A new in vitro assay for cell motility and proliferation

Abstract. We have described and characterized a new micropore membrane assay for migration and proliferation of cells of various tumourigenic potential. The assay was developed to facilitate analysis of some aspects of cancer invasion and metastasis. Tumorigenic and non-tumorigenic C3H/10 T1/2 cells grow in and migrate out of a culture chamber during a 1–11 day period, the shorter periods are used for chambers with 6 μ m thick polycarbonate membranes, the longer ones for 140 μ m thick cellulose nitrate membranes. Cell growth within the chambers, in their micropore membranes and on the outside of the membranes, was assessed with microscopy, electronic cell counting, flow cytometry of propidium iodide (PI) stained cells, and ³H-thymidine ([³H]TdR) incorporation. A complete retrieval of intact cells that have traversed the membraneous chamber wall is possible, and these cells can be recultured or used in other studies. The tumorigenic cells had a steeper growth curve in vitro than the non transformed cells, but the relative sizes of the emigrated subpopulations were not significantly different. The subpopulation of tumorigenic cells that emigrated spontaneously from the chambers was less able than the subpopulation retained to populate secondary chamber cultures, suggesting that the clonogenic (stem) tumour cells are 'slow movers'.     

Continuous flow electrophoretic separation of proteins and cells from mammalian tissues

A new continuous flow electrophoretic separator for cells and macromolecules was built and tested in laboratory experiments and in the microgravity environment of space flight. Buffer flows upward in a 120-cm long flow chamber, which is 6 cm wide X 1.5 mm thick in the laboratory version and 16 cm wide X 3.0 mm thick in the microgravity version. Electrophoretic subpopulations are collected in 197 fractions spanning 16 cm at the upper end of the chamber. The electrode buffer is recirculated through front and back cooling chambers, which are also electrode chambers. Ovalbumin and rat serum albumin were used as test proteins in resolution and throughout tests; resolution of these two proteins at 25% total w/v concentration in microgravity was the same as that found at 0.2% w/v concentration in the laboratory. Band spreading caused by Poiseuille flow and conductance gaps was evaluated using polystyrene microspheres in microgravity, and these phenomena were quantitatively the same in microgravity as in the laboratory. Rat anterior pituitary cells were separated into subpopulations enriched with cells that secrete specific hormones; growth-hormone-secreting cells were found to have high electrophoretic mobility, whereas prolactin-secreting cells were found to have low electrophoretic mobility. Cultured human embryonic kidney cells were separated into several electrophoretic subfractions that produced different plasminogen activators; a medium-high-mobility subpopulation and a medium-low-mobility subpopulation each produced a different molecular form of urokinase, whereas a high- and an intermediate-mobility subpopulation produced tissue plasminogen activator. Canine pancreatic islets of Langerhans cells were separated into subpopulations, which, after reaggregation into pseudoislets, were found to be enriched with cells that secrete specific hormones; insulin-secreting beta cells were found in lowest mobility fractions, whereas glucagon-secreting alpha cells were found in the highest mobility fractions. Results of particle electrophoresis experiments were comparable in microgravity and in the laboratory, since cell densities that overloaded the carrier buffer (resulting in zone sedimentation) were avoided, and a 500-fold increase in protein throughput was achieved without compromising resolution in microgravity.     

Microvascular A-V shunts and the growth of autologous tissue flaps in Millipore chambers.

An animal model was developed using the male Sprague-Dawley rat to establish a protocol and design criteria for the growth of autologous tissue based on a microvascular pedicle. It became apparent that growth within the chamber depended heavily on membrane porosity and the presence of the microarteriovenous shunt. Different membrane porosities ranging from 0.0, 0.25, 1.2, to 8.0 microns were evaluated (n = 48). Optimal growth occurred with the 1.2-microns and the 8.0-microns micropore Millipore. Growth within the chamber consisted of a radial deposition of collagen and neovascularization originating from the arteriovenous (A-V) anastomosis. In contrast, control chambers (no A-V anastomosis), with the preceding range in membrane porosity, experienced little to no cell growth. In addition, the majority of A-V shunts did not remain patent in chambers having 0.0-microns porosity or 0.25-microns porosity. Thus it is apparent that a strong relationship exists between membrane porosity, patency, and in situ vascularization allowing for the proliferation of cells and collagen.     

