Three-dimensional Collagen Gel Culture Research Articles

Statins inhibit matrix metalloproteinase release from human lung fibroblasts

Pleiotropic effects of statins have been reported to include inhibition of matrix metalloproteinase (MMP) release from macrophages and endothelial cells. We evaluated whether statins would inhibit MMP release from human lung fibroblasts, which play a major role in remodelling processes. Monolayer and three-dimensional (3D) collagen gel cultures of fibroblasts were used. Cytokines (tumour necrosis factor-alpha and interleukin-1alpha) were used to induce MMP release and mRNA expression. Collagen degradation induced by cytokines and neutrophil elastase (NE) was evaluated by quantifying hydroxyproline. Atorvastatin inhibited MMP-1 and -3 release and mRNA expression in both culture systems. Similar results were obtained with simvastatin and fluvastatin. In 3D cultures where cytokines also stimulated MMP-9 release, atorvastatin also inhibited MMP-9 release. In 3D cultures, cytokines together with NE induced collagen degradation, which was also inhibited by atorvastatin. The effect of atorvastatin was reversed by mevalonate and geranylgeranyl-pyrophosphate but not by farnesyl-pyrophosphate. The current data suggest that statins may modulate remodelling processes mediated by fibroblasts by inhibiting MMP release.

Decidualization Attenuates the Contractility of Eutopic and Ectopic Endometrial Stromal Cells: Implications for Hormone Therapy of Endometriosis

Decidualization of the endometrium involves the morphological and biochemical reprogramming of the estrogen-primed proliferative endometrial stromal compartment under the continuing influence of progesterone. The aim of this study was to evaluate the involvement of the extracellular matrix contractility of eutopic and ectopic endometrial stromal cells during the tissue remodeling processes associated with decidualization. The effect of decidualization on the contractile profile of the endometriotic cyst stromal cells and eutopic endometrial stromal cells with or without endometriosis in the three-dimensional collagen gel culture was investigated using laser scanning microscopy, collagen gel contraction assays, and Western blot analysis. Decidualized ectopic and eutopic endometrial stromal cells in the three-dimensional collagen gel culture mimicked the morphology of decidual tissue in vivo. In vitro decidualization inhibited the contractility of these eutopic and ectopic endometrial stromal cells. Down-regulation of integrin alpha1beta1 and alpha2beta1 expression, suppression of Ras homology A (Rho A), Rho-associated coiled-coil-forming protein kinase (ROCK)-I and ROCK-II expression, inhibition of the differentiation into the myofibroblastic phenotype, and induction of differentiation into epithelioid decidual phenotype were observed in these cells during decidualization. It is suggested that the attenuation of eutopic endometrial stromal cell-mediated contractility by decidualization is a novel and integral mechanism of the physiological endometrial tissue remodeling process during menstrual cycles. Although ectopic endometrial stromal cells have enhanced contractile profile, decidualization can attenuate the contractility of these cells. These findings may be one of the action mechanisms by which oral contraceptives and progestins ameliorate endometriosis.

Distribution and bioactivity of the Ret-specific D4 aptamer in three-dimensional collagen gel cultures

The success of tyrosine kinase inhibitors in cancer therapy prompted intensive research efforts addressed to the development of new specific diagnostics and therapeutics. Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacologic challenge. The D4 RNA-aptamer, isolated applying the Systematic Evolution of Ligand by Exponential Enrichment procedure on living cells, has been proven a specific inhibitor of the human receptor tyrosine kinase Ret. In our attempts to generate new powerful probes for in vivo applications, in the present study, we addressed the ability of D4 to preserve its biological activity in cells embedded in three-dimensional collagen gels. These matrices provide a microenvironment mimicking the cell organization as seen in vivo, thus representing a suitable tool to approach the use of the aptamer in vivo. By taking advantage of transformed fibroblasts expressing Ret as a model system, we showed that the cells maintain normal phenotype and growth patterns when cultured in three-dimensional matrices and that the D4 aptamer preserves its ability to inhibit Ret on the surface of the cells embedded in collagen. Because the biological activity of RNA aptamers is largely dictated by their folded structure, the results indicate that a folded conformation of D4 responsible of its inhibiting function is preserved in the three-dimensional constructs, thus supporting its use in tumors in vivo.

