection instrument allows fast functional assays that generate full kinetic characterization of a drug. Because the cell population is automatically replaced for each assay, variability is minimized, thus allowing greater accuracy.     

New use of cyanosilane coupling agent for direct binding of antibodies to silica supports. Physicochemical characterization of molecularly bioengineered layers.

A simple protocol to fix biological species to silica-based surfaces (silica microbeads and glass slides), using a bifunctional silane reagent (3-cyanopropyl dimethyl chlorosilane), is presented. This silane reagent was used without further derivatization. This system led to strong, but not covalent, linkage of antibodies through their glycosylated regions (OH groups) to solid supports. The use of a microsized sample revealed that the coupling process depends not only on physicochemical interactions but also on steric phenomena, and in this case, it was shown that a molecule acting as a spacer was required for more efficient cell fixation. Here, monoclonal mouse antibodies against the CD45 molecule expressed on rat lymphocytes (MAR anti CD45 Ab) were linked to lymphocytes, and as spacers, sheep anti-mouse antibodies (SAM Ab) were immobilized on silica surfaces, allowing the cells to stick to the floating hollow silica microbeads by simple incubation. Under such conditions, a single microbead can fix several cells. The potential of this hollow, low-density support is in ultrasound applications, for the destruction by cavitation phenomena of cells selectively fixed onto such a support. Such a study can serve as a basic model for various microbiosystems involving cell manipulation.     

Magnetophoretic cell sorting is a function of antibody binding capacity

Antibody binding capacity (ABC) is a term representing a cell's ability to bind antibodies, correlating with the number of specific cellular antigens expressed on that cell. ABC allows magnetically conjugated antibodies to bind to the targeted cells, imparting a magnetophoretic mobility on each targeted cell. This enables sorting based on differences in the cell magnetophoretic mobility and, potentially, a magnetic separation based on the differences in the cell ABC values. A cell's ABC value is a particularly important factor in continuous magnetic cell separation. This work investigates the relationship between ABC and magnetic cell separation efficiency by injection of a suspension of immunomagnetically labeled quantum simply cellular calibration microbeads of known ABC values into fluid flowing through a quadrupole magnetic sorter. The elution profiles of the outlet streams were evaluated using UV detectors. Optimal separation flow rate was shown to correlate with the ABC of these microbeads. Comparing experimental and theoretical results, the theory correctly predicted maximum separation flow rates but overestimated the separation fractional recoveries.     

Production of acetylcholine receptors from TE671 cells grown on microcarriers

Summary When grown in DMEM supplemented with 10 % fetal calf serum and using Cytodex 3® as microcarriers, TE671 cells entering the stationary phase optimally expressed acetylcholine receptors. These, receptors could be conveniently extracted from cell-saturated, microbeads or monodispersed cells obtained by trypsinization of microbeads. Typically, a 500 ml-batch gave 6–7 pmol of receptors which could be used as antigen to assay anti-acetylcholine receptor antibodies in the sera of myasthenic patients.     

Functional immobilization of plant receptor-like kinase onto microbeads towards receptor array construction and receptor-based ligand fishing

Despite the large number of receptor-like kinases (RLKs) in plants, few of their specific ligands are known. Ligand fishing is one of the most challenging post-genomic technologies. Here, we report a strategy for functional immobilization of plant RLKs on microbeads via covalent linkage. Because of the high density of immobilized RLKs, ligand-receptor interaction can be visualized at high sensitivity using fluorescent-labeled ligands under the confocal laser scanning microscope. Moreover, using a receptor-based affinity chromatography system with RLK microbeads, the ligand of the receptor was directly retrieved at high purity from complex natural samples. Our RLK microbeads and receptor-based ligand fishing approach is a feasible alternative to conventional forward genetics and bioassay-based biochemical purification for identification of novel ligand-receptor pairs in plants.     

Alginate Microencapsulated Hepatocytes Optimised for Transplantation in Acute Liver Failure

MethodsHuman hepatocyte microbeads (HMBs) were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs) after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs) produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality.ResultsThe optimised HMBs were of uniform size (583.5±3.3 µm) and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001). 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×10⁶ cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01). Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function.ConclusionAn optimised protocol to produce GMP grade alginate-encapsulated human hepatocytes has been established. Transplantation of microbeads provided effective metabolic function in ALF. These high quality HMBs should be suitable for use in clinical transplantation.