ATP luminescence-based motility-invasion assay

Directional motility and invasion assays are largely based on the use of Boyden chambers or Transwell culture inserts in which porous membranes separate seeded cells from a chemotactic factor supplied in the medium outside the chamber. The major obstaclefor most currently available assays is that they lack a sensitive, easy, and reliable method of quantifying the nonmotile cell populations. Failure to accountfor all cells within the assay chamber prohibits the determination of percentages of migrated cells. Here we describe an ATP luminescence-based motility-invasion (ALMI) assay that circumvents this problem, enabling investigators to quantify directional cell migration or invasiveness easily. The ALMI assay is based on the detection of ATP in viable cells harvested from inert surfaces that do not generate background signals. We demonstrate how the ALMI assay can be used to assess the effects of various experimental conditions such as growth factor stimulation and ethanol exposure on cell migration. In addition, precoating the membranes with extracellular matrix molecules enabled the measurement of the cell invasion. In conclusion, the ALMI assay provides a reliable and flexible method to quantify cell motility and invasiveness using a luminescence microplate reader.     

Chemotaxis by Entamoeba histolytica

A micropore membrane procedure to assay taxis by Entamoeba histolytica is described and the results of studies of responses to a variety of soluble substances, bacteria, an rat colon washings using this procedure are reported. Trophozoites migrated in blind well chambers through 8-micron pore size polycarbonate membranes but not nitrocellulose membranes up to 12 micron pore size. Amoebae were attracted toward fresh axenic culture medium (TYI-S), an enzymatic hydrolysate of casein (Trypticase), and a partially purified preparation of N-acetylneuraminic acid from egg mucin, but not purified N-acetylneuraminate or a variety of other low molecular weight metabolites. The response was verified as chemotaxis by checkerboard analysis. Amoebae migrated most dramatically toward suspensions of all of seven bacterial species tested, including motile and non-motile, gram-negative and gram-positive rods and cocci. This response was diminished when the bacteria concentration gradient was eliminated. The response to bacteria culture filtrates was less than 10% of that to bacterial suspensions. A response to clarified washings from the rat colon was detected; this was diminished but not eliminated by filter sterilization of the washings. We concluded that some soluble molecules, possibly of intermediate molecular size, whole bacteria, and both soluble and particulate components of the rat colon provide tactic stimuli for E. histolytica. Scanning electron micrographs of trophozoites migrating towards attractants through membranes showed narrow, extended pseudopodia entering the membrane pores, and enlarging spheres exiting as the cells proceeded through.     

A simple micropore system for experimental studies on trichomonad parasites

A simple leak-free micropore chamber containing protozoan parasite species was implanted subcutaneously on the back of hamsters and evaluated for viability and multiplication of protozoan parasites. Trophozoites of defined strains of Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and Tritrichomonas foetus were used; their survival and multiplication in the chambers formed the basis of evaluation. Entamoeba histolytica and G. lamblia did not survive more than 6 hr and succumbed due to cellular adhesion. Trichomonas vaginalis and T. foetus survived 3 and 6 days and multiplied a maximum of 3.6 and 26 times, respectively. This indicated that exchange of body fluids and cells needed for the survival and multiplication of trichomonads readily occurs. This preliminary observation showed that micropore chambers may be useful for chemotherapeutic and immunological studies on trichomonads in ectopic sites.     

Insulin-like growth factor-II/mannose 6 phosphate receptors facilitate the matrix effects of latent transforming growth factor-β1 released from genetically modified keratinocytes in a fibroblast/keratinocyte co-culture system

