Concentrations Of Fetal Calf Serum Research Articles

Effect of serum concentration on adhesion of monocytic THP-1 cells onto cultured EC monolayer and EC-SMC co-culture

The adhesion of monocytes to the endothelium following accumulation of low-density lipoprotein (LDL) in subendothelial spaces is an important step in the development of intimal hyperplasia in arterially implanted vein grafts and atherosclerosis in both animals and humans. However, it is not well known how serum factors affect the adhesion of monocytes. We have studied the effect of fetal calf serum (FCS), which we considered a source of LDL, on the adhesion of monocytes to endothelial cells (ECs) by using human monocytic THP-1 cells and both a monolayer of cultured bovine aortic endothelial cells (EC monoculture) and a co-culture with bovine aortic smooth muscle cells (EC-SMC co-culture). It was found that the addition of FCS to the medium greatly affected the adhesion of THP-1 cells, and the higher the concentration of FCS in the medium, the greater the adhesion of THP-1 cells to endothelial cells. Adhesion of THP-1 cells to an EC-SMC co-culture was approximately twofold greater than that to an EC monoculture, and after adhering to endothelial cells, many THP-1 cells transmigrated into the layer of smooth muscle cells. The results suggest that the elevation of the LDL (cholesterol) level in blood provides a favorable condition for the development of intimal hyperplasia and atherosclerosis by promoting the adhesion of monocytes to the endothelium and their subsequent migration into subendothelial spaces.

Influence of fetal calf serum on differentiation of mesenchymal stem cells to chondrocytes during expansion

Mesenchymal stem cells (MSCs) should be expanded in vitro while maintaining their multilineage potential before differentiation to one mesenchymal lineage for application to regeneration therapy. The effect of fetal calf serum (FCS) on undesirable differentiation during subcultivations for the expansion was investigated. The expression level of the aggrecan gene, which is a marker of chondrogenic differentiation, gradually and markedly increased during the subcultivations of MSCs with the addition of 10% FCS and without additional cytokines. The percentage of cells positive for CD90 and CD166, which are markers of MSCs, decreased, and the percentage of large polygonal cells and the average cell adhesion area increased during the expansion. There was a marked difference in the increase in the aggrecan expression level between the two expansion cultures employing different FCS lots, although their proliferation rates were almost the same. The decrease in FCS concentration resulted in a higher percentage of CD90(+)CD166(+) cells, a lower percentage of large polygonal cells, and a lower level of aggrecan expression. Consequently, FCS components could stimulate MSC differentiation to chondrocytes and a lower concentration could decrease this differentiation.

Galectin-3 Expression and Secretion Links Macrophages to the Promotion of Renal Fibrosis

Macrophages have been proposed as a key cell type in the pathogenesis of renal fibrosis; however, the mechanism by which macrophages drive fibrosis is still unclear. We show that expression of galectin-3, a beta-galactoside-binding lectin, is up-regulated in a mouse model of progressive renal fibrosis (unilateral ureteric obstruction, UUO), and absence of galectin-3 protects against renal myofibroblast accumulation/activation and fibrosis. Furthermore, specific depletion of macrophages using CD11b-DTR mice reduces fibrosis severity after UUO demonstrating that macrophages are key cells in the pathogenesis of renal fibrosis. Disruption of the galectin-3 gene does not affect macrophage recruitment after UUO, or macrophage proinflammatory cytokine profiles in response to interferon-gamma/lipopolysaccharide. In addition, absence of galectin-3 does not affect transforming growth factor-beta expression or Smad 2/3 phosphorylation in obstructed kidneys. Adoptive transfer of wild-type but not galectin-3(-/-) macrophages did, however, restore the fibrotic phenotype in galectin-3(-/-) mice. Cross-over experiments using wild-type and galectin-3(-/-) macrophage supernatants and renal fibroblasts confirmed that secretion of galectin-3 by macrophages is critical in the activation of renal fibroblasts to a profibrotic phenotype. Therefore, we demonstrate for the first time that galectin-3 expression and secretion by macrophages is a major mechanism linking macrophages to the promotion of renal fibrosis.

