Fibroblasts In Collagen Gel Research Articles

Human 3D Ovarian Cancer Models Reveal Malignant Cell-Intrinsic and -Extrinsic Factors That Influence CAR T-cell Activity.

In vitro preclinical testing of chimeric antigen receptor (CAR) T cells is mostly carried out in monolayer cell cultures. However, alternative strategies are needed to take into account the complexity and the effects of the tumor microenvironment. Here, we describe the modulation of CAR T-cell activity by malignant cells and fibroblasts in human three-dimensional (3D) in vitro cell models of increasing complexity. In models combining mucin-1 (MUC1) and TnMUC1 CAR T cells with human high-grade serous ovarian cancer cell spheroids, malignant cell-intrinsic resistance to CAR T-cell killing was due to defective death receptor signaling involving TNFα. Adding primary human fibroblasts to spheroids unexpectedly increased the ability of CAR T cells to kill resistant malignant cells as CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells. However, culturing malignant cells and fibroblasts in collagen gels engendered production of a dense extracellular matrix that impeded CAR T-cell activity in a TGFβ-dependent manner. A vascularized microfluidic device was developed that allowed CAR T cells to flow through the vessels and penetrate the gels in a more physiological way, killing malignant cells in a TNFα-dependent manner. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also enabling evaluation of mechanisms of resistance involving cell-cell and cell-matrix interactions, thus accelerating preclinical research on cytotoxic immune cell therapies in solid tumors. Significance: Three-dimensional in vitro models of increasing complexity uncover mechanisms of resistance to CAR T cells in solid tumors, which could help accelerate development of improved CAR T-cell constructs.

Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium.

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast‐derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D‐organotypic (3D‐OT) cultures were constructed. Various growth factors were added at a range of 0.1‐100 ng/ml. 3D‐OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D‐OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage‐colony stimulating factor (GM‐CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM‐CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and β1‐integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL‐positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM‐CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast‐derived diffusible factor(s).

Investigation of Conjunctival Fibrosis Response Using a 3D Glaucoma Tenon's Capsule + Conjunctival Model.

Surgical techniques such as trabeculectomy aim to treat glaucoma by making an incision into the scleral tissue, to create an alternative drainage pathway for aqueous to flow into the sub-Tenon's/subconjunctival space. However, tissue fibrosis and wound healing occurring after the procedures can reduce the success rate. This study aims to investigate the synergistic effects of aqueous humor in combination with shear stress on the fibrosis response occurring in Tenon's capsule and conjunctival tissue (TCCT) after glaucoma surgery. Two-dimensional (2D) and 3D in vitro TCCT models were constructed by seeding porcine Tenon's capsule + conjunctival fibroblasts in collagen gel. These were used to investigate key growth factors (singular and natural form) with shear stress, which are believed to influence tissue fibrosis after glaucoma surgery. In addition to cell proliferation assessments, a nondestructive assay to quantify neocollagen synthesis in TCCT models, in response to these factors, has been applied up to 14 days. TCCT fibroblast proliferation increased significantly with doses of TGF-β, TNF-α, and VEGF, in comparison with the control. Furthermore, fibroblasts exposed to 50% aqueous humor had significantly increased proliferation and actin expression. Shear stress-induced mechanotransduction was also found to promote metabolic activity across experimental conditions. Neocollagen labeling cross validated the fibrosis process. Shear stress appeared to enhance the influence of key growth factors and further promoted fibrotic response within the model. These findings offer a useful insight for further study into the wound-healing response triggered by aqueous fluid outflow after glaucoma surgery.

三次元培養真皮モデルの収縮過程に及ぼす細胞成長因子の影響

During wound healing process, dermal fibroblasts generate a contraction force stimulating cell infiltration and collagen secretion to accelerate the wound healing process as well as decreasing the surface area of the wound. The disorder of this contraction force has risks to cause a skin disease called “keloid” by excessive cell proliferation and secretion of collagen. Keloid is sometimes accompanied with severe itchiness and pain. However, there are not any ideal treatment methods because the occurrence mechanism has been still unclear. Therefore, it is important to monitor the contraction force during wound healing process and to evaluate the effect of several growth factors working in the healing process on this force. Usually, the dermis tissue is reconstructed by embedding the fibroblasts in collagen gel and cultured in vitro. This dermis tissue model would contract during in vitro culture to simulate the wound contraction phenomena under the healing process. In this study, we developed the culturing device for dermis tissue model to enable monitoring the contraction force. This device allowed the culture of dermis tissue model by simulating the condition of CO2 incubator using the glass heater and gas controller. The effects of ascorbic acid, FGF-2 and cell concentration on contraction process were evaluated using this device. As the results, it was suggested that some growth factors were related to contraction process in three-dimensional dermis tissue model.

