3D Gel Culture Research Articles

Enhancement of Chondrogenic Markers by Exosomes Derived from Cultured Human Synovial Fluid-Derived Cells: A Comparative Analysis of 2D and 3D Conditions.

The goal of this pilot study was to investigate the effects of exosomes derived from synovial fluid-derived cells (SFDCs) cultured under normoxic conditions in a two-dimensional (2D) monolayer or encapsulated within a three-dimensional (3D) matrix for chondrogenic differentiation in vitro and cartilage defect repair in vivo. Synovial fluid samples were obtained from three patients, and SFDCs were isolated and expanded either in a 2D monolayer culture or seeded within a transglutaminase cross-linked gelatin (Col-Tgel) to create a 3D gel culture. Exosomes derived from each environment were isolated and characterized. Then, their effects on cartilage-cell proliferation and chondrogenic differentiation were assessed using an in vitro organoid model, and their potential for enhancing cartilage repair was evaluated using a rat cartilage defect model. SFDCs obtained from different donors reached a state of senescence after four passages in 2D culture. However, transferring these cells to a 3D culture environment mitigated the senescence and improved cell viability. The 3D-cultured exosomes exhibited enhanced potency in promoting chondrogenic differentiation, as evidenced by the increased expression of chondrogenic genes and greater deposition of cartilage-specific extracellular matrix. Furthermore, the 3D-cultured exosomes demonstrated superior effectiveness in enhancing cartilage repair and exhibited better healing properties compared to exosomes derived from a 2D culture. The optimized 3D culture provided a more favorable environment for the proliferation of human synovial cells and the secretion of exosomes compared to the 2D culture. The 3D-cultured exosomes exhibited greater potential for promoting chondrogenic gene expression in vitro and demonstrated improved healing properties in repairing cartilage defects compared to exosomes derived from the 2D culture.

#3679 ANOCTAMIN 1 INHIBITION SUPPRESSES CYSTOGENESIS BY ENHANCING CILIOGENESIS AND THE CILIARY DOSAGE OF POLYCYSTINS

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy which is characterized by abnormal tubular epithelial proliferation and fluid secretion. Anoctamin 1 (ANO1) is a calcium-dependent chloride channel, which has recently been shown to be involved in ADPKD progression. However, how ANO1 contributes to ADPKD is largely unexplored. Method 1. Based on mRNA gene chips, the expression of ANO1 abnormally increased in human ADPKD kidneys. 2. Immunohistochemical staining showed ANO1 location. 3. Real-time PCR and Western blot showed mRNAs and protein of ANO1 in ADPKD patients and animal model-PKD1RC/RC mice. 4. Confocal immunofluorescence showed ANO1 and PC2 location in the cilia. 5. 3D gel culture to verify the function of ANO1 in cysts formation. 6. ANO1 inhibitors and siRNA to discover STAT3 and ERK pathway in ADPKD cell by WB. Results We found that ANO1 is consistently upregulated inhuman and mouse PKD kidneys. Intriguingly, ANO1 located in a vesicle-like patten at the ciliary base but not on the ciliary surface. ANO1 deficiency promotes both ciliogenesis and the ciliary dosage of polycystin-2 in human Pkd1-/- cells, and reduced cyst formation in 3D culture models. Moreover, inhibition of ANO1 abolished the activation of STAT3 and ERK pathways in PKD cells. Conclusion Our data indicate ANO1 may aggravate ADPKD by regulating the cilia length and PC2 expression. Moreover, ANO1 could support cyst growth by activating STAT3 and ERK pathways. Thus ANO1 is a negative regulator for both cilia formation and cilia trafficking of polycystins and provide mechanistic insights regarding the therapeutic potential of ANO1 pathway in ADPKD treatment.

Metastatic behavior analyses of tetraspanin TM4SF5-expressing spheres in three-dimensional(3D) cell culture environment.

Cancer metastasis involves diverse cellular functions via bidirectional communications between intracellular and extracellular spaces. To achieve development of the anti-metastatic drugs, one needs to consider the efficacy and mode of action (MOA) of the drug candidates to block the metastatic potentials of cancerous cells. Rather than under two-dimensional environment, investigation of the metastatic potentials under three-dimensional environment would be much pharmaceutically beneficent, since it can mimic the in vivo tumor lesions in cancer patients, leading to allowance of drug candidates analyzed in the 3D culture systems to lower failure rates during the anti-metastatic drug development. Here we have reviewed on the analyses of metastatic potentials of certain cancer models in 3D culture systems surrounded with extracellular matrix proteins, which could be supported by TM4SF5- and/or EMT-mediated actions. We particularly focused the initial events of the cancer metastasis, such as invasive outgrowth and dissemination from the cancer cell masses, spheroids, embedded in the 3D gel culture systems. This review summarizes the significance of tetraspanin TM4SF5 and Snail1 that are related to EMT in the metastatic potentials explored in the 3D gel systems.