This study was conducted to explore the mechanism of activation of transforming growth factor-β1 (TGF-β1) which is critical to its role in many physiological and pathological conditions. To date, almost all reports concerning TGF-β1 activation delineated that release of mature TGF-β1 from latency associated protein (LAP) is required for its activation. We report that latent TGF-β1 (LTGF-β1) released from TGF-β1 genetically modified keratinocytes grown in the top chamber of a co-culture system functions as a fibrogenic factor through interaction with insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptors of human dermal fibroblasts grown in the lower chamber of this system. Following successful transduction, the pLin-LTGF-β1 vector was amplified in PA317 packaging cells which possess viral structural proteins for vector in the presence of neomycin. Conditioned medium derived from packaging cells containing competent viral particles was then used to transduce either keratinocytes or fibroblasts grown in the upper chamber of a co-culture system, in which a 0.4 μm porous membrane separates the two chambers. In this way, LTGF-β1 produced by transduced cells in the upper chamber is released and diffuses into the lower chambers where dermal fibroblasts are grown. Conditioned medium from the lower chamber was removed 3 days later and used to evaluate the latency and bioactivity of TGF-β1 using enzyme-linked immunosorbent assay (ELISA) and mink lung (Mv1Lu) epithelial growth inhibition assay. Cells were also harvested and used for RNA extraction. The results of these experiments showed that 1) the TGF-β1-LAP complex, which was latent in traditionally used mink lung growth inhibition assay, directly modulated the expression of collagenase, type I, and type III collagen mRNA by dermal fibroblasts; 2) this stimulation was inhibited by M6P in a dose-dependent manner; 3) the TGF-β1-LAP inhibits Mv1Lu epithelial cells only when this complex was incubated with cell membranes isolated from dermal fibroblasts; and 4) LTGF-β1 activation seems to occur through a conformational alteration rather than by release of the mature TGF-β1 from LAP in our co-cultured system. This conformational alteration seems to occur through the interaction of the TGF-β1-LAP complex with the IGF-II/M6P receptors. Thus, the quantity of IGF-II/M6P receptors is important in cellular response to LTGF-β1 in any physiological and pathological conditions. J. Cell. Physiol. 180:61–70, 1999. © 1999 Wiley-Liss, Inc.     

Embryoid bodies cultured in in vivo diffusion chambers show reduced tumorigenicity while retaining expression of F9 antigens

Embryoid bodies of the mouse teratocarcinoma OTT6050 were dissociated into single cells and cultured in diffusion chambers implanted into the peritoneal cavities of mice. The syngeneic host mice, into which the cells of embryoid bodies cultured in the diffusion chambers had been injected, survived much longer than those which received the original cells of embryoid body. But in the case of the F9 cells, obtained in the same culture conditions, only a slight decrease in tumorigenicity was observed. By contrast, the F9 antigenic expression was observed on both F9 and embryoid body cells cultured in diffusion chambers. Judging from the determination of adult-type antigenic expressions, the differentiation of the cells in chamber was negligible. These results suggest that the tumorigenic activity of the embryoid body cells cultured in vivo in a diffusion chamber is almost suppressed, but that they continue in an undifferentiated state.     

Virulence and immunogenicity of types 1 and 3 Neisseria gonorrhoeae in guinea pig subcutaneous chambers.

The relative virulence and immunogenicity of type 1 (T1) and type 3 (T3) cells of Neisseria gonorrhoeae were determined by tests with two different kinds of subcutaneous chambers in guinea pigs. In tests with a tissue nonencapsulated (NE) chamber, T1 gonococci were found to be greater than 1000 times more virulent as well as about 1000 times more immunogenic than T3 cells of the same gonococcal strain. However, T1 and T3 cells were found to be equally virulent for a tissue encapsulated (TE) chamber in guinea pigs. Analysis of fluids from the two types of chambers in a complement-dependent bactericidal assay revealed that the NE chamber fluid contained a substantially higher level of complement activity than fluid from TE chambers. The decline in complement level of chamber fluids due to tissue encapsulation was also confirmed by quantitation with rocket gel electrophoresis. A greater resistance of T1 cells to the bactericidal effects of complement appeared to provide a mechanism by which the T1 cells were most virulent than T3 cells for subcutaneous chambers in guinea pigs. Consequently, the NE chamber implant would appear to provide a more relevant environment for studying the virulence, as well as immunological characteristics of gonococcal strains and experimental immunogens.     

F-actin rings are associated with the ring canals of the Drosophila egg chamber

Staining of Drosophila egg chambers with rhodaminyl-lysine-phallotoxin (RLP), a specific stain for F-actin, has demonstrated the presence of dense F-actin rings associated with the inner surfaces of the ring canals. They were first observed in the distal part of the germarium where rings of four different size classes were found, differing in diameter by up to twofold. The ring sizes are considered to correspond to the ring canals formed at each of four successive incomplete cleavages. During the growth of the egg chamber the actin rings were found to increase in diameter from less than 1 micron to approx. 10 micron. Concomitantly a secondary outer ring of more diffuse material is built up in association with the cell membranes. A well developed array of microfilament bundles was also associated with the nurse cell plasmalemma. In stages where the transfer of the bulk of the nurse cell cytoplasm into the oocyte was occurring the rings came closer together in a central area. In late stage chambers the F-actin rings and the microfilament bundles appeared to be incorporated into large irregular masses of actin, which subsequently disappeared as the mature oocyte formed. The F-actin rings are suggested to act as mechanical strengthening elements for the canal plasmalemma, whilst cytoplasmic transport occurs through the ring canals.     