Survival and developmental competence of buffalo preantral follicles using three-dimensional collagen gel culture system

The aim of the present study was to develop a three-dimensional (3D) collagen gel culture system for the in vitro growth and survival of buffalo preantral follicles with or without growth factors. Buffalo ovaries were collected from a local abattoir and preantral follicles were isolated through microdissection. Isolated preantral follicles were put either in collagen gel coated culture dish or embedded in a microdrop of collagen gel. The culture medium was TCM-199 fortified with fetal calf serum (10%), insulin transferin selenium solution (ITS, 1%), epidermal growth factor (EGF, 20 ng/ml) and follicle stimulating hormone (FSH, 0.5 μg/ml). Follicles were divided into three groups and cultured in the medium described above (group a, control), with addition of insulin like growth factor (IGF-I, 100 ng/ml, group b), or with addition of IGF-I and basic fibroblast growth factor (bFGF, 10 ng/ml, group c). Preantral follicles were incubated at 38.5 °C in 5% CO 2 and maximum humidity. Culture medium was replenished after every 72 h and spent medium was stored at −30 °C for hormone analysis. We found that the extracellular matrix of collagen gel maintained follicle viability and growth by providing surface interaction and increasing attachment of follicles. Preantral follicles embedded in collagen gel droplets had better antrum formation and development as compared to the whole surface coated culture method. Follicles cultured with IGF-I on collagen gel matrix showed a significantly ( P < 0.05) higher survival rate and larger mean diameter of follicles on day 10 of culture with improved growth and mucification as compared to the control group. However, follicles cultured in the combination of IGF-I with bFGF had decreased survival rate and smaller mean follicles diameter than the IGF-I group (b). Progesterone (P 4) accumulation was greater on day 9 of culture in follicles cultured in IGF-I as compared to control; whereas, P 4 was markedly decreased in the combination of IGF-I with bFGF. Follicles of the control group could survive for up to 10–15 days before degenerating, but follicles cultured with growth factors were able to survive up to 20 days and showed signs of early antrum formation. In summary, we have shown that collagen gel was a novel and efficacious 3D microenvironment for the extended culture of buffalo preantral follicles. Supplementation of culture medium with growth factors was found to be essential for antrum formation.

Involvement of RhoA, ROCK I and myosin II in inverted orientation of epithelial polarity

In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin-Darby canine kidney cells in three-dimensional collagen gel culture, blockade of beta1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA-ROCK I-myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.

A New Organotypic Culture of Adipose Tissue Fragments Maintains Viable Mature Adipocytes for a Long Term, Together with Development of Immature Adipocytes and Mesenchymal Stem Cell-Like Cells

Adipose tissue that consists of mature and immature adipocytes is suggested to contain mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. Here we show that three-dimensional collagen gel culture of rat sc adipose tissue fragments maintained viable mature adipocytes for a long term, producing immature adipocytes and MSC-like cells from the fragments, using immunohistochemistry, ELISA, and real time RT-PCR. Bromodeoxyuridine uptake of mature adipocytes was detected. Adiponectin and leptin, and adipocyte-specific genes of adiponectin, leptin, and PPAR-gamma were detected in culture assembly, whereas the lipogenesis factor insulin (20 mU/ml) and inflammation-related agent TNF-alpha (2 nm) increased and decreased, respectively, all of their displays. Both spindle-shaped cell types with oil red O-positive lipid droplets and those with expression of MSC markers (CD105 and CD44) developed around the fragments. The data indicate that adipose tissue-organotypic culture retains unilocular structure, proliferative ability, and some functions of mature adipocytes, generating both immature adipocytes and CD105+/CD44+ MSC-like cells. This suggests that our method will open up a new way for studying both multiple cell types within adipose tissue and the cell-based mechanisms of obesity and metabolic syndrome.

