Separation characteristics of animal cells using a dielectrophoretic filter

The separation characteristics of a wire–wire type dielectrophoretic (DEP) filter were evaluated using animal cells. The separation of cells with different activities was examined using a DEP filter. The specific growth rate of the cells in retention liquid was larger than that in permeation liquid. From the culture results of the separated cells, it becomes clear that the specific growth rate of the cells of the retention liquid was higher than that of the cells of the permeation liquid. Furthermore, as a result of separating cells two kinds of cell suspensions using the DEP filter, the difference between the retention ratios of the two groups of obtained cells was about 20% at maximum.     

Direct Image-Based Enumeration of Clostridium phytofermentans Cells on Insoluble Plant Biomass Growth Substrates

A dual-fluorescent-dye protocol to visualize and quantify Clostridium phytofermentans ISDg (ATCC 700394) cells growing on insoluble cellulosic substrates was developed by combining calcofluor white staining of the growth substrate with cell staining using the nucleic acid dye Syto 9. Cell growth, cell substrate attachment, and fermentation product formation were investigated in cultures containing either Whatman no. 1 filter paper, wild-type Sorghum bicolor, or a reduced-lignin S. bicolor double mutant (bmr-6 bmr-12 double mutant) as the growth substrate. After 3 days of growth, cell numbers in cultures grown on filter paper as the substrate were 6.0- and 2.2-fold higher than cell numbers in cultures with wild-type sorghum and double mutant sorghum, respectively. However, cells produced more ethanol per cell when grown with either sorghum substrate than with filter paper as the substrate. Ethanol yields of cultures were significantly higher with double mutant sorghum than with wild-type sorghum or filter paper as the substrate. Moreover, ethanol production correlated with cell attachment in sorghum cultures: 90% of cells were directly attached to the double mutant sorghum substrate, while only 76% of cells were attached to wild-type sorghum substrate. With filter paper as the growth substrate, ethanol production was correlated with cell number; however, with either wild-type or mutant sorghum, ethanol production did not correlate with cell number, suggesting that only a portion of the microbial cell population was active during growth on sorghum. The dual-staining procedure described here may be used to visualize and enumerate cells directly on insoluble cellulosic substrates, enabling in-depth studies of interactions of microbes with plant biomass.     

An on-line method for the reduction of fouling of spin-filters for animal cell perfusion cultures

The main limitation in the use of spin-filters during perfusion cultures of animal cells was revealed to be filter fouling. This phenomenon involves cell-sieve interactions as well as cell attachment to, and growth on, the filter surface. The cell attachment effect has been analysed in the present study during long-term perfusion simulations with CHO animal cells. It was demonstrated that at low filter acceleration, below 6.2 m/s2, a high perfusion rate of 25 cm/h induced rapid filter pore clogging within 3 days, whereas increasing the filter acceleration to 25 m/s2 increased filter longevity from 3 to 25 days, for filters with a pore size of 8.5 microm. Increasing the filter pore size to 14.5 microm improved filter longevity by 84% with less viable and dead cell deposits on the filter surface. However, it was demonstrated that filter longevity was not necessarily dependent on the amount of cell deposit on the filter surface. In the second part of this study, ultrasonic technology was used to reduce filter fouling. Filter vibration, induced by a piezo actuator, improved filter longevity by 113% during CHO cells perfusion cultures.     

Properties of Trichoderma reesei cells immobilized by the irradiation technique

Aerobic cells of Trichoderma reesei have been immobilized by the radiation polymerization technique using fibrous substances and hydroxyethyl methacrylate. The enzyme [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] productivity and growth of the cells in the immobilized growing cells have been studied. The enzyme (filter paper) activity in the immobilized cells was comparable to that of the intact cells, showing that the cells immobilized with fibrous materials grow and become adhered to the surface of the fibrils. The filter paper activity of the immobilized cells was affected mainly by monomer concentration and the content of the fibrous materials, as well as the irradiation dose. It was demonstrated that in repeated batch culture of the immobilized cells the filter paper activity gave a constant value, and leakage of the cells was not observed.     

