A line of rat ovarian surface epithelium provides a continuous source of complex extracellular matrix

Summary A spontaneously immortalized, yet non-tumorigenic rat ovarian surface epithelial (ROSE 199) cell line, deposits large amounts of extracellular matrix (ECM) in response to crowding. The characteristics and components of ROSE 199-derived cell-free ECM were compared after three different preparative techniques: treatment with 20 mM ammonium hydroxide, with 1% sodium deoxycholate, or by repeated freeze-thaws. The ECMs were analyzed by histochemistry, immunofluorescence, electron microscopy, and Western immunoblotting. Components of ROSE 199 ECM included laminin, fibronectin, and collagen types I and III. Even though ROSE 199 is an epithelial cell line, striated collagen fibers formed a major part of its matrix. Thus, ROSE 199 matrix consists of both basement membrane and stromal matrix components. This matrix supported the adhesion, spreading, and growth of several cell types without altering their morphology or growth pattern, and enhanced the attachment of some cell types that spread on plastic only with difficulty. Immunofluorescence, electron microscopy, and dry weight determinations indicated that a greater proportion of matrix was retained in preparations obtained by ammonium hydroxide or freeze thaw techniques than after sodium deoxycholate treatment. Ammonium hydroxide and freeze-thaw treated matrices were also superior to sodium deoxycholate preparations as evidenced by enhanced initial cellular adhesion and spreading compared to cells plated on plastic. Residual nuclear material did not seem to affect the biological activity of this matrix. ROSE 199 extracellular matrix provides a novel, complex substratum for cell culture and for studies of matrix functions and synthesis.     

Growth and characterization of human skin epithelial cell cultures

Summary In 129 of 140 attempts, human skin cells were successfully cultured on the dermal collagen bed of sterile, dead pigskin. Diploid epithelial cells grew selectively on the collagen bed; fibroblasts grew on the glass surfaces of the culture dishes. The cultures could be subdivided physically up to six times at a 1:2 split ratio, but at least 24 to 48 cell generations were produced over the months the cells could be carried. Much of the cell multiplication resulted in maturation into distinct basal, squamous, granular, and keratinized cell layers. The cultured cells were considered epithelial because of their shape, possession of intercellular bridges, desmosomes and tonofibrils, and because they formed maturating epithelium in vitro and upon transplantation back to the original human donor. As the cells grew they digested the pigskin collagen, thus producing clear zones that could be used to monitor and quantitate cell growth. Multiplication of epithelial cells, rather than migration, was indicated by mitotic figures in colchicine-treated cultures and by DNA synthesis. Expert technical assistance was provided by Nancy Allen (cell culture); William Towler (electron microscopy); James Malone, Nona Scaife, and Joy M. Nicolet (cytogenetics); R. Thomas Campbell and Dorothy Sarver (photography); and V. L. Angerstein, Susan Ekker, and Arnater Yarbrough (histology). This work was supported by The United Fund Cancer Society of Summit County, the Greater Cleveland Associated Foundation (grant no. 3G3490X1), the National Institute of General Medical Services (grant no. 1 R01 GM 21929-01), and the Charles E. Merrill Trust.     

Substrate ifluences human epidermal melanocyte attachment and sperading in vitro

Summary Previous culture systems for melanocytes have employed serum-supplemented medium and uncoated plastic dishes, prohibiting examination of possible substrate influences on cellular morphology and function. We now report, using a sensitive serum-free system and a quantitative procedure for evaluating cellular morphology, that modification of the plating surface affects human epidermal melanocyte attachment rate and subsequent morphology in vitro. Melanocytes attach and spread more rapidly on surfaces coated with fibronectin or Type I/III collagen or on surfaces previously conditioned by human keratinocytes, dermal fibroblasts, melanocytes, or melanoma cells than do melanocytes on untreated control surfaces. Type IV collagen and laminin, although minimally beneficial for cell attachment, do support a characteristics melanocyte morphology that differs from that seen either on the other coated surfaces or on uncoated plastic controls. Addition of fetal bovine serum at the time of inoculation has no appreciable effect on attachment but markedly improves cell spreading on untreated surfaces, while addition of nerve growth factor with or without serum to this system fails to affect cell attachment or spreading. Our data establish that human epidermal melanocytes are indeed capable of responding morphologically to substrate signals. The ability of several biochemically unrelated surfaces to enhance melanocyte attachment rate and spreading suggests that melanocytes have surface receptors with a variety of specificities. This work is relevant to the development of improved culture systems for melanocytes in vitro and to understanding melanocyte behavior in vivo. This work was supported by the USDA Agricultural Research Service, by a grant from Cheesebrough-Ponds, Inc., and by a Dermatology Foundation Fellowship (Dr. Yaar).     