Three-Dimensional Microenvironments Retain Chondrocyte Phenotypes During Proliferation Culture

Although autologous chondrocyte implantation has already been in clinical use, chondrocyte dedifferentiation is problematic during proliferation culture. We attempted a three-dimensional (3D) collagen gel culture under chondrocyte proliferation with repeated passaging to prevent the chondrocytes dedifferentiation. Human auricular chondrocytes were cultured in 3D or conventional monolayer conditions, which reached a 1000-fold increase in cell numbers at passages 3 and 4, respectively. During multiplication, the chondrocytes in 3D culture showed greater suppression of collagen type I (COL1) and preservation of collagen type II (COL2) than those in monolayer. Tissue-engineered cartilage made of 3D cells also abundantly accumulated COL2 or proteoglycan and possessed favorable mechanical properties. The advantage of 3D cells may result from the similarity of microenvironments in cell-to-matrix adhesion or cell-to-cell contacts with that of native cartilage. The up-regulation of integrins and down-regulation of cadherins in the 3D cells mimicked the expression pattern of native cartilage, rather than that of monolayer cells. The silencing of integrin beta1 and Ob-cadherin expression by small interfering ribonucleic acid in the cultured chondrocytes led to the promotion of dedifferentiation and redifferentiation, respectively, indicating that the 3D collagen gel culture provided sufficient cell preparation and reduced chondrocyte dedifferentiation, which is regarded as a feasible strategy in autologous chondrocyte implantation.

Adipocytes and preadipocytes promote the proliferation of colon cancer cells in vitro

Obesity, a risk factor for colon cancer, is associated with elevated serum levels of leptin, a protein produced by adipocytes. The aim of the present study was to clarify the effects of adipose tissue on colon cancer proliferation by using cultured cell lines. To achieve this, colon cancer cells (CACO-2, T84, and HT29) were cocultured with adipose tissue, isolated mature adipocytes, and isolated preadipocytes in a three-dimensional collagen gel culture system. The adipocytes and preadipocytes used were isolated from C57BL/6J and leptin-deficient ob/ob mice. Proliferation of the cancer cells was evaluated by nuclear bromodeoxyuridine uptake. The adipose tissue, mature adipocytes, and preadipocytes isolated from C57BL/6J mice significantly increased the proliferation of the colon cancer cells. This trophic effect of mature adipocytes on the cancer cell lines was observed only for cells from lean littermates and not for those from ob/ob mice. In contrast, the trophic effect of preadipocytes was not abolished in ob/ob mice, and this finding was supported by the result that leptin had a trophic effect on cancer cells. In conclusion, adipocytes were able to enhance the proliferation of colon cancer cells in vitro, partly via leptin, suggesting that adipose tissues, including mature adipocytes and preadipocytes, may promote the growth of colorectal cancer.

Culture system for studying thyroid biology

Abstract. The thyroid is composed of many ball-like structures called thyroid follicles, which are supported by the interfollicular extracellular matrix and a dense network of fenestrated capillaries. The follicles consist of many thyrocytes and a few parafollicular cells (C cells). In this microenvironment, the component thyrocytes are highly integrated in their specific structural and functional polarization. In conventional monolayer and floating culture systems, thyrocytes are unable to organize themselves into the follicles with normal cellular polarity. By contrast, in three-dimensional collagen gel culture, thyrocytes easily form stable follicle structures with normal polarity. However, these culture systems cannot reconstruct follicle structures that consist of both thyrocytes and C cells. To overcome this issue, we developed a new organotypic culture system of thyroid tissue fragments, which contain the two cell types, using three-dimensional collagen gel culture with air–liquid interface. Our thyroid tissue-organotypic culture system retains three-dimensional follicle structures with both thyrocytes and C cells for a long term. In relation to our method, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types. We review here four culture systems above to study thyroid biology.

Adult Kidney Tubular Cell Population Showing Phenotypic Plasticity, Tubulogenic Capacity, and Integration Capability into Developing Kidney

