Pancreatic tumor microenvironment is characterized by abundant fibrosis and aberrant vasculature. Aiming to reproduce in vitro these features, cancer cells have been already co-cultured with fibroblasts or endothelial cells separately but the integration of both these essential components of the pancreatic tumor microenvironment in a unique system, although urgently needed, was still missing. In this study, we successfully integrated cellular and acellular microenvironment components (i.e., fibroblasts, endothelial cells, fibronectin) in a hetero-type scaffold-free multicellular tumor spheroid. This new 3D triple co-culture model closely mimicked the resistance to treatments observed in vivo, resulting in a reduction of cancer cell sensitivity to the anticancer treatment.

Tissues and organs consist of a complex organization of cells, extracellular matrix (ECM), and signaling molecules. In particular, blood vessels and skin are a highly organized hierarchical layer composed of various types of cells and ECM layers. The construction in vitro of three-dimensional (3D) cell-polymeric material composites has created notable advances in tissue regeneration.[1] However, an effective methodology to fabricate a 3D multilayer composed of cells and an ECM layer with the appropriate components and thickness has not yet been achieved. Recently, new technologies such as a cell sheet, [2a] magnetic liposomes, [2b] and a chitosan membrane [2c] have been reported to fabricate layered tissues. Although these methods are intriguing, complicated manipulation is required and the thickness of the ECM layer is not controllable.We focused on a layer-by-layer (LbL) technique, which is an appropriate method to prepare nanometer-sized films on a substrate through the alternate immersion into interactive polymer solutions.[3] The preparation of nanometer-sized multilayer films composed of ECM components on the surface of the first layer of cells provides a cell-adhesive surface for the second layer of cells. Rajagopalan et al. demonstrated a bilayer structure composed of hepatocytes and other cells by preparing a polyelectrolyte multilayer consisting of chitosan and DNA on the hepatocyte surface.[4a]However, chitosan cannot dissolve in neutral buffer solutions and fabrication of polyelectrolyte multilayers onto the cell surface is limited owing to the cytotoxicity of polycations. [4b,c] Furthermore, a highly organized cellular multilayer with more than three layers will be required for the creation of functional artificial tissues that are similar to natural tissues. The use of natural ECM components for nanofilms is significant because the typical ECM presents with celladhesive moieties such as RGD (arginine-glycine-aspartic acid) and other amino acid sequences for cellular functions. [5] In the present study, fibronectin (FN) and gelatin were selected to prepare nanometer-sized ECM films (nano-ECM film) on the cell surface. FN is a flexible multifunctional glycoprotein that plays an important role in cell attachment, migration, differentiation, etc. [6a,b] FN is well known to interact not only with a variety of ECM proteins, such as collagens (gelatins) and glycosaminoglycans, but also with the a 5 b 1 integrin receptor on the cell surface.[6c] Recently, we reported FN-based protein multilayers composed of FN and ECM components, such as gelatin, heparin, and elastin, constructed by LbL assembly.[7] Although FN and gelatin have a negative charge under physiological conditions, they interact with each other because FN has a collagen binding domain.[6b] The preparation of FN-gelatin nanofilms on the surface of the first layer of cells will provide a suitable celladhesive surface that is similar to the natural ECM for the second layer of cells. Herein, we report well-organized, fourlayered architectures...

The effects of hyaluronic acid (HA) on the proliferation and chondroitin sulfate (CS) synthesis of chondrocytes embedded in collagen gels were examined. Articular cartilage was isolated from the humerus, femur, and tibia of 21 10-week-old Japanese white rabbits. Chondrocytes isolated by collagenase digestion were embedded in type I collagen gels and cultured in Dulbecco's modified Eagle's medium (DMEM) with various doses of HA for 4 weeks. Histological and biochemical evaluations were performed at postculture weeks 1, 2, 3, and 4. For biochemical evaluations, isomers such as chondroitin 6-sulfate (delta(di)-6S) and chondroitin 4-sulfate (delta(di)-4S) synthesized by cultured chondrocytes were determined by high performance liquid chromatography (HPLC) combined with fluorometry. Morphological and histological studies demonstrated that HA-treated chondrocytes in collagen gel proliferated profusely while maintaining their phenotype. At postculture week 4, 0.1 mg/ml of HA induced an eightfold increase in cell counts compared with HA pretreatment values, or 1.5-fold more than control group. Synthesis of delta(di)-6S (delta(di)-6S content/cell) in groups treated with 0.01 and 0.1 mg/ml of HA significantly increased, while gel accumulation rates in groups treated with 0.1 and 1.0 mg/ml of HA scored significantly higher values than other groups. In collagen gel culture, HA enhanced the proliferation and delta(di)-6S synthesis of chondrocytes while maintaining their phenotype. In clinical application, since the supply of autologous chondrocytes for transplantation is not unlimited, the HA-treated culture method may be useful for increasing the number of chondrocytes and thus improving the quality of implants.

In vitro fabrication of vascularized three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has attracted much attention owing to their requirement of much amount of nutrition and oxygen, but not yet published. In this manuscript, we report construction of vascularized 3D-iPSC-CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique. The filtration-LbL fabricates nanometer-sized fibronectin and gelatin (FN-G) films onto iPSC-CM surfaces. The FN-G nanofilms induce cell-cell interactions via integrin molecules on cell surfaces, leading to construction of 3D-tissues. The constructed vascularized 3D-iPSC-CM tissues would be a promising tool for tissue regeneration and drug development. We believe that this manuscript has a strong impact and offers important suggestions to researchers concerned with biomaterials and tissue engineering.