Improving stability of human three dimensional skin equivalents using plasma surface treatment.

In developing three-dimensional (3D) human skin equivalents (HSEs), preventing dermis and epidermis layer distortion due to the contraction of hydrogels by fibroblasts is a challenging issue. Previously, a fabrication method of HSEs was tested using a modified solid scaffold or a hydrogel matrix in combination with the natural polymer coated onto the tissue culture surface, but the obtained HSEs exhibited skin layer contraction and loss of the skin integrity and barrier functions. In this study, we investigated the method of HSE fabrication that enhances the stability of the skin model by using surface plasma treatment. The results showed that plasma treatment of the tissue culture surface prevented dermal layer shrinkage of HSEs, in contrast to the HSE fabrication using fibronectin coating. The HSEs from plasma-treated surface showed significantly higher transepithelial electrical resistance compared to the fibronectin-coated model. They also expressed markers of epidermal differentiation (keratin 10, keratin 14 and loricrin), epidermal tight junctions (claudin 1 and zonula occludens-1), and extracellular matrix proteins (collagen IV), and exhibited morphological characteristics of the primary human skins. Taken together, the use of plasma surface treatment significantly improves the stability of 3D HSEs with well-defined dermis and epidermis layers and enhanced skin integrity and the barrier functions.

Fabrication of Fully Positively Charged Layer-by-Layer Polyelectrolyte Nanofilms with pH-Dependent Swelling Properties.

Nanofilms fabricated by layer-by-layer (LbL) assembly from polyelectrolytes (PEs) are important materials for various applications. However, PE films cannot retain the charges along the polymer chains during fabrication, resulting in a low charge density. In this study, the preparation of LbL nanofilms with preserved positive charges via a controllable and efficient approach was achieved. To fabricate fully positively charged (FPC) LbL nanofilms, a polycation, poly-l-lysine, was partially grafted with azide and alkyne groups. Through copper-catalyzed azide-alkyne cycloaddition and the LbL procedure, nanofilms were fabricated with all of the individual layers covalently bonded, improving the pH stability of the nanofilms. Because the resulting nanofilms had a high charge density with positive charges both inside and on the surface, they showed unique pH-dependent swelling properties and adsorption of negatively charged molecules compared with those of traditional polyelectrolyte LbL nanofilms. This kind of FPC nanofilm has great potential for use in sensors, diagnostics, and filter nanomaterials in the biomedical and environmental fields.

Analysis of Homo- and Heterotriple Helix Formation of Collagen Model Peptides and Evaluation of Their Stability in a Biological Environment.

Self-assembled materials have attracted attention and have been extensively studied because the reversibility of noncovalent interactions allows them to possess various properties, such as stimulus responsiveness and self-healing. Collagen model peptides have an amino acid sequence characteristic of the triple helix region of collagen and exhibit repeatable triple helix formation. Many studies of their applications have used homotrimers, and although some studies on heterotrimers have been reported, few have clarified the details. If the characteristics of heterotrimers can be revealed, they are expected to be applied as new self-assembled materials. In this study, we analyzed the detailed self-assembling properties of hetero- and homohelices formed by (proline-proline-glycine)10 (PPG)10 and (proline-hydroxyproline-glycine)10 (POG)10 to evaluate the potential of the helices for biomedical application. Fluorescein isothiocyanate-labeled (PPG)10 (F(PPG)10) and (POG)10 (F(POG)10) were synthesized to analyze the heterotriple helix formation using concentration quenching based on triple helix formation. When (PPG)10 was added to F(POG)10, the fluorescence intensity did not reach a plateau, while the fluorescence intensity reached about 100% in the other pairs such as (POG)10-F(POG)10, (PPG)10-F(PPG)10, and (POG)10-F(PPG)10. The critical triple helix formation concentration was 7 µM for the heterotrimer prepared under 1:2 mixing conditions of (PPG)10 and (POG)10, 320 µM for [(PPG)10]3, and 4 µM for [(POG)10]3, indicating that the triple helix formation concentration of the heterotrimer is almost half that of [(POG)10]3 but 45 times higher than [(PPG)10]3. Furthermore, the heterotrimer formed at 37 °C was stable after 5 days, which was the same as [(POG)10]3. These results suggest that heterotrimers have different association properties from homotrimers and are expected to be applied in nanotechnology and biomaterials as new self-assembled materials.

