Use of extracellular matrix components for cell culture

Extracellular matrix components when used as a substratum in vitro can greatly influence cell behavior. The response observed is dependent on the type of cell and matrix used. Cells in vitro usually respond best to the matrix components with which they are normally in contact in vivo. More differentiated phenotypes are observed and cells generally survive longer on such matrices. In some cases, the presence of such matrices allows cells to be cultured in the absence of serum and growth factors. As more investigators try the matrices and matrix components described, as well as new components and combinations of them, it is anticipated that improvement in the culture of many cells can be expected.     

Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts

Many key processes central to bone formation and homeostasis require the involvement of osteoblasts, cells responsible for accumulation and mineralization of the extracellular matrix (ECM). During this complex and only partially understood process, osteoblasts generate and secrete matrix vesicles (MVs) into the ECM to initiate mineralization. Although they are considered an important component of mineralization process, MVs still remain a mystery. To better understand their function and biogenesis, a proteomic analysis of MVs has been conducted. MVs were harvested by two sample preparation approaches and mass spectrometry was utilized for protein identification. A total of 133 proteins were identified in common from the two MV preparations, among which were previously known proteins, such as annexins and peptidases, along with many novel proteins including a variety of enzymes, osteoblast-specific factors, ion channels, and signal transduction molecules, such as 14-3-3 family members and Rab-related proteins. To compare the proteome of MV with that of the ECM we conducted a large-scale proteomic analysis of collagenase digested mineralizing osteoblast matrix. This analysis resulted in the identification of 1,327 unique proteins. A comparison of the proteins identified from the two MV preparations with the ECM analysis revealed 83 unique, non-redundant proteins identified in all three samples. This investigation represents the first systematic proteomic analysis of MVs and provides insights into both the function and origin of these important mineralization-regulating vesicles.     

Co-localization of integrins and matrix metalloproteinases in the extracellular matrix of chondrocyte cultures

Beta1-integrins were found in the cartilage matrix, suggesting their implication in the assembly of its architectural scaffold, but the mechanism for this event is not yet clear. Matrix metalloproteinases (MMPs) may be involved in an integrin-shedding mechanism and matrix beta1-integrins may act to alter MMP activity. To begin to address this question, this study was designed to determine whether beta1-integrins and MMPs are colocalized in the chondrocytes or in the extracellular matrix of cartilage. We investigated high-density cultures of limb buds of 12-day-old mouse embryos by double immunofluorescence, immunoelectron microscopy and by coimmunoprecipitation assays in order to examine the localization of beta1-integrins and matrix metalloproteinases (MMP-1, MMP-3 and MMP-9) in cartilage. It was found, that all investigated MMPs and beta1-integrins were specifically co-localized in high-density cartilage cultures. Immunogold and immunofluorescence labelling of both beta1-integrins and MMPs were observed not only at the surface of chondrocytes but mainly also in the pericellular space and distributed between collagen fibrils in the extracellular matrix (ECM) as well. Results of immunoprecipitation experiments suggest a functional association of MMPs and beta1-integrins in chondrocytes as already described for other cell types. Further investigations are needed to elucidate the functional association between beta1-integrins and MMPs in chondrocytes.     

LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation

Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling.     

Hydrogels as extracellular matrix mimics for 3D cell culture

Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two‐dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three‐dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have become popular as three‐dimensional cell culture platforms; yet, both of these systems possess limitations. In this perspective, we discuss the use of both synthetic and natural hydrogels as scaffolds for three‐dimensional cell culture as well as synthetic hydrogels that incorporate sophisticated biochemical and mechanical cues as mimics of the native extracellular matrix. Ultimately, advances in synthetic–biologic hydrogel hybrids are needed to provide robust platforms for investigating cell physiology and fabricating tissue outside of the organism. Biotechnol. Bioeng. 2009;103: 655–663. © 2009 Wiley Periodicals, Inc.     

Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro

Objective

To investigate the interactions of chondrocyte metabolism by synovial cells and synovial supernatants in a new perfusion co-culture system.

Methods

Chondrocytes and synovial fibroblasts were obtained from knee joints of slaughtered adult cattle. For experimental studies chondrocytes and synovial fibroblasts were placed together into a perfusion chamber (co-culture) or were placed into two different perfusion culture containers, which were connected by a silicone tube (culturing of chondrocytes with synovial supernatants). A control setup was used without synovial cells. Chondrocyte proliferation was shown by measurement of DNA content. The proteoglycan synthesis was quantified using ³⁵SO₄²⁻-labelling and the dimethylmethylene blue assay. ³H-proline incorporation was used to estimate the protein biosynthesis. Type II collagen synthesis was measured by ELISA, furthermore extracellular matrix deposition was monitored immunohistochemically (collagen types I/II). Regarding to the role of reactive oxygen species LDH release before and after stimulation with hydrogen peroxide was measured.