Phenotypically determined resistance of Neisseria gonorrhoeae to normal human serum: environmental factors in subcutaneous chambers in guinea pigs.

Some gonococci obtained from human urethral exudate or from subcutaneously implanted chambers in guinea pigs show a resistance to killing by human serum which is lost on sub-culture in vitro after a few generations. The environmental factors which may influence the phenotypic expression of resistance to serum killing were investigated in guinea pig chambers and in chamber fluid in vitro. The redox potential in chambers before and after infection was lower than that of heart blood but conditions were not anaerobic; H2O2 increased the redox potential but did not decrease gonococcal serum resistance. The chambers were slightly alkaline before and after infection. When the concentration of glucose (depleted in infected chambers by the abundant polymorphonuclear cells) was restored to excess, the serum resistance of the gonococci was unaffected. Concentrations of free amino acids in chambers changed little during infection. Gonococci adapted to growth in chambers and subsequently rendered serum-sensitive by growing once on agar reverted to serum-resistance after 0.5 to 1 h incubation in chamber fluid in vitro at 37 degrees C but not at 25 degrees C or 4 degrees C. After 16 to 24 h growth at 37 degrees C, resistance was again lost. The reversion to serum resistance did not occur in a complex laboratory medium. Examination of the chamber fluid after growth of gonococci in vitro showed depletion of lactate, glutamine and proline.     

Incubation of environmental samples in a diffusion chamber increases the diversity of recovered isolates

The majority of microorganisms from natural environments cannot be grown in the laboratory. The diffusion-chamber-based approach is an alternative method that allows microorganisms to grow in their natural environment. An inoculum is sandwiched between semipermeable (0.03-mum-pore-size) membranes of the chamber, which is then returned to the source environment. The chamber allows for a free exchange of chemicals with the external milieu by diffusion while restricting the movement of cells. We used freshwater pond sediment to inoculate diffusion chambers and petri dishes. The diffusion chambers were incubated on top of the sediment for 4 weeks. Both chamber and petri dish cultivation resulted in the isolation of numerous representatives of Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. However, the diffusion-chamber-based approach also led to the isolation of species from rarely cultivated groups, such as Deltaproteobacteria, Verrucomicrobia, Spirochaetes, and Acidobacteria. Material from the chambers was also transferred to new chambers in order to learn whether this will increase the recovery of isolates. Several isolates could be obtained only from material transferred through multiple diffusion chambers. This suggests that continuous cultivation in diffusion chambers adapts some microorganisms for growth under otherwise prohibitive in vitro conditions.     

Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.     

Incubation of Environmental Samples in a Diffusion Chamber Increases the Diversity of Recovered Isolates

The majority of microorganisms from natural environments cannot be grown in the laboratory. The diffusion-chamber-based approach is an alternative method that allows microorganisms to grow in their natural environment. An inoculum is sandwiched between semipermeable (0.03-μm-pore-size) membranes of the chamber, which is then returned to the source environment. The chamber allows for a free exchange of chemicals with the external milieu by diffusion while restricting the movement of cells. We used freshwater pond sediment to inoculate diffusion chambers and petri dishes. The diffusion chambers were incubated on top of the sediment for 4 weeks. Both chamber and petri dish cultivation resulted in the isolation of numerous representatives of Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. However, the diffusion-chamber-based approach also led to the isolation of species from rarely cultivated groups, such as Deltaproteobacteria, Verrucomicrobia, Spirochaetes, and Acidobacteria. Material from the chambers was also transferred to new chambers in order to learn whether this will increase the recovery of isolates. Several isolates could be obtained only from material transferred through multiple diffusion chambers. This suggests that continuous cultivation in diffusion chambers adapts some microorganisms for growth under otherwise prohibitive in vitro conditions.     

Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fourth leading cause for cancer-related mortality. Stem cells have been implicated in pancreatic tumor growth, but the specific role of these cancer stem cells in tumor biology, including metastasis, is still uncertain. We found that human pancreatic cancer tissue contains cancer stem cells defined by CD133 expression that are exclusively tumorigenic and highly resistant to standard chemotherapy. In the invasive front of pancreatic tumors, a distinct subpopulation of CD133(+) CXCR4(+) cancer stem cells was identified that determines the metastatic phenotype of the individual tumor. Depletion of the cancer stem cell pool for these migrating cancer stem cells virtually abrogated the metastatic phenotype of pancreatic tumors without affecting their tumorigenic potential. In conclusion, we demonstrate that a subpopulation of migrating CD133(+) CXCR4(+) cancer stem cells is essential for tumor metastasis. Strategies aimed at modulating the SDF-1/CXCR4 axis may have important clinical applications to inhibit metastasis of cancer stem cells.     

Identification of surrogate rodent hosts for larval Onchocerca lienalis and induction of protective immunity in a model system.

The objectives of this project were to screen a variety of inbred rodent species and strains to determine their usefulness as surrogate hosts for the study of the early larval development of Onchocerca lienalis and then to use a selected model to study the induction of protective immunity. In the primary screen, 6 strains of mice, 5 strains of rats, jirds, and multimammate rats were tested. Animals were infected with fresh O. lienalis by subcutaneous implantation of third-stage larvae (L3) contained in diffusion chambers covered with 5.0-microns pore-size membranes. After 7 days the chambers were recovered, and larval viability and growth were assessed. Approximately one-half of inoculated larvae were recovered alive regardless of the host tested. Larvae were implanted in CBA/J and DBA/2J mice in chambers covered with membranes that prevented host cells from entering; survival and growth rates of the larvae were not altered by the absence of cells from the chambers. Cryopreserved larvae were implanted in chambers with 5.0-microns pore-size membranes in CBA/J and DBA/2J mice and Wistar Furth rats for 3-28 days. No statistically significant difference was seen in the larval recoveries on days 3-28 in all 3 hosts. Statistically significant increases in length were seen in the 3 strains from day 3 to day 14, after which growth appeared to cease. Molting from L3 to fourth-stage larvae was observed in all 3 hosts beginning on day 3, with most larvae completing the molt by day 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic study of human scirrhous gastric carcinoma during cultivation in diffusion chambers]

The growth of the epithelial and connective tissue cells was noted in cultivation of human schirrous carcinoma of the stomach in diffuse chambers. In difference to the initial tissue of the tumour in vitro in which no incorporation of thymidine-H3 into the connective tissue cells was noted, these cells proved to be labeled under conditions of growth in the chambers. One hour after the administration of thymidine-H3 the percentage of cells with labeled nuclei averaged 25.1% this considerably exceeding the value of the label index determined under conditions of incubation in vitro of the initial tumour tissue (6.6%). Under conditions of cultivation there was revealed a rapidly proliferating subpopulation of cells with the mitotic cycle duration of 14.8 hours.     

Automated production of cultured epidermal autografts and sub-confluent epidermal autografts in a computer controlled bioreactor

The objective of this work was to engineer an automated system for the production of cultured epidermal autografts and sub-confluent cultured epidermal autografts. Human epidermal cells were grown directly on a transparent FEP film, which was held in place and surrounded by a polycarbonate growth chamber. The growth chambers were stacked to accommodate various surface area requirements. To monitor the development of the grafts, the upper-most growth chamber in the stack was periodically placed on a standard phase contrast microscope. The growth chambers were connected to a multi-channel peristaltic pump, which was controlled automatically to manage fluid-handling operations. Sub-confluent graft production involved removing the epidermal-film composite from the growth chambers and cutting desired graft geometries. Producing cultured epidermal autografts involved (1) removing the confluent epidermal-film composite from the growth chambers, (2) treating the composites with dispase, and (3) clipping the detached cultured epidermis to a synthetic support. Twelve to fifteen days were required to produce sub-confluent grafts (total surface area 3500-4500 cm2 50% confluent) and 18 to 24 d were required to produce standard cultured epidermal autografts (total surface area 3500-4500 cm2). The system reduces the tedious manual labor associated with producing cultured epidermal autografts.