Structural and functional characteristics of primary cell cultures derived from the adrenal gland tissue of neonatal mallard ducklings (Anas platyrhynchos)

Summary Primary cell cultures were prepared from the adrenal glands of one-day-old mallard ducklings (Anas platyrhynchos). The cells attached equally well to uncoated plastic and glass surfaces and on surfaces that had been coated with collagen. The phase of logarithmic growth occurred between the second and the fourth day, and the cells became confluent between the fifth and the sixth day. Staining with Sudan black B and toluidine blue and viewing fixed preparations by transmission electron microscopy indicated that the cultures consisted mostly of steroidogenic cells. A smaller population of chromaffin cells was also present. Scanning electron microscopy showed that most of the cells had long filopodia, and some cells had numerous surface blebs that were interpreted as exocytotic vesicles. When incubated in Krebs-Henseleit buffer containing 1–24 ACTH the cultured cells released three corticosteroids, namely, corticosterone, aldosterone and deoxycorticosterone. These responses occurred within 15 min of exposure to medium containing 1–24 ACTH and continued throughout a 60-min period of continuous stimulation. The minimally effective concentration of 1–24 ACTH was 0.078 ng per ml (0.0234 nM) and, as the concentration was increased up to 10 ng per ml (2.99 nM), the total output of each hormone during the 60-min incubation period increased significantly according to the following semi-logarithmic relationship: Y=a+b log X, where Y=the total output of hormone, X=the concentration of 1–24 ACTH in the medium, and a=the total output of hormone when the medium contained 1.0 ng of 1–24 ACTH per ml. The total outputs of each hormone in the presence of a maximally effective concentration of 1–24 ACTH, however, were low compared to the responses of similarly stimulated tissue slices taken from the neonatal duckling. It is concluded that most of the cells comprising the confluent cultures were derived from steroidogenic cells in the neonatal adrenal. These cells appeared to retain corticotropin receptors during the course of developing into confluent monolayers, but their diminished steroidogenic capacity to respond when stimulated maximally suggests that some generational changes may have occurred.This work was supported by grants to James Cronshaw and W.N. Holmes from the University of California Committee on Research and the National Science Foundation (DIR-8820923), Washington, DC, USA     

Covalent attachment of an Arg-Gly-Asp sequence peptide to derivatizable polyacrylamide surfaces: support of fibroblast adhesion and long-term growth

A synthetic nonapeptide (Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser), which includes the adhesive Arg-Gly-Asp (RGD) sequence, was covalently immobilized on chemically well-defined polyacrylamide gel surfaces utilizing N-succinimidyl active esters. The amount of peptide immobilized varied linearly with the concentration added to the gels. Immobilization was approximately 80% efficient (based on peptide added), resulting in up to 17.5 nmol peptide/cm2 gel surface. Balb/c 3T3 mouse fibroblast cells adhered readily to peptide-derivatized surfaces, even in the absence of serum. Furthermore, surfaces derivatized with 2 nmol peptide/cm2 gel supported long-term fibroblast growth at a rate and to an extent comparable to that on tissue culture plastic. Surfaces derivatized with a control nonapeptide having no RGD sequence were nonsupportive of cell attachment or growth. The immobilization technology used to derivatize the gel surfaces with adhesive nonapeptide can be modified to allow coderivatization with proteins, glycoproteins, glycosides, or other amine-containing compounds to test their effects on long-term cell behaviors.     