Results

The proliferation of chondrocytes shows an increase in monoculture as well as in co-culture or in culture with synovial supernatants more than fivefold within 12 days. ³H-proline incorporation as a marker for chondrocytes biosynthetic activity decreases in co-culture system and in culture with synovial supernatants. A similar effect is seen measuring total proteoglycan content as well as the ³⁵SO₄²⁻ incorporation in chondrocytes. Co-culturing and culturing with synovial supernatants lead to a significant decrease of proteoglycan release and content. Quantification of collagen type II by ELISA shows significant lower amounts of native collagen type II in the extracellular matrix of co-cultured chondrocytes as well as in culture with synovial supernatants. The membrane damage of chondrocytes by hydrogen peroxide is reduced when chondrocytes are co-cultured with synovial fibroblasts.

Conclusion

The co-culture perfusion system is a new tool to investigate interactions of different cell types with less artificial interferences. Our results suggest that synovial supernatants and synovial fibroblasts modulate the biosynthetic activity and the matrix deposition of chondrocytes as well as the susceptibility to radical attack of reactive oxygen species.     

A tissue-like culture system using microstructures: influence of extracellular matrix material on cell adhesion and aggregation

Special microenvironmental conditions are required to induce and/or maintain specific qualities of differentiated cells. An important parameter is the three-dimensional tissue architecture that cannot be reproduced in conventional monolayer systems. Advanced tissue culture systems will meet many of these demands, but may reach their limits, especially when gradients of specific substances over distinct tissue layers must be established for long-term culture. These limitations may be overcome by incorporating microstructures into tissue-like culture systems. The microstructured cell support presented consists of a flat array of 625 cubic microcontainers with porous bottoms, in which cells can be supplied with specific media from both sides of the tissue layer. Permanent cell lines and primary rat hepatocytes have been used to test the culture system. In order to define reproducible conditions for tissue formation and for cell adherence to the structure, several ECM (extracellular matrix) components were tested for coating of microstructured substrata. The described tissue culture system offers great flexibility in adapting the cell support to specific needs.     

On the ultrastructure of hyalin, a cell adhesion protein of the sea urchin embryo extracellular matrix

Hyalin is a large (ca. 350 x 10(3) kD by gel electrophoresis) molecule that contributes to the hyalin layer surrounding the sea urchin embryo. In previous work a mAb (McA Tg-HYL), specific for hyalin, was found to inhibit cell-hyalin adhesion and block morphogenesis of whole embryos (Adelson, D. L., and T. D. Humphreys. 1988. Development. 104:391-402). In this report, hyalin ultrastructure was examined via rotary shadowing. Hyalin appeared to be a filamentous molecule approximately 75-nm long with a globular "head" about 12 nm in diameter that tended to form aggregates by associating head to head. Hyalin molecules tended to associate with a distinct high molecular weight globular particle ("core"). In fractions containing the core particle often more than one hyalin molecule were seen to be associated with the core. The core particle maintained a tenacious association with hyalin throughout purification procedures. The site(s) of McA Tg-HYL binding to the hyalin molecule were visualized by decorating purified hyalin with the antibody and then rotary shadowing the complex. In these experiments, McA Tg-HYL attached to the hyalin filament near the head region in a pattern suggesting that more than one antibody binding site exists on the hyalin filament. From the ultrastructural data and from the cell adhesion data presented earlier we conclude that hyalin is a filamentous molecule that binds to other hyalin molecules and contains multiple cell binding sites. Attempts were made to demonstrate the existence of lower molecular weight hyalin precursors. Whilst no such precursors could be identified by immunoprecipitation of in vivo labeled embryo lysates, immunoprecipitation of in vitro translation products suggested such precursors (ca 40 x 10(3) kD) might exist.     

Sertoli cell glycosylation patterns as affected by culture age and extracellular matrix