Isolation and characterization of bone marrow–derived equine mesenchymal stem cells

To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transduction rate of equine MSCs. Equine MSCs can be culture-expanded. Equine MSCs undergo cryopreservation in liquid nitrogen without altering morphologic characteristics. Furthermore, equine MSCs maintain their ability to proliferate and differentiate after thawing. Immunocytochemically, the expression of the stem cell marker CD90 can be detected on equine MSCs. The multilineage differentiation potential of equine MSCs was revealed by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation. Our data indicate that bone marrow-derived stromal cells of horses can be characterized as MSCs. Equine MSCs have a high transduction rate and migratory potential and adapt to scaffold material in culture. As an autologous cell population, equine MSCs can be regarded as a promising cell population for tissue engineering in lesions of the musculoskeletal system in horses.

Establishment and characterization of a primary canine duodenal epithelial cell culture

Many mechanisms involved in the pathogenesis of chronic enteropathies or host-pathogen interactions in canine intestine have not been elucidated so far. Next to the clinical and in vivo research tools, an in vitro model of canine intestinal cell culture would be very helpful for studies at the cellular level. Therefore, the purpose of this study was to establish and characterize a primary canine duodenal epithelial cell culture. Neonatal duodenum was disrupted with trypsin-ethylenediaminetetraacetic acid (EDTA) and the mucosa scraped off and digested with collagenase and dispase. After centrifugation on a 2% sorbitol gradient, the cells were incubated at 37 degrees C in OptiMEM supplemented with Primocin, epidermal growth factor, insulin, hydrocortisone, and 10% fetal calf serum (FCS). After 24 h, the FCS concentration was reduced to 2.5%, and the temperature decreased to 33 degrees C. With this method, the cultures were growing to confluent monolayers within 5-6 d and remained viable for an average of 2 wk. Their epithelial nature was confirmed by electron microscopy and immunofluorescence staining using antibodies directed against specific cytokeratins, desmosomes, and tight junctions. The intestinal cells proliferated, as evidenced by immunolabeling with a Ki-67 antibody, and cryptal cell subpopulations could be identified. Furthermore, alkaline phosphatase and sucrase activity were detected.

Reduction of protein synthesis and statin-induced cardiomyocyte cell death

The objective of this study was to determine whether an HMG Co A reductase inhibitor (statin) reduces protein synthesis in cardiomyocytes and whether this action maybe an underlying mechanism for statin-induced cell death. Cardiomyocytes from embryonic chick heart were maintained in culture. Cells exposed to lovastatin for 4 h showed a concentration dependent reduction in protein synthesis as assessed by [3H] leucine incorporation and [35S] methionine incorporation. Compared to control, lovastatin 100 microM, which produced a 25% increase in cell death, induced a three-fold reduction in methionine incorporation. [35S] methionine autoradiography showed little (new) protein synthesis at concentrations of lovastatin of 70 microM or higher; an effect that was not limited to specific proteins. Cardiomyocytes treated with lovastatin showed morphologic changes in the nucleoli consistent with insufficient protein synthesis. These cardiomyocytes manifested cell death under conditions of reduced protein synthesis. Interruption of protein synthesis with cycloheximide, a ribosomal RNA transcription inhibitor or reduction in protein substrate availability by lowering the media concentration of fetal calf serum was associated with a concentration-dependent reductions in cell viability. Importantly, stimulation of protein synthesis by higher concentrations of fetal calf serum limited lovastatin-induced cell death. These data suggest that statin-induced inhibition of protein synthesis is an underlying mechanism for statin-induced cell death.

Chemically induced premature chromosome condensation in short-term cultured human peripheral lymphocytes: applications to biodosimetry

We describe here the chemical induction of premature condensed chromosomes in human peripheral lymphocytes after culture for 6 h. Many have attempted this induction without culture or with short-term culture, because this technique permits prompt cytogenetic biodosimetry of radiation accidents. Lymphocytes were separated from blood, incubated in the presence of phytohemagglutinin, ATP, and p34cdc2/cyclin B kinase, then treated with calyculin A during the last hour. The culture medium was supplemented with a lower concentration of fetal calf serum than conventionally used to minimize its possible interference with the effects of these drugs. We obtained, rarely, a suitable morphology of premature chromosome condensation in short-term cultured lymphocytes for conventional chromosome aberration analysis.

Tissue factor expression by a human kidney proximal tubular cell line in vitro: a model relevant to urinary tissue factor secretion in disease?

