Fabrication of Cellular Multilayers with Nanometer‐Sized Extracellular Matrix Films

Abstract

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...

Development of Three-Dimensional Tissue Models Based on Hierarchical Cell Manipulation Using Nanofilms

“…12 We focused on cell adhesive properties of ECM-surrounding cell microenvironment. In general in vitro cell culture method, we obtain only cellular monolayers probably due to absence of sufficient ECM on cell surfaces.…”

Development of Microfluidic Systems for Fabricating Cellular Multilayers

“…In this flow experiment for the LbL process in the microfluidic channel, the values of Q, a, and b at the narrowest region were 0.35 mL/min, 4 mm, and 1 mm, respectively. The mechanical force at 100g that was applied to the cells by centrifugation [ρ × (4/3) × 3.14 × r 3 × 100 × g] was calculated using a cell density (ρ) of 1.05 g/cm 3 , a spherical cell radius (r) of 10 μm, and g of 9.8 N/Kg. 15 The mechanical force (0.4 mdyn) that was applied to a cell by centrifugation under these experimental conditions was higher than the force that was applied to a cell by FSS (0.02 × 3.14 × r 2 = 0.15 μdyn).…”

“…A recent study has demonstrated that gelatin adhesion on the fibronectin (FN) layer was necessary for thickness enhancement of the FN layer. 3 A cellular multilayer was not fabricated by only one coat. 25 Cell adhesion onto cells at a lower layer may be related to the thickness of the BFN layer.…”

“…We designed the PDMS-based microfluidic channel for the LbL method, and formed an inlet center region parallel to the flow direction from the inlet to the outlet. Because the density of mammalian cells (1.05 -1.08 g/cm 3 ) is higher than that of the medium, these round cells were settled by gravitational and/or fluid mechanical forces and then attached to the bottom of the microfluidic channel. 15,16 The volume of the channel region where the cells were coated was approximately 1 mL.…”

“…The deposition of layer-by-layer (LbL) films of the extracellular matrix (ECM) over cultured cells is an important step in preparing cellular multilayers. [2][3][4] Microfluidic devices and systems have been developed to fabricate three-dimensional (3D) cellular structures, including cellular multilayers, and to assess how cells sense changes in their microenvironment. [5][6][7][8][9][10][11] Owing to the availability of soft lithography, a convenient method for microscale fabrication of microfluidic channels has been developed for advancing such research.…”