This study evaluated the responsiveness of Sertoli cell glycosylation in vitro to changes in culture age and to the presence of a reconstituted basement membrane (Matrigel) or collagen IV/laminin substrata. Primary Sertoli cell cultures were prepared from 20-day-old rats and incubated with [3H]mannose, a monosaccharide specific for asparagine-linked oligosaccharides. The cells were harvested on Days 4, 6, or 10 of culture life. A supernatant enriched in cell-surface glycopeptides (the trypsinate) and a cell pellet stripped of surface glycoconjugates were evaluated separately. Glycopeptides derived from a Pronase digest of the two samples were fractionated using concanavalin-A lectin affinity chromatography into three major classes: multiantennary complex-type, biantennary complex-type, and high-mannose-type oligosaccharide structures. The proportion of radiolabeled glycopeptides appearing in each of the three classes did not differ between Days 4 and 6 of culture. In contrast, a significant increase in the percentage of radiolabeled glycopeptides containing multiantennary complex-type oligosaccharides was observed in cells harvested from the 10-day-old cultures. In other experiments, Sertoli cells were grown on various substrata: plastic; collagen IV/laminin; or Matrigel, a reconstituted basement membrane (RBM) composed of laminin, collagen IV, proteoglycan sulfate, entactin, and nidogen. Growth on RBM significantly increased multiantennary complex-type oligosaccharide formation compared to plastic, whereas the high-mannose-type glycopeptides increased in cells grown on collagen IV/laminin. These studies suggest that environmental and physiological conditions such as culture age and the presence of extracellular matrix significantly affect glycosylation patterns in Sertoli cell cultures.     

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.     

Localization of extracellular matrix receptors on the chondrocyte primary cilium.

A single primary cilium is found in chondrocytes and other connective tissue cells. We have previously shown that extracellular matrix (ECM) macromolecules such as collagen fibers closely associate with chondrocyte primary cilia, and their points of contact are characterized by electron-opaque plaques suggesting a direct link between the ECM and the cilium. This study examines the expression of receptors for ECM molecules on chondrocyte primary cilia. Embryonic chick sterna were fluorescently labeled with antibodies against alpha and beta integrins, NG2, CD44, and annexin V. Primary cilia were labeled using acetylated alpha-tubulin antibody. Expression of ECM receptors was examined on chondrocyte plasma membranes and their primary cilia using immunofluorescence and confocal microscopy. All receptors examined showed a punctate distribution on the plasma membrane. alpha2, alpha3, and beta1 integrins and NG2 were also present on primary cilia, whereas annexin V and CD44 were excluded. The number of receptor-positive cilia varied from 8/50 for NG2 to 43/50 for beta1 integrin. This is the first study to demonstrate the expression of integrins and NG2 on chondrocyte primary cilia. The data strongly suggest that chondrocyte primary cilia have the necessary machinery to act as mechanosensors, linking the ECM to cytoplasmic organelles responsible for matrix production and secretion.     

Invasive growth and topoisomerase-switch induced by tumorous extracellular matrix in osteosarcoma cell culture

Osteosarcoma cells are capable of extracellular matrix (ECM) synthesis. The ability of ECM to trigger the proliferation of a novel osteosarcoma cell line (OSCORT) was tested in this study in relation to a known tumor ECM, isolated from Engelbreth-Holm-Swarm (EHS) sarcoma (EHS-ECM). OSCORT was grown in monolayer, in EHS-ECM and in ECM deposited by the cells (OSCORT-ECM). Both EHS-ECM and OSCORT-ECM increased the proliferation and migration of OSCORT cells. Among the ECM biopolymers, heparan sulfate proteoglycan (HSPG) and fibronectin enhanced invasive growth, collagen type IV reduced it, while laminin had no effect. Among the ECM components HSPG and collagen IV increased both the synthesis and activation of collagenase type IV, and all the ECM components substantially increased beta1 integrin levels in the cells. The majority of ECM biopolymers decreased the level of topoisomerase I (except laminin) and elevated topoisomerase II (except fibronectin) in OSCORT. The switch in the ratio between the activities of topoisomerases I and II was mainly due to HSPG. The HSPG synthesized by OSCORT cells is described as agrin, which is a novel finding. The present study showed that HSPG (agrin) showed the most remarkable stimulatory action on the growth and migration of OSCORT cells. HSPG-induced topoisomerase II-induction deserves further experimentation, to discover its relevance to tumor progression.     

Effects of interleukin-1 on fibroblast extracellular matrix, using a 3-dimensional culture system