Osteogenic cells form mineralized particles, a few μm in size, in a 3D collagen gel culture.

Osteogenic cells form mineralized matrices in vitro, as well as in vivo. The formation and shape of the mineralized matrices are highly regulated by the cells. In vitro formation of mineralized matrices by osteogenic cells can be a model for in vivo osteogenesis. In this study, using a three-dimensional (3D) collagen gel culture system, we developed a new in vitro model for the formation of mineralized particles, a few µm in size, by the osteogenic cells. Human osteosarcoma (HOS) cells formed spherical mineralized matrices (about 12 µm) at approximately 7 days when cultured with β-glycerophosphate (β-GP)-containing culture media on 2D tissue culture plates. Alternately, when they were cultured in a 3D collagen gel containing β-GP, they formed mineralized particles with about 1.7 µm in the gel at approximately 3 days. Calcium precipitation in the gel was evaluated by measuring the gel turbidity. This type of mineralization of HOS cells, which formed mineralized particles inside the gel, was also observed in a peptide-based hydrogel culture. The mineralized particles were completely diminished by inhibiting the activity of Pit-1, phosphate cotransporter, of the HOS cells. When mouse osteoblast-like MC3T3-E1 cells, which form large and flat mineralized matrices in 2D osteogenic conditions at approximately 3 weeks of culture, were cultured in a 3D collagen gel, they also formed mineralized particles in the gel, similar to those in HOS cells, at approximately 18 days. Thus, osteogenic cells cultured in the 3D collagen gel form mineralized particles over a shorter period, and the mineralization could be easily determined by gel turbidity. This 3D gel culture system of osteogenic cells acts as a useful model for cells forming particle-type mineralized matrices, and we assume that the mineralized particles in the 3D hydrogel are calcospherulites, which are derived from matrix vesicles secreted by osteogenic cells.

A novel and efficient method for culturing mouse nucleus pulposus cells

BACKGROUND CONTEXTAs degeneration of the nucleus pulposus (NP) is a major cause of intervertebral disc degeneration, research directed toward nucleus pulposus cells (NPCs) is drawing increased attention. However, caused by the difficulties associated with their harvest and culture, there are few reports describing cultivation methods for mouse NP cells (mNPCs). PURPOSETo establish efficient culture methods for mNPCs. STUDY DESIGNIn vitro animal study. METHODSAfter primary 3-dimensional (3D) gel culture of mNPCs and analysis of gene expression, cells digested from the gel were cultured in various bio-coated dishes with and without basic fibroblast growth factor (bFGF), and their growth kinetics and changes in gene expression profiles were evaluated. Next, the mNPCs obtained after sequential 3D gel and 2D culture were subjected to micromass culture and the effects of adding transforming growth factor-β3 (TGF-β3) on their gene expression profile and extracellular matrix (ECM) synthesis were evaluated. RESULTSThe cell morphology and gene expression pattern of mNPCs proliferated in primary 3D collagen gel culture resembled those of mNP. In contrast, mNPCs could not proliferate in conventional monolayer culture. Cell adhesion (colony number) and proliferation (colony size) were greater in fibronectin-coated dishes than in dishes with other bio-coatings. The addition of bFGF enhanced mNPCs proliferation, but the gene expression characteristics of mNPCs were lost as passage number increased. 2D culture with bFGF followed by micromass culture allowed for the recovery of the mNPC gene expression profile in primary 3D-gel culture, and TGF-β3 supplementation during micromass culture enhanced ECM synthesis. CONCLUSIONSWe established novel culture methods for mNPCs. These methods will benefit basic cell-based and molecular research involving these cells.

Downregulation of Hes1 expression in experimental biliary atresia and its effects on bile duct structure.