A new bacterial cellulose substrate for mammalian cell culture

A new substrate for mammalian cell culture was developed using a cellulose membrane produced byAcetobacter aceti. Modification of the ionic charge of the membrane and adsorption of collagen to it promoted cellular adhesion to the membrane surface. The growth of eight kinds of cells on the membrane, was comparable to that achieved in plastic Petri dishes. The membrane was tested for use in the production of recombinant Erythroid Differentiation Factor (EDF)/activin A using genetically engineered Chinese hamster ovary cells. Both the viability of the cells and production of EDF/activin A were maintained for about 1 month, while cultures on plastic dishes lasted only 12 days. It was considered that the mechanism of improved cell viability was related to the ultrastructure of the cellulose membrane.     

Morphogenesis in vitro of dissociated fetal rat small intestinal cells upon an open surface and subsequent to collagen gel overlay

The morphogenesis of cells dissociated from fetal rat intestine at 18 days of gestation was compared in vitro on two different substrates, tissue culture plastic and collagen gel. During the first 2 days in culture growth on both substrates was similar, resulting in the formation of layers of epithelial cells and of small lumina within them. On plastic, the cell layers contracted over time, resulting in the formation of small mounds of cells bearing on their surface small protrusions covered with epithelial cells which had densely packed microvilli on their apical surfaces. When the cultures on collagen gels were overlaid with more collagen gel, vesicles lined with epithelial cells developed. These cells were joined by junctional complexes and displayed an apical brush border which was periodic acid-Schiff and alkaline phosphatase positive. After 1 week in culture, when the vesicles reached their maximum extent, numerous epithelial cells were actively incorporating labeled thymidine and cells were being extruded into the lumen. These results demonstrate that the dissociated intestinal cells have the capacity to form intestine-like organoids in vitro and that surrounding the cells with the collagen gel allows expression of this potential. The collagen gel cultures thus undergo morphogenetic processes which can be modified by the surrounding environment and should provide a useful in vitro model system for the study of intestinal development.     

Improved growth of cultured brain microvascular endothelial cells on glass coated with a biological matrix

An improved method for culturing primary rat brain capillary endothelial cells on glass has been developed, using a corneal extracellular matrix coat. Since the collagen-coated plastic attachment surface conventionally used for primary cultures of brain microvascular endothelium gives a high level of background fluorescence in microfluorimetric studies, an alternative attachment surface was tested involving no plastic element. Five substrata combinations were examined and a new combination of glass and corneal endothelial extracellular matrix coat was found to provide excellent cell adhesion, culture growth and purity. Other established substrata combinations tested for comparison, either involved plastic, or used glass with collagen or carbodiimide and collagen coating but the last two gave poor endothelial cell adhesion and growth. Our method using this new attachment surface combination results in stable and pure endothelial cultures, as verified by immunocytochemistry, which are suitable for fluorimetric investigations.     

Rapid communication Improved growth of cultured brain microvascular endothelial cells on glass coated with a biological matrix

An improved method for culturing primary rat brain capillary endothelial cells on glass has been developed, using a corneal extracellular matrix coat. Since the collagen-coated plastic attachment surface conventionally used for primary cultures of brain microvascular endothelium gives a high level of background fluorescence in microfluorimetric studies, an alternative attachment surface was tested involving no plastic element. Five substrata combinations were examined and a new combination of glass and corneal endothelial extracellular matrix coat was found to provide excellent cell adhesion, culture growth and purity. Other established substrata combinations tested for comparison, either involved plastic, or used glass with collagen or carbodiimide and collagen coating but the last two gave poor endothelial cell adhesion and growth. Our method using this new attachment surface combination results in stable and pure endothelial cultures, as verified by immunocytochemistry, which are suitable for fluorimetric investigations.     

Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins

Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study we evaluate the application of a novel technology for the immobilisation of functional domains of known ECM proteins in a controlled manner on a surface. By examining the adherence of cultured PC12 cells to alternative growth surfaces, we show that surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules. Furthermore, we show that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment. Collectively, these data demonstrate the application of a new technology to enable optimisation of cell growth in the tissue culture laboratory.     

Conditions critical for optimal visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron microscopy.

The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of infected cells which were fixed with glutaraldehyde followed by postfixation in OsO4 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively smooth surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of resolving viral particles on cell surfaces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used.     

Effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells

The use of protein hydroxy ethylmethacrylate (HEMA) hydrogels to control cell morphology and growth, as well as the synthesis of extracellular matrix components, is described in this communication. HEMA hydrogels prepared with collagen support growth of embryonic lung fibroblasts (IMR-90), as well as bovine aortic and pulmonary artery endothelial cells at a level comparable to the respective cells grown on tissue culture surfaces. On the other hand, HEMA hydrogels prepared with solubilized elastin inhibit the fibroblast growth and prevent both types of endothelial cell cultures from achieving their normal morphology. These morphologically altered endothelial cells resume a normal cobblestone-like appearance when subcultivated from the elastin-HEMA hydrogels to tissue culture plastic. When pulsed with [14C]proline, the procollagens synthesized by the endothelial cells on the different surfaces vary, as shown by immunoprecipitation and polyacrylamide gel electrophoresis. On the standard tissue culture plastic, the confluent cells produce mainly type III procollagen in the medium, whereas those endothelial cells grown on collagen and elastin-HEMA hydrogels synthesize primarily type I procollagen (much like sprouting cells on tissue culture plastic), regardless of their morphology.     

Growth of endothelial and HeLa cells on a new multipurpose microcarrier that is positive,negative or collagen coated

A new cell culture microcarrier that can be covalently bonded by cell attachment proteins and can be thin-sectioned for electron microscopy was synthesized. It was easily made by sulfonating cross-linked polystyrene beads for a negative surface charge followed by covalent attachment of polyethylenimine for a positive charge. Cell attachment proteins, e.g. collagen, was covalently bonded directly to the microcarrier using a carbodiimide or after activating the microcarrier surface with glutaraldehyde. HeLa-S3 cells attached, spread and grew to confluence more efficiently on the positive microcarriers and those coated with collagen than on the negative ones. Endothelial cells grew best on those with a negative surface charge. The nature of the microcarrier surface was not the only aspect involved in cell adhesion but also the type of serum proteins adsorbed. Qualitatively different proteins coated the microcarriers depending upon whether the carrier was negative, positive or coated with collagen. Comparison of various types of available microcarriers indicated that the modified cross-linked polystyrene beads used here were best for transmission and scanning electron microscopy. Endothelial cells grown on the microcarriers had the same ultrastructure as cells grown in monolayers in culture dishes. Of a variety of microcarriers tested the modified cross-linked polystyrene beads were the only ones that could be used for both ultrastructural and biochemical techniques.     

A comparison of the effects of different substrata on chondrocyte morphology and the synthesis of collagen types IX and X

Summary Embryonic chick sternal chondrocytes were cultured either within three dimensional gels of type I collagen, type II collagen or agar, or as monolayers on plastic dishes coated with air-dried films of these matrix macromolecules. It was observed that cell shape and cell growth varied markedly between the different culture conditions. Flattened monolayers of cells on plastic or films of type I or type II collagen, proliferated more rapidly and reached a higher final cell density per culture than the more rounded cells found in the cultures on agar films or within three-dimensional gels. Biosynthetic studies demonstrated that in addition to the synthesis of type II collagen, all the cultures were producing collagen types IX and X. Chondrocytes cultured on plastic or films of the different matrix macromolecules all showed a similar expression of types IX and X collagen, independent of whether they displayed a flattened or round cell morphology. In contrast, marked variations in the proportions of the minor collagens, particularly type X collagen, were observed when the cells were cultured within three-dimensional gels. The data suggest that direct interaction of the cell surface with matrix constituents displaying a particular spatial array could be an important aspect in the control of type IX and X collagen expression by chondrocytes. The financial support of the Arthritis & Rheumatism Council and the Medical Research Council is gratefully acknowledged.     

