Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl₂ solution, and the alginate/cell suspension is gelled on contact with the CaCl₂ to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.     

Stabilization of alginate beads using radiation polymerized polyacrylamide.

A technique has been described for the stabilization of calcium alginate beads using radiation polymerized acrylamide. The technique involved dropping a mixture containing the cells (20%), sodium alginate (2%), acrylamide (2.5%) and N-N'-methylene-bis-acrylamide (0.1%) through a syringe needle into cold (-75 degrees C) toluene. The frozen beads obtained were exposed to 60Co gamma-rays (0.5 KGy) and were then thawed in 0.1 M CaCl2 solution. Unlike the calcium alginate beads the conjugate beads were not found to be dissolved when incubated in 3% trisodium citrate solution. Stabilized beads containing entrapped yeast cells could be reused for over 15 batches for the inversion of sucrose without loss in activity or chemical integrity of the beads.     

A new assay and cellular localization for an inducible glucuronyltransferase in the embryonic chick liver

A direct, radioisotopic assay is described for the uridine diphosphate glucuronic acid (UDPGA): p-aminophenol glucuronyltransferase. The assay uses solid phase p-aminophenol-Sephadex as the glucuronyl acceptor and UDP-[¹⁴C]GA as the glucuronyl donor. After incubation with the enzyme, the derivatized Sephadex beads are washed in SDS-urea or with high salt concentrations to remove all labeled material except for that covalently attached to the beads. Sonicated livers from chick embryos exposed to phenobarbital for at least 5 days transfer more than ten times the glucuronic acid to derivatized beads than do uninduced livers of the same developmental age. Glucuronyl-transferase activity can be detected on intact, living cells after 5 days of phenobarbital induction, whereas sonicate activity is detectable within 3 days of induction. Suspensions of living cells can show 25% the activity found in the same suspension after all the cells are lysed by sonication.     

Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells

Microinjected 0.26-micron fluorescent, carboxylated microspheres were found to display classical saltatory motion in tissue culture cells. The movement of a given particle was characterized by a discontinuous velocity distribution and was unaffected by the activity of adjacent particles. The microspheres were translocated at velocities of up to 4.7 micron/s and sometimes exhibited path lengths greater than 20 micron for a single saltation . The number of beads injected into a cell could range from a few to over 500 with no effect on the cell's ability to transport them. Neither covalent cross-linking nor preincubation of the polystyrene beads with various proteins inhibited the saltatory motion of the injected particles. The motion of the injected beads in cultured cells was reversibly inhibited by the microtubule poison nocodazole, under conditions in which actin-rich, nitrobenzoxadiazol - phallacidin -staining structures remain intact. Whole-cell high voltage electron microscopy of microinjected cells that were known to be moving the fluorescent microspheres revealed that the beads were embedded in the cytoplasmic matrix and did not appear to be membrane bound. The enhanced detectability of the fluorescent particles over endogenous organelles and the ability to modify the surfaces of the beads before injection may enable more detailed studies on the mechanism of saltatory particle motion.     

Plasmid inheritability and biomass production: comparison between free and immobilized cell cultures of Escherichia coli BZ18(pTG201) without selection pressure.

Maintenance of the plasmid pTG201 in Escherichia coli BZ18 was studied for both free and immobilized cells during chemostat culture, in the absence of the antibiotic against which resistance was plasmid encoded. Electron microscopic observations of immobilized proliferant cells within carrageenan gel beads showed high cell concentrations and growth into distinct cavities. The plasmid which coded for the catechol 2,3-dioxygenase activity was stably maintained during 80 generations in the case of immobilized cells. A theoretical analysis founded on the compartmentalization resulting from the immobilized growth conditions was described. However, the model still showed a plasmid stability inferior to that determined experimentally. Hypotheses dealing with physiological changes of immobilized cells were presented. In addition, the high cell concentrations obtained in the outer 50 microns of the carrageenan gel beads gave a biomass productivity within this useful volume which was 20 times higher than in free-cell cultures.     