Using in vivo bromodeoxyuridine (BrdU) labeling, a tubular cell population (label-retaining tubular cells [LRTC]) was identified recently in normal adult kidneys, which contributes actively to the regeneration process of the kidney after injury. Here, these LRTC are characterized in vitro. The LRTC population was isolated from BrdU-treated rat kidney by FACS. Both LRTC and non-LRTC underwent proliferation and maintained an epithelial phenotype in the presence of tubulogenic growth factors such as EGF, TGF-alpha, IGF-I, and hepatocyte growth factor. It is interesting that LRTC also proliferated without epithelial markers expression in the presence of soluble factors derived from an embryonic kidney metanephric mesenchyme cell line. The type of extracellular matrix strongly influenced the phenotype of LRTC. Furthermore, in three-dimensional collagen gel culture, LRTC formed tubule-like or tubulocystic structures in response to growth factors (hepatocyte growth factor and fibroblast growth factor) that are known to induce kidney cell tubulogenesis in vitro and/or participate in renal regeneration in vivo. In contrast, non-LRTC did not form these structures. When transplanted into the metanephric kidney, LRTC but not non-LRTC were integrated into epithelial components of nephron, including the proximal tubular cells and the ureteric bud. They also differentiated into fibroblast-like cells. Collectively, these findings suggest that LRTC are an adult kidney tubular cell population that shows phenotypic plasticity, tubulogenic capacity, and integration capability into the developing kidney.

An in vitro model for studying vascular injury after laser microdissection

We have developed an in vitro model for studying vascular injury. After 7-10 days in a three-dimensional collagen gel culture, capillary-like tubes were formed in the collagen gels. We injured these capillary-like tubes with a laser microdissection system or a scrape method with razors and then examined the collagen gel culture by phase contrast and electron microscopy. After laser injury, profuse necrotic cells were observed around the injured capillary-like tubes and within the tubular lumen compared to the razor injury. We then isolated total RNA from these cultures and prepared cDNA for investigations by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Quantitative real time RT-PCR revealed the up-regulation of transcription factor early growth response-1 (Egr-1) after both laser and razor injury, accompanied by the up-regulation of fibroblast growth factor-2 (FGF-2), a proangiogenic factor downstream of Egr-1. The effective laser energy is concentrated on the minute focal spot only. These methods provide a useful in vitro model for studying vascular injury.

Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166/MEMD), a Novel Actor in Invasive Growth, Controls Matrix Metalloproteinase Activity

Activated leukocyte cell adhesion molecule (ALCAM/CD166/MEMD) could function as a cell surface sensor for cell density, controlling the transition between local cell proliferation and tissue invasion in melanoma progression. We have tested the hypothesis that progressive cell clustering controls the proteolytic cascade for activation of gelatinase A/matrix metalloproteinase-2 (MMP-2), which involves formation of an intermediate ternary complex of membrane type 1 MMP (MT1-MMP/MMP-14), tissue inhibitor of metalloproteinase-2 (TIMP-2), and pro-MMP-2 at the cell surface. Surprisingly, truncation of ALCAM severely impaired MMP-2 activation in a nude mouse xenograft model, in which we previously observed diminished primary tumor growth and enhanced melanoma metastasis. Comparative studies of two-dimensional monolayer and three-dimensional collagen-gel cultures revealed that extensive cell-to-cell contacts, wild-type ALCAM, and cell-to-matrix interactions were all indispensable for efficient conversion of pro-MMP-2 to its active form in metastatic melanoma cells. Truncated, dominant-negative ALCAM diminished MMP-2 activation via reduced transcript levels and decreased processing of MT1-MMP. Failure of the proteolytic cascade after selective ALCAM depletion by RNA interference was mainly due to incomplete MT1-MMP processing, which was otherwise promoted by extensive cell-to-cell contacts. These data attribute a novel signaling role to ALCAM in regulation of proteolysis and support its previously postulated sensor function in invasive growth.

Multidifferentiation potential of mesenchymal stem cells in three‐dimensional collagen gel cultures

Mesenchymal stem cells (MSCs) have a great therapeutic potential resulting from their ability to differentiate into multiple tissues when cultured under specific conditions. However, it has not been clearly demonstrated whether or not MSCs exhibit a multidifferentiation potential in three-dimensional collagen gel cultures. This study was conducted to explore the multidifferentiation potential of MSCs cultured in three-dimensional collagen gels. Human MSCs were cultured in 0.3% collagen gel for 20 days in chondrogenic differentiation medium (CDM), and for 14 days in osteogenic differentiation medium (ODM). Increases in GAG deposits, intensity of toluidine blue staining, and mRNA expressions of chondrogenic markers (type II collagen and type X collagen) were found in human MSCs cultured in the collagen gel maintained in CDM. Positive staining for alkaline phosphatase (ALP) activity and alizarin red, and increases in mRNA expressions of osteogenic markers (type I collagen, bone sialoprotein and ALP) were noted in the MSCs maintained in ODM. These findings emphasize that human MSCs have an ability to differentiate into both bone and cartilaginous tissues in three-dimensional collagen gel cultures, indicating potential clinical applications of MSC transplant therapy with collagen gel as a scaffold for bone or cartilage regeneration in complicated tissue defects.

