Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization

We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.     

Transparent polymeric cell culture chip with integrated temperature control and uniform media perfusion

Modern microfabrication and microfluidic technologies offer new opportunities in the design and fabrication of miniaturized cell culture systems for online monitoring of living cells. We used laser micromachining and thermal bonding to fabricate an optically transparent, low-cost polymeric chip for long-term online cell culture observation under controlled conditions. The chip incorporated a microfluidic flow equalization system, assuring uniform perfusion of the cell culture media throughout the cell culture chamber. The integrated indium-tin-oxide heater and miniature temperature probe linked to an electronic feedback system created steady and spatially uniform thermal conditions with minimal interference to the optical transparency of the chip. The fluidic and thermal performance of the chip was verified by finite element modeling and by operation tests under fluctuating ambient temperature conditions. HeLa cells were cultured for up to 2 weeks within the cell culture chip and monitored using a time-lapse video recording microscopy setup. Cell attachment and spreading was observed during the first 10-20 h (lag phase). After approximately 20 h, cell growth gained exponential character with an estimated doubling time of about 32 h, which is identical to the observed doubling time of cells grown in standard cell culture flasks in a CO2 incubator.     

Mathematical Determination of Cell Population Doubling Times for Multiple Cell Lines

Cell cycle times are vital parameters in cancer research, and short cell cycle times are often related to poor survival of cancer patients. A method for experimental estimation of cell cycle times, or doubling times of cultured cancer cell populations, based on addition of paclitaxel (an inhibitor of cell division) has been proposed in literature. We use a mathematical model to investigate relationships between essential parameters of the cell division cycle following inhibition of cell division. The reduction in the number of cells engaged in DNA replication reaches a plateau as the concentration of paclitaxel is increased; this can be determined experimentally. From our model we have derived a plateau log reduction formula for proliferating cells and established that there are linear relationships between the plateau log reduction values and the reciprocal of doubling times (i.e. growth rates of the populations). We have therefore provided theoretical justification of an important experimental technique to determine cell doubling times. Furthermore, we have applied Monte Carlo experiments to justify the suggested linear relationships used to estimate doubling time from 5-day cell culture assays. We show that our results are applicable to cancer cell populations with cell loss present.     

Cell adhesion monitoring using a quartz crystal microbalance: comparative analysis of different mammalian cell lines

The quartz crystal microbalance (QCM) has been widely accepted as a sensitive technique to follow adsorption processes in gas as well as in liquid environments. However, there are only a few reports about the use of this technique to monitor the attachment and spreading of mammalian cells onto a solid support in culture. Using a QCM-setup we investigated the time course of cell attachment and spreading as a function of seeding density for three widespread and frequently used cell lines (MDCK strains I and II and Swiss 3T3-fibroblasts). Results were found to be in good agreement with the geometrical properties of the individual cell types. The shifts of the resonance frequency associated with confluent cell layers on top of the quartz resonators were found to be dependent on the cell species [MDCK-I: (320±20) Hz; MDCK-II: (530±25) Hz; 3T3: (240±15) Hz] reflecting their individual influence on the shear oscillation of the resonator. These findings are discussed with respect to the basic models of materials in contact with an oscillating quartz resonator. We furthermore showed by inhibition-assays using soluble RGD-related peptides, that only specific, integrin mediated cell adhesion is detected using this QCM approach, whereas the sole presence of the cellular body in close vicinity to the resonator surface is barely detectable.     

Studies on the mechanism of cell attachment to a substratum with serum in the medium: further evidence supporting a requirement for two biochemically distinct processes

Treatment of baby hamster kidney cells with cytochalasin B or omission of divalent cations from the culture medium are conditions resulting in an inhibition of cell attachment at rest; however, these conditions do not result in inhibition of cell attachment in a centrifugal field. In marked contrast, treatment of cells with trypsin or with tranquilizers such as fluphenazine results in an inhibition of cell attachment at rest or in a centrifugal field. The evidence is interpreted to indicate that cell adhesion involves at least two biochemical processes: formation of the adhesive bond per se (inhibited by tranquilizers or trypsin) and a mechanical process of cell-to-substratum contact and/or spreading (inhibited by cytochalasin B or omission of divalent cations from the medium).     