A paper membrane filter assay for ciliate chemoattraction

A quantitative bioassay for ciliate chemoattraction based on the Boyden assay is described with the ciliates Tetrahymena thermophila and Tetrahymena pyriformis as test organisms. A chamber is separated into two compartments by a Whatman 3MM filter, and a suspension of starved cells (approximately 10(5) cells/ml) is placed in one compartment and a solution containing attractant in the other. The gradient of chemoattractant across the filter causes the cells to swim through the filter into the attractant-containing compartment where their appearance is determined by electronic cell counting. The assay described is convenient with a signal-to-noise ratio of approximately 10. It is shown here to work with the attractants proteose peptone and platelet-derived growth factor.     

Migration of polarized epithelial cells through permeable membrane substrates of defined pore size.

We have observed that cells of various epithelial lines exhibit the ability to migrate through permeable membrane substrates containing 3.0 microns pores. Scanning and transmission electron microscopic observations of Vero C1008 and Caco-2 cell lines grown on polycarbonate membranes containing 3.0 microns pores revealed extensive penetration of the filter and the establishment of virtually complete monolayers on the opposing surface. The migration of MDCK cells was also observed to occur under the same conditions; however, the extent of MDCK cell growth on the opposing surface was significantly less than observed for Vero C1008 and Caco-2 cells. Morphological differences were apparent between cells growing on the upper and lower faces of the filter membrane, although cells growing on both surfaces exhibited a polarized phenotype. The cells which invaded the filter were collected and maintained by serial passage. The passaged cells exhibited morphological differences and an altered rate of differentiation in comparison to the parental cell type, suggesting that the invasive cells represent a variant of the parental cell population. Studies using filters of different pore sizes indicated that cellular migration also occurs through pores of 2.0 microns diameter, but not through 1.0 micron (or smaller) pores. These observations have significant implications for studies involving the growth of epithelial cells on permeable membrane substrates containing large pores.     

间充质干细胞过滤分离器制备人羊膜间充质干细胞的研究

自然存在的间充质干细胞数量少,限制了其研究应用。依靠自主发明的间充质干细胞过滤分离器,分离制备了人羊膜间充质干细胞,并对制备的干细胞进行了三维培养扩增。结果表明,制备的干细胞形态长势良好,并能诱导分化为类胰岛样组织。与常规方法相比,干细胞收获率提高了8倍以上,且细胞活性状态良好。间充质干细胞过滤分离器可以批量制备高质量的各种间充质干细胞,有利于高效率地建设各种间充质干细胞库,以促进间充质干细胞的研究应用。     

Selective removal of ammonia from animal cell culture broth

Serum-free perfusion cultures of hybridoma TO-405 cells were carried out in spinner flasks coupled with zeolite A-3 packed beads. Ammonia was selectively removed from the culture broth by passing cell free permeate from ceramic cross flow filtration, through the zeolite packed bed. Ammonia concentration in the culture broth was effectively maintained between 1 to 4 mmol/l which was below the inhibitory concentration for cell growth. Maximum cell density levels of 10⁷ cells/ml as well as improved percentage cell viability higher than in serum-supplemented cultures were feasible in this system.The possible effects of shear stress, generated by variation of the flow rates of the broth through the ceramic filter module, on the growth of the hybridoma cells were investigated. Backwashing, by reversing the direction of the permeate, was found necessary to prolong the life of the filter. Variation of the flow rates of the broth through the ceramic module between 0.29 m/s to 0.59 m/s did not cause immediate cell damage but growth was repressed at the higher flow rate.This study also showed that glutamine appears to be one of the factors limiting the growth of the hybridoma cells.     

Use of membrane filters for the enumeration of autotrophic thiobacilli

A new membrane filter technique for field use was developed for the enumeration of either aerobic or anaerobic, autotrophic, sulfur-oxidizing bacteria in waters and soils. Immediately after collection, samples were filtered through sulfur-coated filters and incubated in selective media. Acidification or gas evolution was used as a growth indicator of aerobic and anaerobic thiobacilli, respectively, and related to the initial number of cells deposited on the filter.     