Aim: To study baseline and stimulated tissue factor (TF) production from a normal, albeit immortalised, human kidney proximal tubular cell line (HKC-5), in order to establish a model for investigating...

Characterization of Placenta Derived Adherent Cells (PDAC): A Novel Type of Stem Cells Isolated from Human Placenta.

The placenta is a rich source of ethically non-controversial stem cells. In this work, we describe the development of procedures to isolate and culture adherent stem cells from a full term, postpartum human placenta (Placenta Derived Adherent Cells, PDACs). PDACs are isolated from the placenta by one of several methods including physical disruption of tissue from several different anatomical sites within the placenta that include the amniotic membrane, chorion, cord, placental cotyledons, or any combination thereof. PDACs were established in a medium containing low concentrations of fetal calf serum and limited growth factors. Flow cytometry analysis showed that PDACs isolated from certain sites exhibit phenotypes, including for example CD200+ CD105+ CD73+ CD34− CD45− at percentages ≥70%. We found that PDACs differentiate down the adipocyte, chondrocyte and osteocyte lineages. In an induction medium containing IBMX, insulin, dexamethasone and indomethacin, PDACs turned into fat laden adipocytes in 3 to 5 weeks. Under osteogenic induction culture conditions, PDACs were found to form bone nodules and have calcium depositions in their extracellular matrix. Chondrogenic differentiation of PDACs was performed in micropellets and was confirmed by formation of glycosaminoglycan in the tissue aggregates. In addition, we showed that PDACs suppress an MLR consisting of suitable effectors (T cells or NK cells) and target populations (allogeneic irradiated PBMCs or mature DCs). The range of suppression of T cell proliferation was 50 to 95 %. The observed suppression of the MLR is likely cell-to-cell contact dependent in the range of 20%. In summary, the PDACs described herein exhibit in vitro properties of pluripotent stem cells. The placenta is an excellent raw material for the isolation of stem cells.

ISOLATION AND PRIMARY CULTURE OF NECTURUS MACULOSUS (AMPHIBIA: URODELA) HEPATOCYTES

In order to evaluate their suitability for physiological and ecotoxicological studies, hepatocytes were isolated from the common mudpuppy (Necturus maculosus) using a two-step collagenase perfusion. Hepatocytes in primary culture were investigated for 14 d using light and electron microscopy and biochemical analyses. A typical perfusion yielded 1.7 x 10(5) viable hepatocytes per gram body weight with an average viability of 86 +/- 5%. The majority of isolated cells remained in suspension and formed aggregates. The viability of hepatocytes in primary culture was dependent on a fetal calf serum (FCS) concentration and incubation temperature. Viability was best at 8 degrees C in Leibovitz L-15 medium supplemented with 5% FCS. The ultrastructural characteristics of freshly isolated hepatocytes resembled those of N. maculosus hepatocytes in vivo. Whereas hepatocyte viability remained relatively stable (around 80%) up to 14 d in culture, electron microscopic analyses revealed changes at ultrastructural level. The majority of hepatocytes retained similar structural characteristics to those in vivo up to 4 d. Loss of cellular polarity, fractionation of rough endoplasmic reticulum, formation of autophagosomes, and successive exhaustion of cellular glycogen deposits were observed with increased time in culture. Functional integrity, as estimated by tyrosine aminotransferase induction, decreased during the culture period. Ultrastructural and biochemical analyses indicate the need for further improvement of culture conditions. Nevertheless, isolated hepatocytes in primary culture for up to 4 d can be recommended as a model for physiological and toxicological studies in lower vertebrates.

A new fibroblast like cell line from the fry of golden mahseer Tor putitora (Ham)

We report the development of a diploid cell line (TP-1) for the first time from golden mahseer Tor putitora, a world famous food and game fish. Thirty-day-old fry were used to prepare a primary cell culture employing trypsin digestion. The optimal conditions of nutrition and incubation for growth of the dissociated cells were determined. The [ 3H] thymidine uptake assay was employed to investigate the effect of different concentrations of fetal calf serum (FCS) and fish muscle extract (FME) on the growth of TP-1 cells and to study the cell proliferation rate at different time intervals. It was found that L-15 supplemented with 20% FCS and 10% FME at an incubation temperature of 28 °C resulted in optimal growth. The concentration of fetal calf serum was reduced to 10% after 10 subcultures. After confluency, the cells were subcultured with a split ratio of 1:2. The cells showed a fibroblast-like morphology and reached confluency on the fourth day after subculture. The cells were successfully cryopreserved and revived at passage numbers 5, 8, 15 and 18. The cells were characterized for chromosome number (2 n = 100) at 10 and 20 passages. The cell cycle analysis by FACS (Fluorescence Activated Cell Sorter) revealed that most of the cells on the first and fourth day of culture were in S-phase, indicating a high growth rate.