Probing the role of scaffold dimensionality and media composition on matrix production and phenotype of fibroblasts.

Porous sponges, hydrogels, and micro/nanofibrous matrix are most commonly used three dimensional (3D) biomaterials in tissue engineering; however, reciprocal interaction between internal dimensionality of biomaterials and fibroblasts remains largely unexplored. Such studies would have potential to generate valuable insights about wound healing, tissue morphogenesis and homeostasis. To the best of our knowledge this is the first study to evaluate functionality of porous collagen matrix and collagen gels for in vitro culture of fibroblasts while investigating the role of culture media composition in modulating morphology, phenotype, extracellular matrix (ECM)-related gene expression and protein synthesis by fibroblasts. Encapsulation of fibroblasts in collagen gel was found to be more effective for ECM production compared to scaffold-based culture, as evidenced by enhanced collagen type I, elastin, lysyl oxidase, aggrecan gene expression. High glucose media induced spindle like morphology of typical in vivo fibroblasts and enhanced collagen production compared to other media. This variation in biosynthesis in different glucose concentrations was possibly due to endogenous activation of TGF-β or by an increase in ATP consuming anabolic pathways in high glucose concentration.

Interleukin-1-mediated effects of normal oral keratinocytes and head and neck squamous carcinoma cells on extracellular matrix related gene expression in fibroblasts

The composition of tumor stroma and the activity of tumor associated fibroblasts are important for tumor growth. Interactions between carcinoma cells and fibroblasts regulate the turnover of extracellular matrix (ECM). Here, the in vitro effects of oral squamous cell carcinoma (SCC) cells (UT-SCC-30 and UT-SCC-87) on fibroblast expression of genes for ECM components and connective tissue growth factor (CTGF/CCN2), were compared to those of normal oral keratinocytes (NOK). Cocultures with fibroblasts in collagen gels and keratinocytes with the two cell types separated by a semi permeable membrane were used, and relative gene expression was measured with real-time PCR. All investigated genes were regulated by NOK and the SCCs. The downregulation of pro-collagens α1(I) and α1(III) was more pronounced in cocultures with NOK, while the expression of CCN2 and fibronectin was downregulated by both NOK and the SCCs to a similar extent. UT-SCC-87, but not UT-SCC-30, secreted significantly more IL-1α than NOK. A recombinant interleukin-1 receptor antagonist reversed many of the observed effects on fibroblast gene expression suggesting involvement of IL-1 in cocultures with NOK as well as with SCCs. The observed differential effects on fibroblast gene expression suggest that NOK are more antifibrotic compared to UT-SCC-30 and UT-SCC-87. These findings may contribute to a better understanding of the mechanisms behind ECM turnover in tumors.

Culture of 3T3 fibroblasts in collagen gels in the presence of ACE inhibitors

Conference submission on the development of a culture of 3T3 fibroblasts in collagen gels in the presence of ACE inhibitors presented at the annual congress of the british toxicology society

The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures

BackgroundStromal-epithelial interactions mediate breast development, and the initiation and progression of breast cancer. In the present study, we developed 3-dimensional (3D) in vitro models to study breast cancer tissue organization and the role of the microenvironment in phenotypic determination.MethodsThe human breast cancer MCF7 cells were grown alone or co-cultured with primary human breast fibroblasts. Cells were embedded in matrices containing either type I collagen or a combination of reconstituted basement membrane proteins and type I collagen. The cultures were carried out for up to 6 weeks. For every time point (1-6 weeks), the gels were fixed and processed for histology, and whole-mounted for confocal microscopy evaluation. The epithelial structures were characterized utilizing immunohistochemical techniques; their area and proliferation index were measured using computerized morphometric analysis. Statistical differences between groups were analyzed by ANOVA, Dunnett's T3 post-hoc test and chi-square.ResultsMost of the MCF7 cells grown alone within a collagen matrix died during the first two weeks; those that survived organized into large, round and solid clusters. The presence of fibroblasts in collagen gels reduced MCF7 cell death, induced cell polarity, and the formation of round and elongated epithelial structures containing a lumen. The addition of reconstituted basement membrane to collagen gels by itself had also survival and organizational effects on the MCF7 cells. Regardless of the presence of fibroblasts, the MCF7 cells both polarized and formed a lumen. The addition of fibroblasts to the gel containing reconstituted basement membrane and collagen induced the formation of elongated structures.ConclusionsOur results indicate that a matrix containing both type I collagen and reconstituted basement membrane, and the presence of normal breast fibroblasts constitute the minimal permissive microenvironment to induce near-complete tumor phenotype reversion. These human breast 3D tissue morphogenesis models promise to become reliable tools for studying tissue interactions, therapeutic screening and drug target validation.