A rapid and convenient preparation of [4-3H]NADP and stereospecifically tritiated NADP3H

The glass-binding properties of a number of purified glycoproteins capable of promoting attachment and spreading of a variety of types of animal cells in culture have been examined. Two such factors in human serum, fibronectin and serum spreading factor, exhibited strong affinities for glass beads and could be eluted from glass-bead columns under similar conditions. A number of other glycoproteins of human serum that do not promote cell adhesion did not bind to glass beads under conditions that resulted in binding of serum spreading factor or fibronectin. At a sufficiently low ratio of serum volume to glass-bead volume, human serum could be simultaneously depleted of serum spreading factor, fibronectin, and cell spreading-promoting activity by glass-bead affinity chromatography. Laminin, another cell spreading-promoting glycoprotein, possessed glass-binding properties similar to those of serum spreading factor and fibronectin while chondronectin, a fourth cell spreading-promoting factor of more limited specificity of biological activity and distribution in vivo, did not exhibit a strong interaction with glass beads under the same conditions. These observations suggest that glass-bead column affinity chromatography may prove useful as a general method for isolation and study of glycoprotein factors promoting attachment and spreading of cells in culture.     

Adhesion of mouse blastocysts to uterine epithelium in culture: a requirement for mutual surface interactions

Blastocysts readily adhered to inert materials in culture, but they resisted adhesion to living cells even after several days under conditions which encouraged cell aggregation. As far as could be determined by observing their spreading behavior on polylysine- and polyglutamate-coated dishes, the mechanism of adhesion of blastocysts to inert surfaces was similar to that of freshly dissociated cells and cell lines. However, their adhesion to vesicles of isolated uterine epithelium, which was encouraged by hanging drop culture, was by a different mechanism that involved microvilli on both the embryonic and maternal surfaces. This interactive step, which was similar to that seen during attachment in vivo, was followed by a brief period of close trophoblast-epithelial contact which led ultimately to phagocytosis of sloughed epithelium. Blastocysts showed a clear preference for adhesion to cultured epithelium in vesicles that had begun to collapse. In this case the cells showed a columnar profile with sharply defined microvillous apexes, unlike the flattened cells in fully expanded vesicles or on culture dishes. We conclude that the preimplantation adhesion of mouse blastocysts requires specific changes on both the embryonic and maternal surfaces to overcome the mutual nonadhesiveness typical of epithelia. The relatively rapid adhesion of blastocysts to a culture dish, on the other hand, is more typical of the well-known spreading behavior of cells on a highly attractive surface.     

Establishment and characterization of the NEYS cell line derived from carcinosarcoma of human ovary with special reference to the susceptibility test of anticancer drugs

A cell line designated as NEYS was established from ovarian carcinosarcoma (stage IIIc) of a 56-year-old Japanese woman. The extirpated original tumor was carried in growth medium at 0 °C to the culture room. The primary culture was done on 20 August 2003. The cell line was composed of angular adhesive cells and showed neoplastic and pleomorphic features, such as bizarre aggregation of chromatin granules, an irregular thickening nuclear membrane and multiple large nucleoli. They grew as multi-layered cultures without contact inhibition. The cells proliferated moderately, and population doubling time was about 56 h. The chromosome number showed an underdiploidy of aneuploidy. The modal chromosome numbers were 37 (36%) and 38 (26%). The cultures produced carcinoembryonic antigen (27.4 ng/mL), carbohydrate antigen 19-9 (210 U/mL), and carbohydrate antigen 125 (526 U/mL). The NEYS cells did not give rise to transplant tumors in nude mice, and showed no susceptibility against cisplatin (CDDP), CPT-11, carboplatin, Paclitaxel, Taxotere and 5-FU. This cell line is useful for studies on the histogenesis of carcinosarcoma and susceptibility of cancer drugs in human ovarian carcinosarcoma. The immunohistochemical and ultrastructual analysis demonstrated that NEYS cells showed epithelial and mesenchymal differentiation, and supported the metaplasis theory as the cause of carcinosarcoma.     