Role of serum factors in epithelial cell responses to Helicobacter pylori infection in vitro

Gastric epithelial cell lines have been utilized extensively as tools to define aspects of the interactions between Helicobacter pylori and host epithelial cells. Fetal calf serum (FCS) is employed as a growth stimulant, but it is unclear whether this agent may in itself alter host responses. Two gastric epithelial cell lines were utilized to ascertain the effects of varying FCS concentration on cellular responses following H. pylori infection. Media containing 0%, 5% or 10% FCS was added to cell lines prior to infection with H. pylori of defined genotype. Cellular interleukin (IL)-8 production was measured as a marker of cellular response. Effects of altered FCS upon cell viability were also determined by trypan blue exclusion. Interleukin-8 production by AGS cells following H. pylori infection was not altered by variation of media FCS concentration. However, KATO-III cells produced greater amounts of IL-8 when media was FCS-free than at 5% or 10% FCS. Although cellular viability was not altered in AGS cells exposed to varied concentrations of FCS, viability was decreased in serum-free KATO-III cells, but not when cells were kept at 5% FCS. Serum-derived factors alter cellular responses to H. pylori infection in a cell-line-dependent manner and impaired cellular viability may relate to this effect. However, the mechanisms for these observations are unclear and further work is now required to determine the nature of these important interactions.

Photodynamic inactivation of fibroblasts by a cationic porphyrin

An important determinant of the clinical applicability and value of antimicrobial photodynamic inactivation (PDI) is the cytotoxicity of the treatment to human cells. We evaluated the in vitro cytotoxicity of PDI to human dermal fibroblasts using 5-phenyl-10,15,20-tris(N-methyl-4-pyridyl)porphyrin chloride (TriP[4]) as the photosensitiser. The fibroblasts were exposed to a PDI regime that is known to be sufficient for the inactivation of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The PDI experiments were carried out in phosphate-buffered saline (PBS) and in 6.25%, 12.5%, 25% and 50% fetal calf serum (FCS)/PBS suspensions. Cell viability subsequent to exposure was evaluated after 0 h, 6 h and 18 h using the methylthiazoletetrazolium (MTT) assay and compared to pretreatment values. At a TriP[4] concentration previously demonstrated to induce a 5 log(10)-unit reduction in a viable count for S. aureus, 79% of the fibroblasts were photo-inactivated. Increasing the FCS concentration in the medium protected the fibroblasts against PDI. Based on our in vitro results, we propose that in vivo PDI of S. aureus holds potential; however, PDI of P. aeruginosa and C. albicans will probably require such a strong PDI regime that it will induce substantial damage to fibroblasts.

Increased Cardiomyocyte Differentiation from Human Embryonic Stem Cells in Serum‐Free Cultures

Human embryonic stem cells (hESCs) can differentiate into cardiomyocytes, but the efficiency of this process is low. We routinely induce cardiomyocyte differentiation of the HES-2 cell line by coculture with a visceral endoderm-like cell line, END-2, in the presence of 20% fetal calf serum (FCS). In this study, we demonstrate a striking inverse relationship between cardiomyocyte differentiation and the concentration of FCS during HES-2-END-2 coculture. The number of beating areas in the cocultures was increased 24-fold in the absence of FCS compared with the presence of 20% FCS. An additional 40% increase in the number of beating areas was observed when ascorbic acid was added to serum-free cocultures. The increase in serum-free cocultures was accompanied by increased mRNA and protein expression of cardiac markers and of Isl1, a marker of cardiac progenitor cells. The number of beating areas increased up to 12 days after initiation of coculture of HES-2 with END-2 cells. However, the number of alpha-actinin-positive cardiomyocytes per beating area did not differ significantly between serum-free cocultures (503 +/- 179; mean +/- standard error of the mean) and 20% FCS cocultures (312 +/- 227). The stimulating effect of serum-free coculture on cardiomyocyte differentiation was observed not only in HES-2 but also in the HES-3 and HES-4 cell lines. To produce sufficient cardiomyocytes for cell replacement therapy in the future, upscaling cardiomyocyte formation from hESCs is essential. The present data provide a step in this direction and represent an improved in vitro model, without interfering factors in serum, for testing other factors that might promote cardiomyocyte differentiation.