Gingival fibroblast inhibits MMP-7: Evaluation in an ex vivo aorta model

Matrix metalloproteinases (MMP) play a deleterious role in numerous vascular diseases. In contrast, gingival matrix remodelling is adequately regulated by the gingival fibroblast (GF). Here, we aimed to evaluate the GF activity on MMP-7 expression and secretion in coculture with aorta rings. We evaluated MMP-7 transcription and secretion in rabbit aorta rings cultured or not with gingival fibroblasts in collagen gels. GF induced an increase of TIMP-1 transcription and secretion, followed, similarly to other MMPs, by the formation of TIMP-1/MMP-7 complexes. There was also a decrease of MMP-7 mRNA by RT-PCR in aorta rings cocultured with gingival fibroblasts. Interestingly, in contrast with other MMPs (which were not influenced at a transcription level), GF stimulated the release of TGF-β1, which in turn inhibited the transcription and synthesis of MMP-7, as shown by neutralizing MMP-7 inhibition due to gingival fibroblast by overexpressing decorin (a TGFβ1 inhibitor) or by silencing TGFβ1 using siRNA. We showed that healing properties of the GF could be transposed to another organ, i.e., ex vivo aneurism model, implicating a down-regulation of MMP-7.

A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels, which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2-8 weeks, the gels contain bilayered mammary epithelial structures, including luminal and myoepithelial cells, their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations.

009 Differential Phenotypical Expression of Healing vs. Normal Fibroblasts

Fibroblasts from a healing ligament likely have differential phenotypical expression from those in an intact ligament, because fibroblasts at the healing site need to repair/regenerate the wounded matrix around them caused by injury, whereas normal fibroblasts just maintain an intact matrix. This study was designed to determine the differences in proliferation, collagen production, α-smooth muscle actin (α-SMA) expression, and contraction between healing and normal fibroblasts. Transected and sham-operated rat medial collateral ligaments (MCL) were used to obtain healing and normal fibroblasts, respectively. It was noted that healing and normal MCL fibroblasts in culture were seen to have a similar morphology. However, healing fibroblasts in monolayer culture proliferated 1.4-fold faster at 48 hours and had 1.7-fold greater protein expression of α-SMA than normal fibroblasts. In addition, it was noted that the proliferation of healing fibroblasts in collagen gels was not significantly different from that of normal fibroblasts at 24 hours, but it was at 48 hours. Furthermore, in collagen gels, healing fibroblasts produced 10% more type I collagen than normal fibroblasts and generated 1.6- and 1.7-fold larger contractile forces at 15 and 20 hours, respectively, than their normal counterparts. Taken together, the results of this study show that healing fibroblasts possess a differential proliferation, α-SMA protein expression, and contraction than normal fibroblasts. This study also highlights the importance of using healing fibroblasts to study the cellular and molecular mechanisms of ligament healing. Supported by the NIH grant AR049921 and Whitaker Foundation Grant (JHW)

Shedding of Syndecan-1 by Stromal Fibroblasts Stimulates Human Breast Cancer Cell Proliferation via FGF2 Activation

The cell surface heparan sulfate proteoglycan syndecan-1 is induced in stromal fibroblasts of breast carcinomas and participates in a reciprocal feedback loop, which stimulates carcinoma cell growth in vitro and in vivo. To define the molecular mechanism of carcinoma growth stimulation, a three-dimensional co-culture model was developed that combines T47D breast carcinoma cells with immortalized human mammary fibroblasts in collagen gels. By silencing endogenous syndecan-1 induction with short interfering RNA and expressing mutant murine syndecan-1 constructs, it was determined that carcinoma cell mitogenesis required proteolytic shedding of syndecan-1 from the fibroblast surface. The paracrine growth signal was mediated by the syndecan-1 heparan lfate chains rather than the ectodomain of the core protein and required fibroblast growth factor 2 and stroma-derived factor 1. This paracrine pathway may provide an opportunity for the therapeutic disruption of stromaepithelial signaling.