Serial propagation of mammalian cells on gelatin-coated microcarriers

The ability to serially propagate mammalian cells in microcarrier cultures is essential for large-scale operation. The success of such serial propagation depends on viable dissociation of cells from microcarriers and the normal growth and product formation after subsequent reinoculation. The high pH treatment developed for dissociating cells from DEAE-derivatized microcarriers was not as effective for a number of cell strains cultivated on gelatin-coated microcarriers. By prewashing the cell-laden microcarriers with buffer containing a chelating agent, bovine kidney cells, BK, human embryonic foreskin fibroblasts, FS-4, and continuous human kidney cells, TCL-598 which produces prourokinase, were viably dissociated from commercially available gelatin-coated microcarriers, Cytodex-3. Cells dissociated from microcarriers reattached and grew on micro-carriers subsequent to inoculation into subcultures. However, after subculturing, cells may attach at different rates to newly added beads and to conditioned microcarriers which cells had previously grown. It resulted in an uneven cell distribution on microcarriers and inferior growth kinetics. This effect was more profound for BK and FS-4 cells which are propagated with a low multiplication ratio. Specifically, BK cells attach to conditioned beads at a faster rate than to new beads, while FS-4 cells attach to new beads faster than to conditioned beads. Thus, for these two cell strains, a separator was used to separate the microcarriers from the suspension of dissociated cells before subsequent inoculation. For TCL-598 cells, which are propagated at a high multiplication ratio, this dissociation technique can be applied directly without the separation of dissociated cells and conditioned microcarriers. All the three cell lines tested exhibit normal growth kinetics in serial propagation on microcarriers. Furthermore, the production of prourokinase by TCL598 cells serially propagated on microcarriers was comparable to that inoculated from roller bottles.     

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.     

A scintillation proximity assay for human interleukin-5 (hIL-5) high-affinity binding in insect cells coexpressing hIL-5 receptor alpha and beta subunits

The high-affinity receptor for human interleukin-5 (hIL-5) is composed of alpha and beta subunits. A baculovirus expression system was established in Sf9 cells capable of expressing hIL-5 receptor alpha and beta subunits simultaneously. By using wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads to capture 125I-labeled hIL-5-bound Sf9 cells, a SPA was developed and used to measure hIL-5 high-affinity binding. The hIL-5 receptors expressed in the Sf9 cells represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 0. 24 nM and a density of 2.95 x 10(5) sites/cell. This is the first study in which the high-affinity Kd value similar to that for hIL-5 binding to human eosinophils was achieved using a recombinant expression system. The SPA compared favorably with the filter binding assay with regard to various binding parameters. We also found that several lectins, when coated on SPA beads, were even more effective than WGA-coated SPA beads for capturing the insect cells. We conclude that the baculovirus expression system was highly efficient in producing the high-affinity hIL-5 receptors and that the SPA was a simple and sensitive assay that could be readily adapted into a high-throughput screening format. The SPA described here could be a prototype for binding assays for other multimeric receptors.     

Tuning structural durability of yeast-encapsulating alginate gel beads with interpenetrating networks for sustained bioethanol production

Microorganisms have become key components in many biotechnological processes to produce various chemicals and biofuels. The encapsulation of microbial cells in calcium cross-linked alginate gel beads has been extensively studied due to several advantages over using free cells. However, industrial use of alginate gel beads has been hampered by the low structural stability of the beads. In this study, we demonstrate that the incorporation of interpenetrating covalent cross-links in an ionically cross-linked alginate gel bead significantly enhances the bead's structural durability. The interpenetrating network (IPN) was prepared by first cross-linking alginate chemically modified with methacrylic groups, termed methacrylic alginate (MA), with calcium ions and subsequently conducting a photo cross-linking reaction. The resulting methacrylic alginate gel beads (IPN-MA) exhibited higher stiffness, ultimate strength and ultimate strain and also remained more stable in media either subjected to high shear or supplemented with chelating agents than calcium cross-linked alginate gel beads. Furthermore, yeast cells encapsulated in IPN-MA gel beads remained more metabolically active in ethanol production than those in calcium cross-linked alginate gel beads. Overall, the results of this study will be highly useful in designing encapsulation devices with improved structural durability for a broad array of prokaryotic and eukaryotic cells used in biochemical and industrial processes.     