Effects of mycophenolic acid on endothelial cells

Mycophenolate mofetil (MMF) is a potent immunosuppressant that inhibits the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo synthesis of guanosine nucleotides. MMF has been used widely in solid-organ transplantation. Increased evidence indicated that MMF exhibited beneficial effects on various types of vasculitis, for reasons that were not fully understood. Endothelial cells play a pivotal role in the pathogenesis of vasculitis. Endothelium may not only be the main target for injury, but also be able to amplify the inflammatory response by adhesion molecule expression, leukocyte adhesion, cytokine production and angiogenesis. In the present study, the effect of mycophenolic acid (MPA), the active metabolite of MMF, on human umbilical vein endothelial cells (HUVECs) was investigated. MPA markedly inhibited tumor necrosis factor-α (TNFα)-induced intercellular adhesion molecule-1 (ICAM-1) mRNA and surface expression, suppressed TNFα-induced neutrophils adhesion to endothelial cells, and reduced TNFα-induced interleukin-6 (IL-6) secretion. The inhibitory effects of MPA on ICAM-1 surface expression and IL-6 secretion were not attenuated by addition of guanosine, implying that inhibition of these processes were not due to intracellular guanosine nucleotides depletion. MPA also decreased angiogenesis of endothelial cells in three-dimensional collagen gel culture system, reduced the migration in a wounded monolayer of endothelial cells, and inhibited the proliferation of endothelial cells. In conclusion, MPA exhibited multifarious effects on endothelial cells including inhibition of ICAM-1 expression, neutrophil attachment, IL-6 secretion, and the process of angiogenesis, which might contribute to the efficacy of MMF in the treatment of vasculitis.

Anti-angiogenic effects of thalidomide: expression of apoptosis-inducible active-caspase-3 in a three-dimensional collagen gel culture of aorta.

The anti-angiogenic properties of thalidomide have led to the use of the agent as a remedy for multiple myeloma. Nevertheless, the anti-angiogenic moiety of thalidomide remains unidentified. In this study we examined the anti-angiogenic effects of thalidomide in an in vitro model using a three-dimensional collagen gel culture. Angiogenesis was significantly inhibited when the culture was treated with thalidomide plus cytochrome P-450 (CYP2B4), and the migrating cells and tubules were positive for active-caspase-3 in an accompanying immunohistochemical investigation. Transmission electron microscopic observation also confirmed that active-caspase-3-positive cells demonstrated apoptotic characteristics. This study is the first to morphologically demonstrate the effect of thalidomide in directly inducing the apoptosis of new tubules and migrating cells on a three-dimensional collagen gel culture of aorta. Taken together with earlier findings, our new results indicate that the thalidomide-induced inhibition of angiogenesis involves apoptosis in addition to the suppression of TNF-alpha and inhibition of cell migration from aorta explants, i.e., the factors important for capillarogenesis.

Thyrocytes, but not C cells, actively undergo growth and folliculogenesis at the periphery of thyroid tissue fragments in three-dimensional collagen gel culture.

In the thyroid, new follicle formation from preexisting follicles (mother follicle-derived folliculogenesis) has been poorly understood. To address this issue, we analyzed mother follicle-derived folliculogenesis, using a thyroid tissue-organotypic culture that retains three-dimensional follicles with both thyrocytes and C cells over a long period. Three types of mother follicle-derived folliculogenesis occurred only at the periphery of the tissue fragments embedded in collagen gel: (1) solid nest, (2) budding and (3) lumen-dividing types. Immunohistochemistry showed that neogenic follicles rarely had calcitonin-positive C cells. Electron microscopy showed that their component thyrocytes expressed normal polarity. In growth, bromodeoxyuridine uptake of thyrocytes at the tissue periphery was about 5 times the uptake that occurred at the center. C cells had no uptake. Thyrotropin (TSH, 10 mU/ml) and free calcium (0.17~1.95 mM), which are associated with the biological behavior of thyrocytes and C cells, respectively, did not affect the events described above. The data indicate that thyrocytes, but not C cells, actively undergo growth and three types of mother follicle-derived folliculogenesis at the tissue periphery in a TSH- or free calcium-independent manner. This suggests that the tissue periphery, which may escape from contact inhibition of cell growth, is the regenerative site.