Difference in the apoptotic response of fetal versus adult human lung fibroblasts in collagen gels

Difference in the apoptotic response of fetal versus adult human lung fibroblasts in collagen gels

Collagenase-3 (MMP-13) Enhances Remodeling of Three-Dimensional Collagen and Promotes Survival of Human Skin Fibroblasts

Collagenase-3 (MMP-13) is a matrix metalloproteinase capable of cleaving a multitude of extracellular matrix proteins in addition to fibrillar collagens. Human MMP-13 is expressed by fibroblasts in chronic cutaneous ulcers, but not in normally healing adult skin wounds. However, MMP-13 is produced by fibroblasts in adult gingival and in fetal skin wounds characterized by rapid collagen remodeling and scarless healing. Here, we have examined the role of human MMP-13 in remodeling of three-dimensional (3D) collagenous matrix by primary adult human skin fibroblasts. The high level of human MMP-13 expression by fibroblasts achieved by adenoviral gene delivery resulted in potent enhancement of remodeling and contraction of 3D collagen. Fibroblasts expressing MMP-13 in 3D collagen possessed altered filamentous actin morphology with patch-like actin distribution in cell extensions. The expression of MMP-13 promotes survival and proliferation of fibroblasts in floating collagen gel, and results in activation of Akt and extracellular signal-regulated kinase-1/2 by these cells. The results provide evidence for a novel role for human MMP-13 in regulating dermal fibroblast survival, proliferation, and interaction in 3D collagen, which may be an important survival mechanism for fibroblasts in chronic skin ulcers and contribute to scarless healing of adult gingival and fetal skin wounds.

Healing and Normal Fibroblasts Exhibit Differential Proliferation, Collagen Production, α-SMA Expression, and Contraction

This study determines the differences in proliferation, collagen production, alpha-smooth muscle actin (alpha-SMA) expression, and contraction between healing and normal fibroblasts. Transected and sham-operated rat medial collateral ligaments (MCL) were used to obtain healing and normal fibroblasts, respectively. It was found that healing fibroblasts in monolayer culture proliferated 1.4-fold faster at 48 h and had 1.7-fold greater protein expression of alpha-SMA than normal fibroblasts. In addition, it was noted that the proliferation of healing fibroblasts in collagen gels was not significantly different from that of normal fibroblasts at 24 h, but it was at 48 h. Furthermore, in collagen gels, healing fibroblasts produced more type I collagen than normal fibroblasts and generated 1.6- and 1.7-fold larger contractile forces at 15 and 20 h, respectively, than their normal counterparts. Taken together, the results of this study show that healing fibroblasts possess a differential proliferation, alpha-SMA protein expression, and contraction than normal fibroblasts.

Evaluation of Lateral Mechanical Tension in Thin-Film Tissue Constructs

Fibroblast-populated collagen matrices provide a simplified tissue model for wound healing and development processes. A technology (CELLDRUM Technology) evaluating lateral mechanical tension in fibroblast-populated collagen matrices (tissue constructs) with a thickness of 1 mm was introduced. Defined mechanical boundary conditions together with the known number and orientation of the cells revealed precise data on the average tension exerted by a single cell. Circular cell-populated collagen gels were manufactured inside the CELLDRUM on top of a flexible membrane. The collagen matrix was then excited by a sound pulse. The resulting resonance oscillation was monitored by a laser-based deflection sensor and frequency and damping were analyzed giving information on mechanical properties of the tissue construct. Several evaluation experiments were performed. Calf serum enhanced contractile forces of fibroblasts dose dependently. After the gels were treated with cytochalasin D for 24 h, the cell forces were reduced by 42% of control. The remaining tension was attributed to the extracellular matrix remodeling occurring during cell growth and to other cytoskeletal structures like microtubules and intermediate filaments. We also found that only after a few hours of culture fibroblast-seeded collagen gels began developing significant mechanical tension. A mechanical tension profile of proliferating fibroblasts in collagen gels over culture time was obtained.