Improving immobilized biocatalysts by gel phase polymerization

A new method is presented for the treatment of gel-type supports, used for immobilizing microbial cells and enzymes, to obtain high mechanical strength. It is particularly useful for ethanol fermentation over gel beads containing immobilized viable cells, where the beads can be ruptured by gas production and the growth of cells within the gels. This method consists of treating agar or carrageenan gel with polyacrylamide to form a rigid support which retains the high catalytic activity characteristic of the untreated biocatalysts. The size and shape of the biocatalyst is unaffected by this treatment. The method involves the diffusion of acrylamide, N,N'-methylenebisacrylamide and beta-dimethylaminopropionitrile (or N,N,N',N'-tetramethyl-ethylenediamine) into the performed biocatalyst beads followed by the addition of an initiator to cause polymerization within the beads. Treated gels have been used for the continuous fermentation of glucose to ethanol in a packed column for over two months. During this operation, the gel beads maintained their rigidity, and the maximum productivity was as high as 50 g h(-1) L(-1) gel. There was no appreciable decay of cell activity.     

Effects of different fragments of the fibronectin molecule on latex bead translocation along neural crest migratory pathways

Previous studies from this laboratory have utilized latex beads as probes of embryonic migratory pathways. After microinjection into embryos at the time of neural crest migration, uncoated latex polystyrene beads were found to translocate to ventral sites and to settle in the vicinity of endogenous neural crest derivatives. However, latex beads coated with fibronectin did not translocate ventrally, but remained associated with cells surrounding the implantation site. Fibronectin is a large glycoprotein with a variety of biological activities and multiple binding domains. Here, the binding activities which might be responsible for immobilization of the fibronectin-coated beads are examined. Latex beads were coated with three types of fragments of the fibronectin molecule representing different functional domains: (i) a 66-kDa fragment containing collagen-binding activity; (ii) a mixture of 45- and 32-kDa fragments containing heparin-binding activity; and (iii) a 120-kDa fragment containing cell-binding activity. The beads coated with fibronectin fragments were injected into the newly formed trunk somites of avian embryos. After injection, beads coated with either the heparin- or the collagen-binding domain translocated ventrally and distributed analogously to uncoated latex beads. In contrast, the majority of beads coated with the fibronectin cell-binding domain did not translocate but remained associated with dermamyotomal cells surrounding the injection site. The cell-binding fragment, however, was not as effective as the intact fibronectin molecule in preventing translocation of the beads. The results suggest that the cell-binding domain is primarily responsible for restriction of fibronectin beads from the ventral neural crest pathway. Because intact fibronectin is more effective at immobilizing beads than is the cell-binding fragment, other binding domains of fibronectin, more efficient coating with intact fibronectin, or crosslinking of intact fibronectin molecules may also play some role in immobilization of the beads at the implantation site.     

Preparation of Encapsulated Microbial Cells for Environmental Applications

An improved method for the encapsulation of bacteria into microspheres of alginate, agarose, or polyurethane is described. Cell suspensions were passed through a low-pressure nozzle into an aqueous phase where matrix polymerization or gelation yielded beads 2 to 50 μm in diameter. Trials with a chlorophenol-degrading Flavobacterium species showed that cells entrapped by these procedures were as catabolically active as free cells. These types of beads should have numerous applications in the fields of environmental science and engineering.     

Pure gelatin microcarriers: Synthesis and use in cell attachment and growth of fibroblast and endothelial cells