Human endothelial cells cultured on microporous filters used for leukocyte transmigration studies form monolayers on both sides of the filter

Summary A growing number of studies on the mechanism of leukocyte transendothelial migration use endothelial cells grown on microporous filters as an in vitro model of endothelium. Ultrastructural examination of such a model system previously demonstrated that human pulmonary artery endothelial cells (HPAEC) formed confluent monolayers on both sides of the 3-μm-pore filter (Mackarel et al., 1999). To determine whether this was a characteristic specific to pulmonary artery endothelial cells, the growth characteristics of a human pulmonary microvascular endothelial cell type (HMVEC-L) and the widely used human umbilical vein endothelial cells (HUVEC) on 3-μm microporous filters were examined by transmission electron microscopy (TEM). Similar to HPAEC, HMVEC-L and HUVEC were also found to grow on both sides of the filter. All three endothelial cell types were capable of migrating through the 3 μm pores of the filter to form a monolayer on the filter underside. The endothelial cells on the underside were orientated in an inverted position with the luminal surface facing away from the filter. Such ‘bilayer’ formation was observed at a range of seeding densities and in different culture media. Despite the presence of a bilayer of endothelial cells, TEM demonstrated that neutrophils migrated successfully across the cell-filter-cell system. Previous transmigration reports in which an in vitro model similar to ours was used have often assumed only one layer of endothelial cells. The observations reported here indicate that while endothelial cells on microporous filters are useful models for examining leukocyte-endothelial interactions, they are not appropriate for studies examining endothelial cell ‘sidedness.’     

Branching morphogenesis of mouse salivary epithelium in basement membrane-like substratum separated from mesenchyme by the membrane filter

Branching morphogenesis of mouse salivary gland has been studied with organ-culture system. We developed a novel transfilter culture system for analyzing branching morphogenesis of the salivary epithelium. The submandibular salivary epithelium from early 13-day mouse fetus, clotted with Matrigel and separated from the mesenchyme by membrane filter, showed extensive growth and branching morphogenesis, morphological differentiation of lobules and stalks, and a typical cleft shape. The epithelium showed little growth and no branching without Matrigel clot or without the mesenchyme. This branching morphogenesis was induced even when the pore size of the filter was reduced to 0.05 microns. Use of type I collagen gel instead of Matrigel mostly induced incomplete morphogenesis with various histological abnormalities. These results suggest that the salivary epithelium can undergo branching morphogenesis in the absence of the mechanical action of mesenchymal cells although it needs an appropriate extracellular matrix and some mesenchymal factors transmitted through the filter.     

Bioreactor considerations for secondary metabolite production from plant cell tissue culture: Indole alkaloids from Catharanthus roseus

Batch shake flask studies with Catharanthus roseus demonstrated that alkaloid production commenced only after growth had slowed or ceased. To obtain high alkaloid productivities for extended periods, a hormone-free production medium was used. To develop a readily scalable process, both immobilized and suspended cell systems were studied. In the immobilized cell systems, growth, glucose utilization, and alkaloid production were suppressed; for the case of membrane entrapped cells this suppression was observed to be reversible. Based on the oxygen requirements of the cells, and the oxygen transfer capabilities of a pneumatically agitated bubble column, conditions were established that allowed the growth and production dynamics observed in shake flasks to be reproduced in the air sparged column reactor. The requirement for the aseptic exchange of growth for production medium was satisfied by using a coarse cotton filter and coupling filtration to aeration. With this filtration system, media can be rapidly and completely exchanged and the filter can be quickly and effectively backwashed. By coupling filtration to aeration, a two-stage batch operation can be employed while requiring only a single bioreactor. Studies with this system demonstrated its capabilities for alkaloid production.     

Hypertrophic Chondrocytes in the Rabbit Growth Plate Can Proliferate and Differentiate into Osteogenic Cells when Capillary Invasion Is Interposed by a Membrane Filter