This study describes the alterations induced by Interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta) on fibroblast-synthesized extracellular matrix. Fibroblasts were grown between pieces of dentin or in collagen-coated Terasaki wells for 3 or 6-9 weeks to create 3-dimensional cell-containing matrices constituted primarily of proteoglycans and collagens, respectively. Following incubation with IL-1 alpha or IL-1 beta (10(-9) M) at 37 degrees C for 24 or 72 hr, samples were prepared for light and electron microscopy. Both IL-1 alpha and IL-1 beta induced collapse of the extracellular matrix by 72 hr, as manifested by a decrease of the cross-sectional area and an increased density of the matrices. Three-week matrices were reduced 26% and 45% by using IL-1 alpha and IL-1 beta, respectively. Comparable values obtained by using 6-week matrices were 14% and 30%. Cells within the matrix, normally stellate in shape with numerous extended processes, attained a more rounded or spindle shape with few and reduced processes and showed apparent alterations at cell matrix attachment sites and rearrangement of the cytoskeleton. Elongated cells at the top of the matrix appeared more compressed. The alterations were more pronounced in cultures incubated with IL-beta than with IL-1 alpha. Immunocytochemistry of extracellular matrix components revealed a decrease in staining intensity of chondroitin and dermatan sulfate in the 3-week matrix following IL-1 beta incubation. There was also a decrease in collagen type 1 staining of 9-week matrices treated with IL-1 alpha or IL-1 beta. These studies show that IL-1 has an effect on fibroblast-synthesized extracellular matrix and indicate that the effects of IL-1 alpha and IL-1 beta may differ. The resulting collapse of the matrix appears at least in part to be due to changes in proteoglycans and collagens.     

Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: a pragmatic approach

The common technique of growing cells on tissue culture plastic (TCP) is gradually being supplanted by methods for culturing cells in two-dimensions (2-D) on matrices with more appropriate physical and biological properties or by encapsulation of cells in three-dimensions (3-D). The universal acceptance of the new 3-D paradigm is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. In this Prospect, I argue that the standard for 3-D cell culture should be bio-inspired, biomimetic materials that can be used "as is" in drug discovery, toxicology, cell banking, and ultimately in medicine. Such biomaterials must therefore be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end-users-physicians and patients-must dictate the key design criteria. Herein I describe the development of one such material that meets these requirements: a covalently crosslinked, biodegradable, simplified mimic of the extracellular matrix (ECM) that permits 3-D culture of cells in vitro and enables tissue formation in vivo. In contrast to materials that were designed for in vitro cell culture and then found unsuitable for clinical use, these semi-synthetic hyaluronan-derived materials were developed for in vivo tissue repair, and are now being re-engineered for in vitro applications in research.     

Lysophospholipid regulates release and activation of latent TGF-beta1 from chondrocyte extracellular matrix

Transforming growth factor beta-1 (TGF-beta1) is released from the extracellular matrix of rat growth plate chondrocytes and activated by stromelysin-1 (matrix metalloproteinase 3, MMP-3), an enzyme that is stored in matrix vesicles. MMP-3 is released from these extracellular organelles by the direct action of 1alpha,25(OH)2D3 via activation of phospholipase A2 (PLA2), resulting in local production of lysophospholipids and matrix vesicle membrane destabilization. This effect of 1alpha,25(OH)2D3 is greater in matrix vesicles from growth zone chondrocyte cultures and PLA2 activity is higher in the growth zone in vivo, suggesting that it may depend on chondrocyte maturation state in the endochondral lineage. Previous studies have shown that latent TGF-beta1 can be activated by mild detergents in vitro, suggesting that lysophospholipids may act in vivo in a similar manner. To test this hypothesis, we determined if rat costochondral growth plate cartilage cells produce lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) in a maturation state-dependent manner and if LPC or LPE could release and activate latent TGF-beta1 from the extracellular matrix produced by these cells. Rat growth plate chondrocytes produced both lysophospholipids, with growth zone cells producing higher levels of LPE via PLA1, and resting zone cells producing higher levels of LPC via PLA2. LPC and LPE directly increased activation of recombinant human latent TGF-beta1 in a biphasic manner with a peak at 2 microg/ml. Phosphatidylcholine, phosphatidylethanolamine, and LPE plasmalogen (LPEP), but not choline, also activated TGF-beta1. Latent TGF-beta1 incubated with LPC or LPE, but neither lysophospholipid alone, stimulated [3H]-thymidine incorporation of resting zone cells, indicating the TGF-beta1 released was biologically active. LPC and LPE also released TGF-beta1 in a dose- and time-dependent manner when incubated with cell-free extracellular matrices produced by the cells. These results indicate that LPC and LPE have important roles as regulators of rat growth plate chondrocytes by directly and indirectly activating TGF-beta1 stored in the extracellular matrix.     

Gene trap screening for cell surface and extracellular matrix molecules produced by chondrocytes

We have developed a simple and unique strategy for identifying cell surface and extracellular matrix molecules produced by chondrocytes. Our strategy comprises two methods, retrovirus-based signal sequence gene trapping and culturing of Swarm rat chondrosarcoma chondrocytes. After infection with a retrovirus vector and isolation of hygromycin-resistant clones, trapped genes could be easily identified by the 5' rapid amplification of cDNA ends (5' RACE) method. Furthermore, the treatment of isolated clones with gadolinium chloride enabled us to determine whether the trapped gene expression was dependent on the state of chondrogenic differentiation.