Transforming growth factor-β1 stimulates collagen matrix remodeling through increased adhesive and contractive potential by human renal fibroblasts

Renal tubulointerstitial fibrosis is the common final pathway leading to end-stage renal failure. Tubulointerstitial fibrosis is characterized by fibroblast proliferation and excessive matrix accumulation. Transforming growth factor-β1 (TGF-β1) has been implicated in the development of renal fibrosis accompanied by α-smooth muscle actin (α-SMA) expression in renal fibroblasts. To investigate the molecular and cellular mechanisms involved in tubulointerstitial fibrosis, we examined the effect of TGF-β1 on collagen type I (collagen) gel contraction, an in vitro model of scar collagen remodeling. TGF-β1 enhanced collagen gel contraction by human renal fibroblasts in a dose- and time-dependent manner. Function-blocking anti-α1 or anti-α2 integrin subunit antibodies significantly suppressed TGF-β1-stimulated collagen gel contraction. Scanning electron microscopy showed that TGF-β1 enhanced the formation of the collagen fibrils by cell attachment to collagen via α1β1 and α2β1 integrins. Flow cytometry and cell adhesion analyses revealed that the stimulation of renal fibroblasts with TGF-β1 enhanced cell adhesion to collagen via the increased expression of α1 and α2 integrin subunits within collagen gels. Fibroblast migration to collagen was not up-regulated by TGF-β1. Furthermore, TGF-β1 increased the expression of a putative contractile protein, α-SMA, by human renal fibroblasts in collagen gels. These results suggest that TGF-β1 stimulates fibroblast–collagen matrix remodeling by increasing both integrin-mediated cell attachment to collagen and α-SMA expression, thereby contributing to pathological tubulointerstitial collagen matrix reorganization in renal fibrosis.

Molecular dissection of the fibroblast-traction machinery.

Motile fibroblasts generate forces that can be expressed as cell migration or as traction, the drawing-in of extracellular matrix. Traction by cultured fibroblasts can induce a rapid concerted reorganization of collagen gel, creating a pattern of collagen alignment similar to that seen in tendons and ligaments. In such fibrous connective tissues, after pattern morphogenesis is complete, ongoing traction may be responsible for the maintenance of proper form and function. The molecules that generate and transmit forces have been catalogued; however, how these nanometer-scale molecules contribute to millimeter-scale patterns has not been directly tested. Here, we placed pairs of explants of human periodontal ligament fibroblasts in collagen gels, where ligament-like straps of anisotropic collagen formed on the axes between them. We scrutinized the traction apparatus using electron microscopy, video microscopy, and computer-based pattern analysis, augmented with pharmacologic inhibitors of cytoskeletal function. Patterning was marked by the co-alignment of collagen, fibroblasts, and their actin cytoskeletons, all parallel to the axis between explants. The pattern was diminished by depolymerizing actin filaments or by blocking myosin activity, but was accentuated by depolymerizing microtubules. The plasma membrane also seems to contribute to the traction force. These molecular components combine to exert a sub-maximal traction force on the matrix, suggesting that the force may be regulated to ensure tissue tensional homeostasis.

Multilineage Differentiation of Rhesus Monkey Embryonic Stem Cells in Three‐Dimensional Culture Systems

In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

Secretory Leukocyte Protease Inhibitor Is a Novel Inhibitor of Fibroblast-mediated Collagen Gel Contraction

Cultured epithelial cells, including those from the oral epithelium, have been successfully applied in the promotion of scarless wound healing. Factors released from the epithelial cells are thought to contribute significantly to the beneficial effects. In the conditioned medium of human oral epithelial cells, we found a factor that inhibited fibroblast-mediated collagen gel contraction, an in vitro model of wound healing and scar formation. Biochemical analysis identified the factor to be human secretory leukocyte protease inhibitor (SLPI). Fibroblasts transfected with SLPI cDNA showed reduced gel-contracting activity. SLPI purified from the conditioned medium inhibited gel contraction in a dose-dependent manner, and anti-SLPI antibody counteracted this activity. Upon SLPI treatment, human skin fibroblasts in collagen gel became shorter in length and were inhibited in pseudopodia extension. Furthermore, after SLPI treatment, α1-integrin immunoreactivity decreased, and cyclic AMP levels increased. Excessive gel contraction was observed when fibroblasts treated with TGF-β1 and fibroblasts from hypertrophic and from keloid scar tissue were cultured in collagen gel. SLPI was also effective in inhibiting gel contraction in the above three models of scar formation. These results suggest that SLPI may be useful in promoting scarless wound healing.