Summary A new type of microcarrier was described using bead emulsion-polymerization techniques. An aqueous solution of gelatin and glutaraldehyde was dispersed in a hydrophobic phase of mineral oil, using Triton X-114 as an emulsifier, and polymerization was initiated. The resultant spherical beads, composed entirely of gelatin, showed excellent mechanical stability to ethanol drying, sterilization, and long-term use in microcarrier spinner cultures. The solid gelatin microcarriers supported the growth of L-929 fibroblast, swine aorta endothelial, human umbilical endothelial, and HeLa-S₃ cultures with no adverse effects on cell morphology or growth. The beads were transparent in growth medium and attached cells were clearly visualized without staining. The beads were also compatible with techniques for scanning electron microscopy. Collagenase could be used to entirely digest the gelatin beads, leaving the cells free from microcarriers and suspended in solution while retaining 98% cell viability. The results further showed that after collagenase treatment the cells would populate fresh gelatin microcarriers and grow to confluence. Cell attachment kinetics revealed that the endothelial cells attached to the gelatin beads at the same rate as to tissue culture plates, whereas the fibroblast cells attached to the beads more slowly. However, once the fibroblast cells were attached to the gelatin microcarriers they spread and grew normally. This research was supported in part by the National Institutes of Health (GN 29127) and Ventrex Laboratories, Portland, Maine.     

Characterization of an encapsulation device for the production of monodisperse alginate beads for cell immobilization

An encapsulation device, designed on the basis of the laminar jet break-up technique, is characterized for cell immobilization with different types of alginate. The principle of operation of the completely sterilizable encapsulator, together with techniques for the continuous production of beads from 250 microm to 1 mm in diameter, with a size distribution below 5%, at a flow rate of 1-15 mL/min, is described. A modification of the device, to incorporate an electrostatic potential between the alginate droplets and an internal electrode, results in enhanced monodispersity with no adverse effects on cell viability. The maximum cell loading capacity of the beads strongly depends on the nozzle diameter as well as the cells used. For the yeast Phaffia rhodozyma, it is possible to generate 700 microm alginate beads with an initial cell concentration of 1 x 10(8) cells/mL of alginate whereas only 1 x 10(6) cells/ml could be entrapped within 400 microm beads. The alginate beads have been characterized with respect to mechanical resistance and size distribution immediately after production and as a function of storage conditions. The beads remain stable in the presence of acetic acid, hydrochloric acid, water, basic water, and sodium ions. The latter stability applies when the ratio of sodium: calcium ions is less than 1/5. Complexing agents such as sodium citrate result in the rapid solubilization of the beads due to calcium removal. The presence of cells does not affect the mechanical resistance of the beads. Finally, the mechanical resistance of alginate beads can be doubled by treatment with 5-10 kDa chitosan, resulting in reduced leaching of cells.     

Characteristics of a BHK cell variant defective in the cell-substratum contact process

In this paper we document the phenotypic characteristics of a novel BHK cell adhesion variant designated FN-2. Unlike parental cells, FN-2 cells did not attach to fibronectin (pFN)-coated dishes, even after 4-hr incubations on dishes treated with 100 micrograms/ml of pFN. Mixing experiments with the variant and parental cells revealed that the parental cells attached normally in the presence of a ninefold excess of variant cells and the variant cells failed to attach in the presence of a ninefold excess of parental cells. Therefore, the defect in FN-2 cells could not be explained by secretion of a factor inhibiting attachment or lack of secretion of a factor required for attachment. Also, the inability of FN-2 cells to attach to pFN-coated dishes could not be explained by an absence of cell pFN receptors since the variant cells bound normal numbers of small (ca. 0.8 micron) pFN-coated latex beads, although they phagocytosed the beads poorly compared to parental cells. Also, the variant cells were not able to bind large (5.7 or 16.8 microns) pFN-coated beads. When tested on dishes coated with ligands that, unlike fibronectin, have a high affinity for cell surface receptors, e.g., lectins and anti-BHK antibodies, FN-2 cells were observed to attach at a rate similar to that of parental cells but spread much more slowly. The phenotypic characteristics of FN-2 cells suggest that they are deficient in what previously has been called the "cell contact" process in cell adhesion. It is proposed that the cell contact process is the initial formation by an individual cell of a sufficient number of cell-substratum bonds to resist the shear forces operationally used to define "attachment," and that more cell-substratum bonds are necessary for cell attachment to large substrata (dishes or large beads) than for attachment to small substrata (small beads). The molecular defect in FN-2 cells was studied by electroblotting analysis. A high molecular weight (ca. 370 kd) glycoprotein detected by blotting with anti-BHK antibodies and ConA that was present in parental cell membranes was reduced or absent in the variant cells.     