AIMTo analyze the expression and function of the Notch signaling target gene Hes1 in a rhesus rotavirus-induced mouse biliary atresia model.METHODSThe morphologies of biliary epithelial cells in biliary atresia patients and in a mouse model were examined by immunohistochemical staining. Then, the differential expression of Notch signaling pathway-related molecules was investigated. Further, the effects of the siRNA-mediated inhibition of Hes1 expression were examined using a biliary epithelial cell 3D culture system.RESULTSBoth immature (EpCAM+) and mature (CK19+) biliary epithelial cells were detected in the livers of biliary atresia patients without a ductile structure and in the mouse model with a distorted bile duct structure. The hepatic expression of transcripts for most Notch signaling molecules were significantly reduced on day 7 but recovered to normal levels by day 14, except for the target molecule Hes1, which still exhibited lower mRNA and protein levels. Expression of the Hes1 transcriptional co-regulator, RBP-Jκ was also reduced. A 3D gel culture system promoted the maturation of immature biliary epithelial cells, with increased expression of CK19+ cells and the formation of a duct-like structure. The administration of Hes1 siRNA blocked this process. As a result, the cells remained in an immature state, and no duct-like structure was observed.CONCLUSIONOur data indicated that Hes1 might contribute to the maturation and the cellular structure organization of biliary epithelial cells, which provides new insight into understanding the pathology of biliary atresia.

Fibroblast VEGF-receptor 1 expression as molecular target in periodontitis.

Degradation of extracellular matrices is an integral part in periodontitis. For antagonizing this pathophysiological mechanism, we aimed at identifying gene expression profiles in disease progression contributing periodontitis-associated fibroblasts (PAFs) versus normal gingival fibroblasts to determine their molecular repertoire, and exploit it for therapeutic intervention. Applying an exploratory analysis using a small number of microarrays in combination with a three dimensional (3D) in vitro culture model that incorporates some aspects of periodontitis, PAFs were initially characterized by gene-expression analyses, followed by targeted gene down-regulation and pharmacological intervention in vitro. Further, immunohistochemistry was applied for phosphorylation analyses in tissue specimens. PAFs were characterized by 42 genes being commonly up-regulated >1.5-fold, and by five genes that were concordantly down-regulated (<0.7-fold). Expression of vascular endothelial growth factor (VEGF)-receptor 1 (Flt-1) was highly enhanced, and was thus further explored in in vitro culture models of periodontal fibroblasts without accounting for the microbiome. Phosphorylation of the VEGF-receptor 1 was enhanced in PAFs. Receptor inhibition by a specific VEGF-receptor inhibitor or intrinsic down-regulation by RNAi of the VEGF-receptor kinase in 3D gel cultures resulted in significant reduction in collagen degradation associated with increased tissue inhibitor of metalloproteinase expression, suggesting that Flt-1 may contribute to periodontitis. Based on the finding that VEGF-receptor kinase inhibition impaired collagen degradation pathways, Flt-1 may represent a candidate for therapeutic approaches in periodontitis.

Effects of Wnt-10b on proliferation and differentiation of murine melanoma cells

In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-β-gal-positive senescent cells in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage.

Mesenchymal stem cell response to TGF-β1 in both 2D and 3D environments.

Smooth muscle cells (SMC) are critical in stabilizing developing vascular networks, and transforming growth factor β1 (TGF-β1) has been shown to promote SMC differentiation from stem cells. Previously, our lab has developed a chemically modified fibrin-based hydrogel that induces endothelial cell (EC) phenotype and network formation from human mesenchymal stem cells (hMSCs) without exogenous cytokines. Additionally, we have shown that this hydrogel system is capable of releasing growth factors in a controlled manner. In the present work, the effects of TGF-β1 on hMSCs in both monolayer and fibrin-based gel culture systems were demonstrated. The objective was to enhance SMC properties through TGF-β1 signaling for vessel stability while maintaining EC gene expression and morphology. Proliferation was decreased with higher TGF-β1 concentration in both monolayer and 3D gel cultures. EC genes were predominantly downregulated in the presence of TGF-β1 in monolayer cultures, while SMC genes were generally upregulated. In fibrin-based gels, several SMC genes were significantly upregulated at high concentrations of TGF-β1. Even at elevated TGF-β1 concentrations, no significant differences were seen in EC genes for hMSCs in gels compared to controls. Network formation and growth occurred in PEGylated fibrin gels loaded with TGF-β1 and were not significantly different from gels without loaded growth factor. Additionally, production of smooth muscle α-actin (SMA) was significantly increased in gels loaded with TGF-β1. These results demonstrate a simultaneous response of hMSCs to both the 3D biomatrix and cytokine signaling cues.