Differential regulation of smooth muscle markers in human bone marrow-derived mesenchymal stem cells

To study smooth-muscle differentiation and de-differentiation of human bone marrow-derived mesenchymal stem cells (MSCs), which have been shown to enter the circulation and to contribute to vascular repair and atherosclerosis. Human MSCs from bone marrow were cultured with 20% fetal calf serum (FCS) or with 10% FCS and various concentrations of dimethyl sulfoxide (DMSO). Expression of smooth muscle markers was determined by Western blot analysis and immunofluorescence. For signalling studies, involvement of the mammalian target of rapamycin (mTOR) pathway was tested by treatment with rapamycin. MSCs cultured with 20% FCS acquired a smooth muscle-like appearance and expressed the smooth muscle (sm) markers sm-alpha-actin, desmin, sm-calponin and myosin light chain kinase (MLCK). DMSO induced a spindle-like morphology with marked reduction of stress fibers. As judged by Western blot analysis, treatment with 2.5% DMSO strongly downregulated expression of sm-calponin (-85%), short MLCK (-98%) and sm-alpha-actin expression (-51%). Reduced calponin expression was detected by day 2 of treatment with 0.5-2.5% DMSO. After withdrawal of DMSO, MSCs regained high expression of sm-calponin. Treatment with 6 nmol/l rapamycin partly antagonized the effect of DMSO, indicating the involvement of mTOR in regulation of the smooth muscle phenotype of MSCs. DMSO strongly downregulates the smooth muscle markers sm-calponin, short MLCK and sm-alpha-actin in human MSCs, indicating a transition from a smooth muscle-like phenotype to an undifferentiated state by an mTOR-dependent mechanism. Regulating the phenotype of human MSCs may be of relevance for novel therapeutic approaches in atherosclerosis and intimal hyperplasia after vascular injury.

Regulation of Outside-in Signaling in Platelets by Integrin-associated Protein Kinase Cβ

Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin alpha(IIb)beta(3) in platelets, but the responsible PKC isoforms and mechanisms are unknown. Alpha(IIb)beta(3) interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether alpha(IIb)beta(3) might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKC beta co-immunoprecipitated with alpha(IIb)beta(3) in response to the interaction of platelets with soluble or immobilized fibrinogen. PKC beta recruitment to alpha(IIb)beta(3) was accompanied by a 9-fold increase in PKC activity in alpha(IIb)beta(3) immunoprecipitates. RACK1, an intracellular adapter for activated PKC beta, also co-immunoprecipitated with alpha(IIb)beta(3), but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKC beta recruitment to alpha(IIb)beta(3) and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKC beta spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKC beta I to associate with alpha(IIb)beta(3) and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the beta(3) cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to beta(3). These studies demonstrate that the interaction of alpha(IIb)beta(3) with activated PKC beta is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through alpha(IIb)beta(3). Furthermore, the studies extend the concept of alpha(IIb)beta(3) as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.

Induction of proliferation and differentiation of cultured urothelial cells on acellular biomaterials.

To determine the optimum conditions for the proliferation of urothelial cells, leading to the confluent coverage of large surfaces of biocompatible membranes, and for their terminal differentiation. Porcine and human urothelial cells were cultured on different matrices under different growth conditions. Proliferative activity and the viability of cells were evaluated using fluorescent markers for nuclei and cytoplasm. Growth and differentiation were assessed by histological, histochemical and immunohistochemical methods. Under fibroblastic induction and supplementation of 5% fetal calf serum (FCS), urothelial cells showed more proliferation than in other conditions tested. Terminal differentiation of superficial cells was achieved by lowering the concentration of FCS to 1% at the air-liquid interface. The mitogenic effects of the extracellular matrix content of biological membranes and fibroblastic inductive factors are synergistic with each other, and can compensate for a low FCS concentration and the absence of other additives. Lowering the FCS concentration to 1% inhibits the proliferation of urothelial cells and permits their terminal differentiation.

Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation

Little work in the literature focuses on the cryopreservation of primary hepatocytes as monolayer cultures, yet this technique offers many distinct advantages over other cryopreservation systems, including high recovery, high post-thaw nutrient penetration, and low numbers of trapped dead cells. This article investigates the cryopreservation of primary rat hepatocytes at −78 °C attached as monolayers to collagen coated culture dishes, and describes efforts to increase post-thaw viability and function through manipulation of the freeze/thaw protocol. Different concentrations of foetal calf serum (FCS) with 10% (v/v) dimethyl sulphoxide (ME 2SO) were tested as cryopreservation media, and high cryoprotectant serum levels were found to be important in maintaining membrane integrity and function in the cryopreserved rat hepatocyte monolayer cultures. Cultures cryopreserved with 90% (v/v) FCS plus 10% (v/v) ME 2SO maintain 79.7 ± 6.5% of the monolayer area as viable cells with normal morphology (by image analysis), 112.7 ± 14.2% protein concentration, 55.4 ± 4.2% carboxyfluorescein diacetate de-acetylation, 27.2 ± 7.5% kaempherol glucuronidation (a measure of UDP-glucuronosyl transferase activity), and 39.3 ± 7.3% testosterone hydroxylation (a measure of cytochrome P-450 activity) compared with non-cryopreserved controls. This method of cryopreservation may provide a simple, convenient means of long-term storage of hepatocytes for in vitro metabolism studies.

The effect of medium composition on interleukin-2 production by murine EL-4 thymoma cells

Due to the role of interleukin-2 (IL-2) in the mediation of immune response, this cytokine has been used in the treatment of some types of cancer and infectious diseases. However, relatively high levels of this cytokine are required to achieve significant activity. The aim of this work was to study a culture medium composition designed to increase the production of IL-2 by suspended murine EL-4 cells. The cultivations were carried out aiming at producing IL-2 in stirred bioreactors. The effects of concentration of glutamine, phorbol-12-myristate-13-acetate (PMA), concanavalin A (Con A), Pluronic F68, and fetal calf serum (FCS) on cell viability and IL-2 production were evaluated. PMA alone was more efficient in IL-2 production than it was in association with Con A. The maximum IL-2 production was around 162 ng/mL with 856 ng/mL PMA and 1.45% (v/v) FCS.

Modulation of Sub-RPE deposits in vitro: a potential model for age-related macular degeneration.

Sub-RPE deposits form in a variety of conditions most notably in age-related macular degeneration. The purpose of this study was to generate sub-RPE deposits in vitro and to test the hypotheses that high protein concentrations or retinal homogenate increase deposit formation and that a challenge with tumor necrosis factor (TNF)-alpha or metalloproteinase (MMP)-2 decreases such deposits. ARPE-19 cells were grown on plastic and on collagen type I-coated membrane inserts in media containing various concentrations of fetal calf serum (FCS), bovine serum albumin, or porcine retinal homogenate. In addition, cells grown on membrane inserts were treated with TNF-alpha or MMP-2. Sub-RPE deposits were assessed by electron microscopy and classified into fibrillar, condensed, banded, and membranous subtypes. The area of the micrograph occupied by each type was estimated with a point-counting technique. MMP-2 activity was assessed in tissue culture supernatants by zymography. With increasing time in culture, total deposit formation did not change, but the amount of condensed material deposited by ARPE-19 cells increased while the fibrillar component decreased. Albumin challenge resulted in an increased amount of deposit, predominantly of the membranous type. Challenge with retinal homogenate led to a greater net deposit formation with significant increases in the condensed and banded forms. Cells treated with TNF-alpha or MMP-2 showed a dramatic reduction in all types of sub-RPE deposit. Zymography demonstrated that unchallenged cells produced predominantly MMP-2. Retinal homogenate challenge reduced the total amount of active MMP-2 produced, and TNF-alpha stimulated MMP-9 production. Sub-RPE deposits formed in vitro share ultrastructural features with those seen in vivo. Deposit formation can be modulated by challenge with retinal homogenate, TNF-alpha, or MMP-2. Significantly, the results provide proof of the principle that sub-RPE deposits can be formed and modified in vitro.