The fate of hypertrophic chondrocytes during endochondral ossification remains controversial. It has long been thought that the calcified cartilage is invaded by blood vessels and that new bone is deposited on the surface of the eroded cartilage by newly arrived cells. The present study was designed to determine whether hypertrophic chondrocytes were destined to die or could survive to participate in new bone formation. In a rabbit experiment, a membrane filter with a pore size of 1 µm was inserted in the middle of the hypertrophic zone of the distal growth plate of ulna. In 33 of 37 animals, vascular invasion was successfully interposed by the membrane filter. During 8 days, the cartilage growth plate was enlarged, making the thickness 3-fold greater than that of the nonoperated control side. Histological examination demonstrated that the hypertrophic zone was exclusively elongated. At the terminal end of the growth plate, hypertrophic chondrocytes extruded from their territorial matrix into the open cavity on the surface of the membrane filter. The progenies of hypertrophic chondrocytes (PHCs) were PCNA positive and caspase-3 negative. In situ hybridization studies demonstrated that PHCs did not express cartilage matrix proteins anymore but expressed bone matrix proteins. Immunohistochemical studies also demonstrated that the new matrix produced by PHCs contained type I collagen, osteonectin, and osteocalcin. Based on these results, we concluded that hypertrophic chondrocytes switched into bone-forming cells after vascular invasion was interposed in the normal growth plate.     

Mouse mammary epithelium produces a soluble heat-sensitive macromolecule that inhibits differentiation of 3T3-L1 preadipocytes.

Coculture of normal mouse mammary gland (NMMG) epithelial cells with 3T3-L1 preadipocytes resulted in inhibition of triglyceride accumulation. This inhibition was also observed when the NMMG cells were grown in inserts and placed within a 100-mm dish containing confluent 3T3-L1 cells. As the number of NMMG-containing inserts was increased, there was a progressive decline in triglyceride content of the 3T3-L1 cells. Conditioned medium from NMMG cells also resulted in a dose-dependent inhibition of adipocyte formation, and when concentrated 10-fold by passage through a filter with a cutoff of 30 kDa, all of the inhibitory activity was recovered. Heating the concentrated conditioned medium at 98 degrees C for 30 min resulted in complete loss of activity. Of several peptides tested, transforming growth factor-beta, platelet-derived growth factor, tumor necrosis factor, interleukin 6, and basic fibroblast growth factor showed inhibitory activity, whereas epidermal growth factor, insulin-like growth factor I, and transforming growth factor-alpha did not.     

Incorporation of P and Growth of Pseudomonad UP-2 on n-Tetracosane

Cultures of the marine pseudomonad UP-2 growing on n-tetracosane contained both free cells and cells bound to the solid hydrocarbon. After separation by filtration through a Whatman no. 1 filter, the numbers of free and bound cells were estimated from the amount of P incorporated into each fraction and the determined value of P incorporation per viable cell in the filtrate (free cells). During the early exponential growth phase, over 80% of the cells were bound to large pieces of n-tetracosane; as the culture approached the stationary phase, the number of bound cells remained constant, whereas free cells continued to accumulate. Pulse-labeling experiments indicated that cells grew both on the surface of the solid and in the aqueous medium. During the growth cycle, a portion of the n-tetracosane which was initially nonfilterable was recovered in the filtrate in a form which was largely cell associated. This cell-associated n-tetracosane was preferentially utilized and could completely account for the observed growth of free cells.     

Enhancement of human muscle growth in diffusion chambers by bone marrow cells

Samples of minced human muscle were cultured in millipore diffusion chambers incubated in the peritoneal cavities of mice. In about half the chambers the minced muscle samples were mixed with autogenous bone marrow cells which lead to improved myogenic growth. A similar but less marked effect was produced by mononuclear cells from the patients' blood. No growth enhancement occurred when the muscle and marrow cells were separated by a filter in double chambers. In addition to accelerated myogenesis, the chambers with added bone marrow cells had a much lower incidence of infection. This work may have practical clinical implications for the treatment of muscle injuries. Local implantation of autogenous marrow cells (+/- minced muscle) may prove useful in improving myogenic regeneration.     

Synchronous Growth and Sporulation of Bacillus megaterium

Filtration of late log-phase cultures of Bacillus megaterium ATCC 19213 grown on defined sucrose salts medium (SS) or SS plus glutamate medium (SSG) through nine layers of Whatman no. 40 filter paper in a fritted-glass disc Büchner funnel resulted in filtrates containing cells which showed synchronous growth and proceeded to sporulation. SS cells completed one synchronous division after filtration; sporulation ensued after the cessation of growth. SSG cells completed two synchronous divisions and sporulation occurred during the second division. A high degree of synchrony of vegetative growth of SSG cells was evident by the stepwise pattern of growth, by the doubling of cell numbers at each division, the high division index, and by the rapid formation of sporulation cell types and homogeneity of cell types in the filtered cultures when compared with asynchronous cultures. Because the described system gives both good growth and sporulation synchrony, the method should be useful in delineating early events in sporulation and their regulation.     