Antimicrobial testing for surface‐immobilized agents with a surface‐separated live–dead staining method

Modification of a traditional live–dead staining technique based on fluorescence microscopy has yielded an improved method capable of differentiating surface‐immobilized antimicrobial agents from those agents acting via solution diffusion processes. By utilizing an inoculation chamber comprised of 50 µm polystyrene spheres as spacers between test substrate and coverslip control surfaces, three distinct bacterial cell populations can be probed by fluorescence microscopy for antimicrobial activity: (1) cells adhered to the coverslip, (2) cells adhered to the substrate, and (3) mobile cells in solution. Truly immobilized antimicrobial agents were found efficacious only at the substrate surface, while elutable agents were effective against all three populations. Glass surfaces derivatized with either quaternized poly dimethylaminoethylmethacrylate (pDMAEMA) or 3‐(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (Si‐QAC) were compared with bare glass control surfaces after contact and 4 h incubation with Staphylococcus aureus. pDMAEMA surfaces were both antimicrobial and immobilized, whereas the Si‐QAC surfaces were only observed to be antimicrobial via active diffusion. In contrast to conventional thinking, Si‐QAC surfaces showed no kill after removing all Si‐QAC elutables via rinsing procedures. The semi‐quantitative surface‐separated live–dead staining (SSLDS) technique provides mechanistic insight and represents a significant improvement relative to current microbiological test methods for evaluating immobilized, antimicrobial agents. Biotechnol. Bioeng. 2011; 108:231–236. © 2010 Wiley Periodicals, Inc.     

Mucin synthesis and secretion by cultured tracheal cells: effects of collagen gel substratum thickness

Summary We previously demonstrated that human tracheobronchial epithelial (TBE) cells synthesize mucin and form mucous granules in culture when they are maintained on a collagen gel (CG) substratum, but not on a plastic tissue culture surface or a thin collagen-coated surface (Wu et al., Am. J. Respir. Cell Mol. Biol., 3:467–478; 1990). This observation led us to examine the effects of CG thickness on cell growth and differentiation in primary human/monkey TBE cell cultures. Using the same CG preparation, culture dishes with different thicknesses of CG substratum were prepared. In general, equivalent degrees of cell attachment and proliferation were observed in all cultures maintained on a collagen gel, independent of the thicknesses of CG substratum. However, a greater degree of mucin synthesis and secretion by the cells was observed as the thickness of the CG substratum was increased. Cultures maintained on a thick collagen gel (1 mm) exhibited greater apical membrane complexity, more pseudostratification, and more mucous granules than did cultures maintained on a thin CG substratum. The optimal culture surface for airway mucous cell differentiation contains more than 1-mm thickness of collagen gel substratum.     

Ultrastructural and tissue-culture studies on the role of fibronectin,collagen and glycosaminoglycans in the migration of neural crest cells in the fowl embryo

Summary The initial migration of neural crest (NC) cells into cell-free space was studied by transmission electron microscopy at trunk levels of fowl embryos, some of which were fixed in the presence of ruthenium red. Migrating NC cells occurred in zones which contained fewer ruthenium-red stained 15–40 nm diameter granules than other regions. The ruthenium-red stained granules were linked by similarly stained thin ( 3 nm diameter) microfibrils. The granules resemble proteoglycan and the microfibrils may be hyaluronate. NC cells contacted thicker ( 10 nm diameter) fibrils and interstitial bodies, which did not require ruthenium red for visualization. Cytoplasmic microfilaments were sometimes aligned at the point of contact with the extracellular fibrils, which may be fibronectin and collagen.Phase-contrast time-lapse videotaping and scanning electron microscopy showed that NC cells of the fowl embryo in vitro migrated earlier and more extensively on glass coated with fibronectin-rich fibrous material and adsorbed fibronectin molecules than on glass coated with collagen type I (fibres and adsorbed molecules). NC cells became completely enmeshed in fibronectin-rich fibres, but generally remained on the surface of collagen-fibre gels. When given a choice, NC cells strongly preferred fibronectin coatings to plain glass, and plain glass to dried collagen gels. NC cells showed a slight preference for plain glass over glass to which collagen was adsorbed. Addition to the culture medium of hyaluronate (initial conc. 20 mg/ml), chondroitin (5 mg/ml) and fully sulphated chondroitin sulphate and dermatan sulphate (up to 10 mg/ml) did not drastically alter NC cell migration on fibronectin-rich fibrous substrates. However, partially desulphated chondroitin sulphate (5mg/ml) strongly retarded the migration of NC cells.The in vivo and in vitro studies suggest that fibronectin may dictate the pathways of NC cell migration by acting as a highly preferred physical substrate. However, the utilization of these pathways may be reduced by the presence of proteoglycans bearing undersulphated chondroitin sulphate.Abbreviations NC neural crest - ECM extracellular material - GAG glycosaminoglycan - FN fibronectin - CIG cold insoluble globulin - TEM transmission electron microscopy - SEM scanning electron microscopy - DMEM-H HEPES buffered Dulbecco's modified Eagle's medium - FCS foetal calf serum - CEE chick embryo extract - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