Osteogenic cell contact with biomaterials influences phenotype expression

Relationship between (1) osteoblast adhesion and spreading, and (2) phenotype expression was investigated. Cellular adhesion and spreading were estimated after short time (24 h), whereas proliferation and other osteoblast functions – after 7 days. Primary human osteogenic cells were seeded on the samples of titanium (T), surgical steel (S) and tissue culture polystyrene (PS), and incubated at 37 °C. After 24 h a number of samples were stained with crystal violet and Hoechst; the average single cell area (spreading) and adhering cell number was measured on each sample. The remaining cultures were supplemented with dexamethasone (10 nM) and -glycerophosphate (5 mM), and incubation was continued for 7 days. The cells on each sample were counted and the following tests were performed: XTT mitochondrial activity assay, total protein content, alkaline phosphatase activity (ALP), Sirius Red test for collagen, osteocalcin and calcium concentration. After 24 h significantly greater cell spreading (p < 0.05) and number (p < 0.05) were on T than on S. After 7 days significantly higher on T than on S were: ALP activity (p < 0.000001), collagen (p < 0.0015) and calcium concentration (p < 0.03). XTT results were bigger on S than on T. In control – XTT results were higher than on the metals; collagen and ALP were lower than on T, and calcium level was significantly lower than on T and S (p < 0.025). After 7 days there were no differences in cell number between T and S. Cell number (24 h) correlated with ALP activity (7 days) on steel (coefficient of correlation, CC = 0.866) and titanium (CC = 0.742). The spreading correlated on steel and on titanium with calcium concentration (CC = 0.645 on S, CC = 0.696 on T) and collagen level (CC = –0.638 on S, CC = –0.69 on T). Conclusions: Better conditions for osteoblast phenotype expression on T after 7 days of culture coincided with greater adhesion and spreading of cells after 24 h on T, as compared with S. The initial contact of cells with underlying surface may influence osteoblast functions and possibly, bone regeneration and implant osteointegration in vivo. Early cell spreading may be an indicator of further expression of osteoblast phenotype and may be important for application of osteogenic cells in reconstructive surgery.     

Interferon effects on the growth and division of human fibroblasts.

The overall rate of proliferation of human fibroblasts in culture is reduced at interferon concentrations greater than 40 international reference units (U)/ml. Inhibition is near maximal at 640 U/ml, at which concentration the doubling time between 24 and 72 h after beginning of treatment is increased 2–3 times over the control value. Inhibition of cell proliferation was not readily reversible upon removal of interferon and refeeding of cultures. Study of the mitotic behavior of individual cells showed that the first intermitotic interval after beginning of treatment with interferon (640 U/ml) was prolonged in about two-thirds of the cells. In this fraction, many cells failed to divide again after the second post-treatment mitosis, while others exhibited a progressively increasing intermitotic interval with subsequent divisions. One-third of the interferon-treated fibroblasts initially divided at a rate similar to the rate of proliferation of control cells, but subsequently these cells also slowed down and finally stopped dividing. After treatment at 640 U/ml for 3 days, the rates of DNA, RNA, and protein synthesis were depressed to 86, 75, and 64% of control values, respectively. However, the interferon-treated fibroblasts had grown larger than control cells as indicated by the following parameters: cell attachment area, 165%; volume, 131%; DNA content, 130% and protein content, 150%. Thus, interferon does not prevent cell growth, but interferes with cell division.     

Thrombospondin-induced attachment and spreading of human squamous carcinoma cells

Thrombospondin (TSP) induced the attachment and spreading of human squamous carcinoma cells on plastic culture dishes and dishes coated with type I or type IV collagen. Increased adhesion was detected as early as 15 min after treatment. Dose-response studies indicated that 1-5 micrograms of TSP per 35 mm (diameter) culture dish was sufficient to induce a response and that a half-maximal response occurred at 10 micrograms of TSP/dish. The squamous carcinoma cells synthesized TSP as indicated by biosynthetic labeling experiments. TSP was secreted (or shed) into the culture medium by these cells and also became bound to the cell surface. TSP also promoted adhesion of human keratinocytes, fibroblasts and fibrosarcoma cells but did not induce attachment or spreading of human melanoma or glioma cells, although these cells did respond to laminin.     

Cell division, ciliary regeneration and cyclic AMP in a unicellular system

The average speed of nuclear translocation of 3T3 cells, recorded in a time-lapse film of a perfused culture, was negatively correlated with the number of contacting cells, and, to a lesser degree, with the amount of a cell's perimeter in contact with other cells. When a cell was in contact with five or more other cells, its speed was reduced by 50%, on the average, although the variation in individual cell speed was considerable at each level of contact. A partial correlation analysis showed that any extracellular soluble factors governed by the local cell density had little or no effect on speed, relative to the prominent effect of the number of cell-cell contacts, and hence that 3T3 cells display true contact inhibition of speed. This confirms the original demonstration by Abercrombie and Heaysman (1952), who studied chick embryo heart fibrpolasts. In our study, the relationships between average speed and age of the culture was such that a possible independent contribution of a time-associated factor other than contact to the diminution in average speed, although not necessary to account for the data, could not be excluded. The same intercellular contacts found to inhibit speed in this study were previously reported to cause no immediate prolongation of individual cell generation times, despite the fact that the filmed culture was undergoing so-called “contact” inhibition of cell division. In the present study, moreover, no correlation was observed between the average speeds of individual cells and their generation times. Hence, postconfluence inhibition of cell division and contact inhibition of speed of cell movement seem to be independent phenomena.     