Expression of single-chain Fv gene specific for gamma-seminoprotein by RTS and its biological activity identification

Fabricating a single-chain variable fragment specific for human seminoprotein is very important in antibody-directed enzyme prodrug therapy and NMR imaging for prostate cancer. Here a single-chain Fv specific for gamma-seminoprotein was expressed by RTS. Its activity and the efficiency of entry into prostate cancer cells are investigated by immunoprecipitation and Western blotting and immunofluorescent staining, as well as entry of conjugated magnetic beads into cells. Results showed that ScFv peptides specific for gamma-seminoprotein were successfully prepared, which can bind with the prostate cells specifically and can bring magnetic beads into prostate cancer cells within 15 min, the amount of magnetic beads inside prostate cancer cells increased as the culture time prolonged. ScFv-conjugated magnetic beads did not enter into control cells. In conclusion, the ScFv peptide against human gamma-seminoprotein with biological activity was successfully fabricated, which can take magnetic beads to prostate cancer cells specifically and not to the control cells. This ScFv peptide against human gamma-seminoprotein should be useful in improving the detection and therapy of prostate cancer at early stages and NMR imaging.     

Cell surface action of thrombin is sufficient to initiate division of chick cells

Thrombin covalently linked to carboxylate-modified polystyrene beads initiated division of quiescent chick embryo (CE) cells either in medium containing low levels of serum or in serum-free medium. Release of thrombin was monitored by measuring acid-precipitable radioactivity released from 125I-thrombin beads into the medium during incubation with cells. Even if all of the acid-precipitable material released from the beads were active thrombin, it was not sufficient to account for any of the observed cell division, and was 10-30 fold less than the amount necessary to produce the increase in cell number caused by the thrombin beads. Two other kinds of experiments also showed that material released into the medium did not account for the observed initiation of cell division. First, medium taken from cultures incubated with thrombin beads did not initiate cell division when added to new quiescent cultures. Second, in coverslip experiments where populations of cells with an without thrombin feads shared the same medium, only bead-contacted cells divided. Several results suggested that the material which was released from the thrombin beads resulted from cell-associated proteolysis rather than from "leakage" of intact thrombin from the beads. For example, after incubating 125I-thrombin beads with or without CE cells, we were unable to detect any intact thrombin released into the medium. In addition, most of the material released from the beads was acid-soluble and was only released in the presence of CE cells. A few thrombin beads were endocytosed by CE cells, but they were surrounded by an intact plasma membrane. Thus they did not directly interact with the cytoplasm. The close association of many of the beads with the cell surface and the presence of a few beads in endocytic vesicles made it important to consider the possibility that thrombin might be released from the beads directly into the cells. This possibility was explored using ultrastructural (EM) autoradiography. With this technique (where one grain represented 700--900 thrombin molecules), we found that beads inside the cells had approximately the same number of grains as beads not in contact with cells. This suggested that little, if any, additional radioactive material had been released from the beads which were in contact with the cells. In addition, we were unable to detect any grains in the cytoplasm which could be attributed to released thrombin, even using an amount of 125I-thrombin beads which was 8 fold greater than the amount which produced maximal cell division. Taken together, these results provide direct evidence that thrombin action at the cell surface is sufficient to initiate division of CE cells.     

固定化乳酸乳球菌连续生产Nisin的研究

以海藻酸钙为材料 ,固定乳酸乳球菌 (Lactococcuslactissubsp .lactis)SM5 2 6 ,研究不同条件对Nisin合成的影响。结果表明 ,利用 2 %海藻酸钠在 1 0mmol LCaCl2 条件下 ,得到的固定化细胞颗粒稳定性较好 ,可维持 90h无破裂 ;在发酵过程中SYS3培养基中的无机盐成分尤其磷酸盐对固定化颗粒有破坏作用 ;用mSYS3培养基代替SYS3 ,通过 72h三批次循环的半连续培养 ,Nisin活性为 85 0IU mL ,无明显的细胞渗漏现象。连续化生产 70h ,Nisin活性达 1 1 5 0IU mL ,相当于游离细胞的发酵水平。     

Mast cells: an unexpected finding in the modulation of cutaneous wound repair by charged beads