Factors affecting the differentiation of epithelial transport and responsiveness to hormones

Under standard culture conditions, epithelial cells grow with their basal surface attached to the culture dish and their apical surface facing the medium. Morphological and functional markers are located in the appropriate plasma membrane, and transepithelial transport occurs in a variety of cultured epithelia. As a result of the polarity of the cells and the presence of tight junctions between cells, on standard tissue culture dishes there is restricted access of growth medium to the basolateral surface of the epithelium, which is the surface at which nutrient exchange normally occurs. Greater differentiation of epithelial cultures can be achieved by growing primary cultures or continuous cell lines on permeable surfaces such as porous bottom cultures dishes in which the porous bottom is formed by a filter or membrane of collagen, or on floating collagen gels. In many cultures, differentiation varies with the time after the culture was seeded. Certain chemicals that accelerate differentiation in nonepithelial cells also accelerate the differentiation of epithelial cultures. Ultimately, defined media and specific substrates for cell attachment should lead to further differentiation of epithelia in culture.     

Specific association of iduronic acid-rich dermatan sulphate with the extracellular matrix of human skin fibroblasts cultured on collagen gels.

Human skin fibroblasts cultured on collagen gels produced two dermatan sulphate species, one, enriched in iduronic acid residues, that bound specifically to the collagenous fibres of the gel, the other, enriched in glucuronic acid, that accumulated in the culture medium. Collagen-binding and collagen-non-binding dermatan sulphates were also produced by cells grown on plastic surfaces, but in these cultures each constituent was released into the growth medium. Net synthesis of dermatan sulphate was 3-fold higher in cells maintained on collagen gels. In contrast, heparan sulphate synthesis was not influenced by the nature of the culture surface. The concentration of heparan sulphate in surface-membrane extracts was similar for cells grown on plastic and on collagen gels, but cells cultured on collagen showed a notable increase in the content of surface-membrane dermatan sulphate. The patterns of synthesis and distribution of sulphated glycosaminoglycans observed in skin fibroblasts maintained on collagen gels may reflect differentiated cellular functions.     

In vitro behaviour of human osteoblasts on dentin and bone

This investigation studied how the behaviour of isolated osteoblasts on standard tissue culture polystyrene compared with cells cultured on cut surfaces of dentin, a natural calcified material. Cellular attachment, viability and growth were monitored in parallel cultures of human osteosarcoma cell lines (MG63, HOS TE85, SaOS-2) and primary human osteoblast-like cells (HOBs). Culture plastic was either left untreated or roughened with abrasive paper of various grit sizes (4000-1200 grit) in order to obtain a level of roughness comparable to that of the dentin slices. Cell counting and intracellular BCECF staining showed that after an initial incubation of 2 h, the primary cells attached and spread out more quickly on the different substrates than the three cell lines. The primary cells also showed a stronger mitochondrial staining and viability on dentin. During subsequent culture morphological differences appeared with the cells on dentin displaying more cellular extensions. All three cell lines proliferated more slowly on dentin than on plastic. In contrast, the primary HOBs were not significantly affected in their growth by the different substrates. Total and specific alkaline phosphatase (AP) activity of the cell lines was not significantly affected by the different substrates after short-term adhesion, but it was increased for the primary cells on the dentin. However, after 2-3 days of culture, AP was decreased on the dentin slices for both the cell lines and primary HOBs. Plasma treatment of the roughened plastic did not alter cellular viability or AP activity, suggesting that grinding of the surface did not affect the property of the culture plastic to support cell attachment and growth. In conclusion, the results show that not only do osteoblastic cells behave differently on a natural calcified substrate surface than on standard culture plastic, but also that differences were evident between the various cell types, in particular the primary HOB versus the continuous cell lines.