Production of factors required for cell attachment and spreading is a constitutive property in mouse A9 cells.

It is demonstrated here that cells in a suspension culture of an established mammalian cell line release non-dialyzable factors into their growth medium. These factors are capable of promoting the adhesion and spreading of these cells on a generally non-attachable substratum and also promote spreading on an adhering substrate. Evidence is presented which demonstrates that the spreading promotion activity of the condition medium is dependent on the cell density of the culture from which it was derived. Dilution of the conditioned medium results in a proportionate dilution of the spreading promotion activity. The results clearly demonstrate that the production of this spreading promotion factor is continued even in the absence of cell to substrate attachment.     

Unique morphology of HeLa cell attachment, spreading and detachment from microcarrier beads covalently coated with a specific and non-specific substratum

A SEM and TEM evaluation of adhesion of HeLa-S3 cells to suspensions of culture microcarriers coated with various substrata revealed two unique cell morphologies. One is similar to that for cells attaching to culture dishes and the other one only appeared with microcarriers stirred under high shear conditions. The usual appearance of a spreading cell is to change from a sphere to the shape of a 'fried egg'. This proceeded in HeLa cells by a radial extension of the filopodia in between which the cytoplasm subsequently filled. Fluorescent antibody staining of actin suggested that more actin was present at the periphery of the spreading edges of the cell than inwards. The above morphology was characteristic of HeLa cell attachment to gelatin-coated microcarriers. However, the morphology of the attachment to microcarriers coated with non-biological substances such as negatively charged sulfonate groups or positively charged polyethyleneimine or even with the attachment protein laminin was quite different. Here the cells attached and began to spread as with gelatin-microcarriers, however, the spreading was not radial but occurred from one or two major regions of the cell periphery. The cell then appeared to constrict with the formation of a substratum attached pedestal upon which the cell body was perched. With time the cell pinched-off from pedestal. Evidence indicated that the pedestal was quite fragile. Furthermore, fluorescent antiactin staining indicated that the initial spreading region contained abundant actin which was depleted upon pedestal formation and detachment. The above in addition to previous kinetic measurements provided the information to classify cell substrate attachment materials into two distinct types. One is specific substrata which promote normal attachment and spreading and appear to interact with specific cell surface proteins. The other is non-specific substrata which in high shear conditions induces pedestal formation followed by pinching-off of the cells. Had previous attachment assays been done under high shear as done with the microcarriers and HeLa cells it is likely that substrata classified as specific might be reclassified into non-specific.     

Relations Between Individual Cellular Motions and Proliferative Potentials in Successive Cultures of Human Keratinocytes

In the successive cultures of human keratinocyte cells, cellular motions of extension and rotation were analyzed based on observation of the individual cells, to evaluate the proliferative potential in a whole cell population. In lag phases of the serial cultures, an extension index of individual cells, R_E, was defined as an average spreading rate divided by initial cell area for each cell. The mean value of R_E was found to relate to prolongation of lag time; namely it decreased with increasing passage number in the successive cultures approaching cellular senescence. During the courses of the cultures, the rotation rate of paired cells was also measured through time-lapse observation. The mean value of rotation rate, , decreased with an increase in doubling time caused by the progress of cellular age, reaching an almost constant value of h^-1 in the cultures with prolonged doubling time of over 59 h. It was concluded that the indices determined from the motions of individual cells, R_E and , were correlated with the lag time and doubling time, respectively, which are growth parameters varied with the vitality of the cells approaching cellular senescence.     

Immobilized amines and basic amino acids as mimetic heparin-binding domains for cell surface proteoglycan-mediated adhesion.