Biofabrication of engineered blood vessels for biomedical applications.

To successfully engineer large-sized tissues, establishing vascular structures is essential for providing oxygen, nutrients, growth factors and cells to prevent necrosis at the core of the tissue. The diameter scale of the biofabricated vasculatures should range from 100 to 1,000 µm to support the mm-size tissue while being controllably aligned and spaced within the diffusion limit of oxygen. In this review, insights regarding biofabrication considerations and techniques for engineered blood vessels will be presented. Initially, polymers of natural and synthetic origins can be selected, modified, and combined with each other to support maturation of vascular tissue while also being biocompatible. After they are shaped into scaffold structures by different fabrication techniques, surface properties such as physical topography, stiffness, and surface chemistry play a major role in the endothelialization process after transplantation. Furthermore, biological cues such as growth factors (GFs) and endothelial cells (ECs) can be incorporated into the fabricated structures. As variously reported, fabrication techniques, especially 3D printing by extrusion and 3D printing by photopolymerization, allow the construction of vessels at a high resolution with diameters in the desired range. Strategies to fabricate of stable tubular structures with defined channels will also be discussed. This paper provides an overview of the many advances in blood vessel engineering and combinations of different fabrication techniques up to the present time.

This review covers several aspects and advancements of engineered blood vessel biofabrication, which are essential for establishment of large-sized tissues in different areas of biomedical applications.

Construction of enzyme digested holes on hydrogel surface inspired by cell migration processes.

The construction of in vitro capillary network models for drug testing and toxicity evaluation has become a major challenge in the field of tissue engineering. Previously, we discovered a novel phenomenon of hole formation by endothelial cell migration on the surface of fibrin gels. Interestingly, the hole characteristics, such as depth and number, were strongly influenced by the gel stiffness, but the details of hole formation are not to be clarified. In this study, we tried to understand the effect of hydrogel stiffness on the hole formation by dropping collagenase solution onto the surface of the hydrogels because the endothelial cell migration was made possible by the metalloproteinases' digestion. We found that smaller hole structures were formed on stiffer fibrin gels, but larger ones were formed on softer fibrin gels after the hydrogel digestion of the collagenase. This is consistent with our previous results in experiments on hole structures formed by endothelial cells. Furthermore, deep and small hole structures were successfully obtained by optimizing the volume of collagenase solution and incubation time. This unique approach inspired by endothelial cell hole formation may provide new methods of fabricating hydrogels with opening hole structures.

Fabrication of Molecular Blocks with High Responsiveness to the Cancer Microenvironment by Ursodeoxycholic Acid.

In cancer therapy, a drug delivery system (DDS) has been widely studied to achieve selective drug accumulation at the tumor site. However, DDS still has a major drawback in that it requires multistep processes for intracellular delivery, resulting in low efficiency of drug delivery. To overcome this problem, we recently reported a molecular block (MB) that disrupts cancer cell membranes in the cancer microenvironment using deoxycholic acid (DCA). However, the MB showed considerable cytotoxicity even at neutral pH, possibly due to the structural hydrophobic property of DCA. Herein, we focused on selecting the most suitable bile acid for an MB that possessed high responsiveness to the cancer microenvironment without cytotoxicity at neutral pH. Cell viabilities of the free bile acids such as DCA, chenodeoxycholic acid (CDCA), cholic acid (CA), and ursodeoxycholic acid (UDCA) were evaluated at neutral pH (pH = 7.4) and a cancer acidic environment (pH = 6.3-6.5). The half-maximal inhibition concentration (IC50) value of UDCA at pH = 7.4 showed an approximately 7.5-fold higher IC50 value than that at pH = 6.3, whereas the other bile acids yielded less than a 4-fold IC50 value difference between the same pHs. Biocompatible poly(vinyl alcohol) (PVA) was functionalized with UDCA (PVA-UDCA) for the synthesis of higher responsiveness to the cancer microenvironment without cytotoxicity at neutral pH. Importantly, 56% pancreatic cancer cell death was observed at pH = 6.5, whereas only 10% was detected at neutral pH by the PVA-UDCA treatment. However, PVA-DCA indicated almost the same cancer cell death property, independent of pH conditions. These results suggest PVA-UDCA shows great potential for a new class of MB.