Effect of a hepatocyte growth factor/heparin-immobilized collagen system on albumin synthesis and spheroid formation by hepatocytes

A hepatocyte growth factor (HGF)/heparin-immobilized collagen system was used as a synthetic extracellular matrix for hepatocyte culture. The albumin synthesis, nucleus numbers and morphology of the hepatocytes were determined separately to evaluate the hepatocyte number and hepatocyte-specific function under this system. The benefits of the HGF/heparin-immobilized collagen system for hepatocyte culture were confirmed by three types of culture methods in vitro, namely 2D film cultures, 2D gel cultures and 3D gel cultures. In 2D collagen film cultures, hepatocytes exhibited the highest albumin synthesis (1.42 microg/well/day) in HGF/heparin-immobilized collagen films at 7 days of culture. Heparin inhibited hepatocyte adhesion while HGF promoted hepatocyte migration, and spheroid formation was easily detected in HGF/heparin-immobilized collagen films. In 2D collagen gel cultures, albumin synthesis of around 15 microg/well/day was detected and maintained for more than 18 days on HGF/heparin-immobilized collagen gels. Similar findings were obtained in 3D HGF/heparin-immobilized collagen gel cultures, which exhibited albumin synthesis of up to 30 microg/well/day. The albumin synthesis by hepatocytes was two-fold higher in 3D gel cultures compared with 2D gel cultures, and was maintained for over 2 weeks compared with 2D film cultures using the HGF/heparin-immobilized collagen system. Taken together, the HGF/heparin-immobilized collagen system was effective for albumin synthesis by hepatocytes in both 2D film cultures and 3D gel cultures, and therefore shows good potential for tissue engineering use.

αv and β1 integrins regulate dynamic compression-induced proteoglycan synthesis in 3D gel culture by distinct complementary pathways

Our goal was to test the hypothesis that specific integrin receptors regulate chondrocyte biosynthetic response to dynamic compression at early times in 3D gel culture, during initial evolution of the pericellular matrix, but prior to significant accumulation of further-removed matrix. The study was motivated by increased use of dynamic loading, in vitro, for early stimulation of tissue engineered cartilage, and the need to understand the effects of loading, in vivo, at early times after implantation of constructs. Bovine articular chondrocytes were seeded in 2% agarose gels (15x10(6)cells/mL) and incubated for 18 h with and without the presence of specific integrin blockers (small-molecule peptidomimetics, function-blocking antibodies, and RGD-containing disintegrins). Samples were then subjected to a 24-h dynamic compression regime found previously to stimulate chondrocyte biosynthesis in 3D gel as well as cartilage explant culture (1 Hz, 2.5% dynamic strain amplitude, 7% static offset strain). At the end of loading, proteoglycan (PG) synthesis ((35)S-sulfate incorporation), protein synthesis ((3)H-proline incorporation), DNA content (Hoechst dye 33258) and total glycosaminoglycan (GAG) content (dimethyl methylene blue (DMMB) dye binding) were assessed. Consistent with previous studies, dynamic compression increased PG synthesis and total GAG accumulation compared to free-swelling controls. Blocking alphavbeta3 abolished this response, independent of effects on controls, while blocking beta1 abolished the relative changes in synthesis when changes in free-swelling synthesis rates were observed. This study suggests that both alphavbeta3 and beta1 play a role in pathways that regulate stimulation of PG synthesis and accumulation by dynamic compression, but through distinct complementary mechanisms.

Inhibition of in vitro chondrogenesis in RGD-modified three-dimensional alginate gels

The goal of this study was to investigate the effects of adhesion to the arginine-glycine-aspartic acid (RGD) sequence on the chondrogenesis of bone marrow stromal cells (BMSCs). Synthetic RGE- and RGD-containing peptides were conjugated to sodium alginate, and bovine BMSCs were seeded onto 2D alginate surfaces or encapsulated in 3D gels. BMSCs spread specifically on RGD-modified surfaces, and spreading was inhibited by a soluble RGD peptide and by anti- β1 and anti- αv β3 integrin blocking antibodies. After 7 days in 3D gel culture, the chondrogenic supplements (TGF- β1 and dexamethasone) significantly stimulated chondrocytic gene expression (collagen II, aggrecan, and Sox-9) and matrix accumulation (collagen II and sGAG) in RGE-modified gels, but this response was inhibited in the RGD-modified gels. Inhibition of sGAG synthesis increased with increasing RGD density, and synthesis was partially rescued by adding a soluble RGD peptide. Addition of an anti- αv β3 integrin blocking antibody had no effect on chondrogenesis, while an anti- α5 antibody reduced sGAG accumulation. Overall, this study demonstrates that interaction with the RGD motif significantly inhibits the initial chondrogenesis of BMSCs within 3D alginate gels. These results provide new insights into the role of cell–matrix interactions in regulating chondrogenesis and highlight the importance of choosing appropriate biomaterials for tissue engineering therapies.