Diamines covalently coupled to glass substrates promoted human foreskin fibroblast adhesion in the absence of serum. These diamine-derivatized substrates were produced by coupling ethylene diamine, N-methylaminoethylamine, and N,N-dimethylaminoethylamine (NNDMAEA), to sulfonyl chloride-activated glass. Electron spectroscopy for chemical analysis demonstrated that the diamines were coupled via their primary amine ends to produce a surface-bound secondary amine linked to a free amino moiety via a two-carbon spacer. NNDMAEA-modified substrates containing free tertiary amines supported the highest degree of cell spreading (73 +/- 7% actively spreading cells) and the most extensive cytoskeletal organization. Both the free tertiary and surface-bound secondary amines were shown to be required for cell spreading. Lysine- and arginine-grafted substrates supported cell spreading and cytoskeletal organization similar to that on NNDMAEA-modified substrates. Although some stress fibers were observed within spread cells on these substrates, focal contacts did not form. Heparinase treatment did not inhibit cell attachment or spreading to the diamine-derivatized substrates, however chondroitinase ABC inhibited cell attachment and spreading on all substrates; heparinase inhibited spreading on lysine- and arginine-derivatized substrates to a lesser extent. These results imply that cell attachment to these substrates was mediated primarily by cell surface chondroitin sulfate proteoglycans. This study demonstrates that covalently grafted NNDMAEA, lysine, and arginine can mimic the adhesion-promoting activity of the glycosaminoglycan-binding domains of cell adhesion proteins. This study also demonstrates that the interaction with these proteoglycans depends in a very sensitive manner on the particular structure of the immobilized amine.     

Human osteoblast cell spreading and vinculin expression upon biomaterial surfaces

Any biomaterial implanted within the human body is influenced by the interactions that take place between its surface and the surrounding biological milieu. These interactions are known to influence the tissue interface dynamic, and thus act to emphasize the need to study cell-surface interactions as part of any biomaterial design process. The work described here investigates the relationship between human osteoblast attachment, spreading and focal contact formation on selected surfaces using immunostaining and digital image processing for vinculin, a key focal adhesion component. Our observations show that a relationship exists between levels of cell attachment, the degree of vinculin-associated plaque formation and biocompatibility. It also suggests that cell adhesion is not indicative of how supportive a substrate is to cell spreading, and that cell spreading does not correlate with focal contact formation.     

Haemocytes of larval Malacosoma disstria (Lepidoptera: Lasiocampidae) and factors affecting their adhesion to glass slides

Abstract.  Although haemocytes of the forest pest lepidopteran, Malacosoma disstria (L.) have been studied, the physico-chemical factors and signalling components affecting their non-self activities have not been examined. Both the ameboid and stellate forms of plasmatocytes and the granular cells from fifth-instar larvae adhere best to glass slides with phosphate-buffered saline (PBS), with maximum granular cell binding within a pH range of 6.0–7.0 and plasmatocyte binding at pH 6.0. The divalent cations, calcium and magnesium, do not affect granular cell attachment. However, calcium in Galleria -anticoagulant and PBS and, to a lesser extent, magnesium in the anticoagulant, increase plasmatocyte-glass contact. Based upon the use of selective type I protein kinase A inhibitor (Rp-8-Br-cAMPS) and activator (Sp-8-Br-cAMPS), active protein kinase A inhibits the adhesion of both haemocyte types. Similarly, protein kinase C inhibited by Gö 6976 enhances haemocyte adhesion whereas the enzyme activator, phorbol-myristate-acetate, impairs attachment.     

Analysis of the composite response of shear wave resonators to the attachment of mammalian cells

The suitability of the quartz crystal microbalance (QCM) technique for monitoring the attachment and spreading of mammalian cells has recently been established. Different cell species were shown to generate an individual response of the QCM when they make contact with the resonator surface. Little is known, however, about the underlying mechanisms that determine the QCM signal for a particular cell type. Here we describe our results for different experimental approaches designed to probe the particular contributions of various subcellular compartments to the overall QCM signal. Using AC impedance analysis in a frequency range that closely embraces the resonators' fundamental frequency, we have explored the signal contribution of the extracellular matrix, the actin cytoskeleton, the medium that overlays the cell layer, as well as the liquid compartment that is known to exist between the basal plasma membrane and the culture substrate. Results indicate that the QCM technique is only sensitive to those parts of the cellular body that are involved in cell substrate adhesion and are therefore close to the resonator surface. Because of its noninvasive nature, sensitivity, and time resolution, the QCM is a powerful means of quantitatively studying various aspects of cell-substrate interactions.