Coculture of newborn rat skin epidermal cells with Swiss 3T3 cells: effect of the phases of 3T3 cells on attachment, growth and keratin synthesis of epidermal cells.

Swiss albino mouse 3T3 cells in various states were inoculated onto one side of Millipore filters. The other side of the filter was then coated with type I collagen and inoculated with newborn rat skin epidermal cells. On coculture of these cells, the attachment, growth and keratin synthesis of epidermal cells were found to depend on the state of the 3T3 cells: 3T3 cells in the stationary phase of growth were the most effective, followed by those in the logarithmic growth phase, those in the lag phase and plasmolyzed fibroblasts being only slightly effective. The effects of 3T3 cells in different states correlated well with their abilities to synthesize type IV collagen, but not type I collagen: with an increase in type IV collagen synthesis by the 3T3 cells, attachment of epidermal cells to the cell support, and their growth and synthesis of keratins increased. This culture system is concluded to mimic conditions in skin in vivo, and therefore to be suitable for studies on the effects of fibroblasts on the growth of epidermal cells.     

An improved polyurethane support system for monitoring growth in plant cell cultures

An improved culture system for plant cells that employs filter paper resting on polyurethane saturated with liquid medium is described. It combines a simplified version of the system outlined by Weber and Lark [1979, Theor Appl Genet 55: 81–86] with the method of growth estimation described by Horsch et al. [1980, Can J Bot 58: 2402–2406]. The growth of plated cells or callus can be conveniently monitored through repeated non-destructive fresh weight measurements of the filter paper and adhering cells, thereby allowing the construction of a complete growth curve over the course of an experiment. Experiments with 3 Nicotiana genotypes (N. plumbaginifolia Viv., N. tabacum L. ‘SC 58’ and N. tabacum ‘WI 38’) and 3 Vitis vinifera L. genotypes (‘Chenin Blanc’, ‘Dogridge’ and ‘White Riesling’) clearly demonstrate higher growth rates of plated cells on polyurethane supports compared with agar. Further experiments with N. plumbaginifolia illustrate the use of polyurethane supports for culturing cells at low pH (4.0) and the recovery of spent medium for monitoring changes in pH. These features will greatly facilitate quantitative studies of mineral nutrition and metal toxicity in cultured cells. Polyurethane supports also allow the incorporation of conditioned medium or feeder cells to support the growth of cells at low densities and facilitate the rapid recovery of variant cells.     

Co-culture of primary pulmonary cells to model alveolar injury and translocation of proteins

Summary Primary rat alveolar type II cells and early passage rat lung fibroblasts were co-cultured on opposite sides of a collagen-coated polycarbonate filter. This is an approach to “model”, in part, an alveolar wall to study mechanisms of cytotoxicity and translocation of bioactive materials from the alveolar space to the lung interstitium. Type II cells were recovered from adult rat (Fischer 344) lungs by enzyme digestion and “panning”. Lung fibroblasts were separated from the same species, cultured initially in 10% fetal bovine serum and used in the co-culture system at early passage. The type II cells formed a monolayer of defferentiated epithelium which provided a barrier on the upper side of the collagen (human type IV)-coated filter. The fibroblasts on the bottom of the filter replicated logarithmically in the presence of serum, could be rendered quiescent in defined medium and then returned to rapid growth phase with the reintroduction of serum. The intact epithelial monolayer excluded trypan blue, albumin, platelet-derived growth factor, and alpha₂-macroglobulin from the lower compartment of the culture chamber. Altering the integrity of the monolayer by a variety of means allowed translocation of these materials through the collagen-coated filters. Particularly interesting was the effect of taurine chloramine which caused subtle changes in the alveolar epithelium and allowed subsequent translocation of albumin. In addition, we showed that rat alveolar macrophages remain viable with some spreading on the surface of the epithelial monolayer. This co-culture system will have future application in the study of how reactive oxygen species might affect the epithelial barrier, and whether macrophage-derived growth factors can influence fibroblast proliferation if the monolayer is intact or injured.