Reconstruction of tubular structures in three-dimensional collagen gel culture using proximal tubular epithelial cells voided in human urine

Reconstruction of tubular structures in three-dimensional collagen gel culture using proximal tubular epithelial cells voided in human urine

Angiogenesis and fibroblast growth factors (FGFs) in a three-dimensional collagen gel culture.

Small pieces of mouse aorta were cultured in collagen gels, and the formation of capillary-like tubes from the aortic explant was observed under phase-contrast and transmission electron microscopes. Migration of fibroblastic cells from the aortic explant occurred in 2 days. After about 10 days of culture, capillary-like tubes from the aortic explant were formed in the collagen gels. An immunohistochemical study on the collagen gel culture revealed the expression of fibroblast growth factor 2 (FGF-2) near the aortic explant at the active initial stage, with random migration of fibroblastic cells expressing FGF-9. mRNA was isolated from these cultures, and reverse transcription-polymerase chain reaction (RT-PCR) of the cultures revealed the expressions of both FGF-2 and FGF-9. Based on our results, we propose that FGF-9 is also related to angiogenic events.

Coculture of endothelial cells and mature adipocytes actively promotes immature preadipocyte development in vitro.

Adipose tissue consists of mature adipocytes and endothelial cells, which are all supported by the extracellular matrix. Adipose tissue development is closely associated with angiogenesis. However, the adipocyte-endothelial cell interaction is unclear. To address this issue, we examined the effects of endothelial cells on the growth, apoptosis, and differentiation of mature adipocytes in three-dimensional collagen gel culture of the adipocytes with or without rat lung endothelial (RLE) cells. Spindle-shaped preadipocytes, an immature type of adipocyte, developed more actively around the adhesion sites of RLE cells to mature adipocytes in the coculture (rate of preadipocytes: 18.9+/-4.3%) than in the culture of adipocytes alone (2.0+/-5.1%). With respect to growth, RLE cells induced about a three-fold increase in bromodeoxyuridine uptake of mature adipocytes alone, while RLE cells did not influence the uptake of preadipocytes. RLE cells also did not affect the apoptotic indices by immunohistochemistry for single-stranded DNA in mature adipocytes or preadipocytes. These phenomena were not reproduced by RLE cell-conditioned medium, or by certain endothelial cell-produced cytokines. Our in vitro study is the first demonstration that endothelial RLE cells promote the active development of preadipocytes together with increased growth of mature adipocytes. These results suggest that endothelial cells are involved in the enlargement mechanism of adipose tissue mass through their direct adhesion to mature adipocytes.

Red blood cells stimulate human lung fibroblasts to secrete interleukin-8.

Following lung injury, red blood cells (RBC) may interact with extracellular matrix (ECM). Fibroblasts, the resident cell in the ECM, have the capacity to produce and secrete a variety of mediators including interleukin-8 (IL-8). In the present study we hypothesized that RBC, or soluble factors released from them, may stimulate IL-8 production by fibroblasts. Fibroblasts were cultured in a three-dimensional collagen gel culture system in the presence or absence of RBC or conditioned medium from RBC (RBC-CM). IL-8 release from fibroblasts was significantly increased when cultured with RBC or RBC-CM and both tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) further stimulated this IL-8 secretion. The enhanced production of IL-8 within fibroblasts was accompanied by increased IL-8 mRNA expression. To evaluate whether RBC-fibroblast interaction may lead to recruitment of neutrophils, a functional migration assay was performed. RBC and RBC-CM, in the presence of IL-1beta and TNF-alpha, increased the transmigration of neutrophils. Our results indicate that RBC, when interacting with ECM, may participate in the recruitment of inflammatory cells by stimulating fibroblasts to secrete IL-8. This might be an important mechanism regulating tissue repair after injury.