Increased numbers of mast cells are affiliated with a broad spectrum of pathologic skin conditions, including ulcers, atopic dermatitis, neurofibromatosis, hemangiomas, keloids, and hypertrophic scars. It has been proposed that mast cells play a primary pathophysiologic role in these disorders and that their presence represents not merely a secondary event. While investigating their recent hypothesis that positively charged cross-linked diethylaminoethyl dextran (CLDD) beads potentiate cutaneous wound healing, the authors serendipitously observed increased numbers of mast cells in the deep dermis of wounds treated with CLDD beads. The authors propose that mast cells may play an important role in the modulation of healing seen with CLDD beads. Incisional wounds were studied in 30 Sprague-Dawley rats partitioned into two groups that were killed 7 or 14 days after wounding. The wounds were treated with positively, negatively, or neutrally charged CLDD beads. Physiologic saline served as a control. At the designated times after incisional wounding, biopsy specimens were tested for wound breaking strength or processed for histologic testing, fixed in 4% paraformaldehyde, and stained with Giemsa and Goldner-Masson trichrome. Mast cells were counted under light microscopy in a blinded fashion and were expressed as the number of cells per millimeter squared. Significant increases in the number of mast cells were observed in the deep dermis of incisional wounds after implantation with positively or negatively charged CLDD beads. In contrast, neutrally charged beads had no effect on mast cell numbers. At 7 days, the incisions treated with positively charged beads averaged 2.1 times more mast cells compared with those treated with physiologic saline or neutrally charged beads, whereas the incisions treated with negatively charged beads displayed 3.2 times more mast cells. By day 14, the incisions treated with positively charged beads averaged 2.5 times more mast cells than those wounds treated with saline or neutrally charged beads; the incisions treated with negatively charged CLDD beads had 3.4 times more mast cells. The 7-day tensiometric data indicated that wounds treated with negatively charged CLDD beads had increased breaking strength compared with wounds treated with neutrally charged beads or saline (1.8 and 1.7 times, respectively; p = 0.01 and p = 0.02). Wounds treated with positively charged beads also showed increased breaking strength compared with wounds treated with neutrally charged beads or saline (1.5 and 1.4 times greater); however, this did not reach statistical significance. There was no apparent difference in breaking strength when neutrally charged beads were compared with those treated with saline. At 14 days, there was no statistically significant difference in wound breaking strength between different treatments. These findings are clinically germane to the assessment of proposed therapeutic applications of CLDD beads for a variety of impaired wound-healing states. Furthermore, if increased mast cell populations are intimately linked to hypertrophic scar and keloid formation, the results of the authors' study suggest that CLDD bead therapy of cutaneous wounds may lead to pathologic wound healing in humans.     

Artificial induction of autophagy around polystyrene beads in nonphagocytic cells

Autophagy is an intracellular event that acts as an innate cellular defense mechanism to kill invading bacteria such as group A Streptococcus in nonphagocytic epithelial-like cells. The cellular events underlying autophagosome formation upon bacterial invasion remain unclear due to the biochemical complexity associated with uncharacterized bacterial components, and the difficulty of predicting the location as well as the timing of where/when autophagosome formation will take place. To overcome these problems, we monitored autophagosome formation in living nonphagocytic cells by inducing autophagy around artificial micrometer-sized beads instead of bacteria. Beads conjugated with bio-reactive molecules provide a powerful tool for examining biochemical properties in vitro. However, this technique has not been applied to living cells, except for phagocytes, because the beads cannot be easily incorporated into nonphagocytic cells. Here we report that micrometer-sized polystyrene beads coated with transfection reagents containing cationic lipids can be incorporated into nonphagocytic cells, and that autophagy can be efficiently induced around the beads in these cells. Monitoring the process of autophagosome formation for pH-sensitive fluorescent dye (pHrodo)-conjugated beads by fluorescence live cell imaging combined with correlative light and electron microscopy, we found that autophagosomes are formed around the beads after partial breakdown of the endosomal membrane. In addition, the beads were subsequently transferred to lysosomes within a couple of hours. Our findings demonstrate the cellular responses that lead to autophagy in response to pathogen invasion.