"Out-of-the-box" Granular Gel Bath Based on Cationic Polyvinyl Alcohol Microgels for Embedded Extrusion Printing.

Embedded extrusion printing provides a versatile platform for fabricating complex hydrogel-based biological structures with living cells. However, the time-consuming process and rigorous storage conditions of current support baths hinder their commercial application. This work reports a novel "out-of-the-box" granular support bath based on chemically crosslinked cationic polyvinyl alcohol (PVA) microgels, which is ready to use by simply dispersing the lyophilized bath in water. Notably, with ionic modification, PVA microgels yield reduced particle size, uniform distribution, and appropriate rheological properties, contributing to high-resolution printing. Following by the lyophilization and re-dispersion process, ion-modified PVA baths recover to its original state, with unchanged particle size, rheological properties, and printing resolution, demonstrating its stability and recoverability. Lyophilization facilitates the long-term storage and delivery of granular gel baths, and enables the application of "out-of-the-box" support materials, which will greatly simplify experimental procedures, avoid labor-intensive and time-consuming operations, thus accelerating the broad commercial development of embedded bioprinting.

CXCL12 promotes CCR7 ligand-mediated breast cancer cell invasion and migration toward lymphatic vessels.

Chemokines are a family of cytokines that mediate leukocyte trafficking and are involved in tumor cell migration, growth, and progression. Although there is emerging evidence that multiple chemokines are expressed in tumor tissues and that each chemokine induces receptor-mediated signaling, their collaboration to regulate tumor invasion and lymph node metastasis has not been fully elucidated. In this study, we examined the effect of CXCL12 on the CCR7-dependent signaling in MDA-MB-231 human breast cancer cells to determine the role of CXCL12 and CCR7 ligand chemokines in breast cancer metastasis to lymph nodes. CXCL12 enhanced the CCR7-dependent in vitro chemotaxis and cell invasion into collagen gels at suboptimal concentrations of CCL21. CXCL12 promoted CCR7 homodimer formation, ligand binding, CCR7 accumulation into membrane ruffles, and cell response at lower concentrations of CCL19. Immunohistochemistry of MDA-MB-231-derived xenograft tumors revealed that CXCL12 is primarily located in the pericellular matrix surrounding tumor cells, whereas the CCR7 ligand, CCL21, mainly associates with LYVE-1+ intratumoral and peritumoral lymphatic vessels. In the three-dimensional tumor invasion model with lymph networks, CXCL12 stimulation facilitates breast cancer cell migration to CCL21-reconstituted lymphatic networks. These results indicate that CXCL12/CXCR4 signaling promotes breast cancer cell migration and invasion toward CCR7 ligand-expressing intratumoral lymphatic vessels and supports CCR7 signaling associated with lymph node metastasis.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of coxsackievirus and adenovirus receptor.

Porphyromonas gingivalis is a major pathogen of human periodontitis and dysregulates innate immunity at the gingival epithelial surface. We previously reported that the bacterium specifically degrades junctional adhesion molecule 1 (JAM1), causing gingival epithelial barrier breakdown. However, the functions of other JAM family protein(s) in epithelial barrier dysregulation caused by P. gingivalis are not fully understood. The present results show that gingipains, Arg-specific or Lys-specific cysteine proteases produced by P. gingivalis, specifically degrade coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, at R145 and K235 in gingival epithelial cells. In contrast, a gingipain-deficient P. gingivalis strain was found to be impaired in regard to degradation of CXADR. Furthermore, knockdown of CXADR in artificial gingival epithelium increased permeability to dextran 40 kDa, lipopolysaccharide and peptidoglycan, whereas overexpression of CXADR in a gingival epithelial tissue model prevented penetration by those agents following P. gingivalis infection. Together, these results suggest that P. gingivalis gingipains breach the stratified squamous epithelium barrier by degrading CXADR as well as JAM1, which allows for efficient transfer of bacterial virulence factors into subepithelial tissues. TAKEAWAYS: P. gingivalis, a periodontal pathogen, degraded coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, in gingival epithelial tissues. P. gingivalis gingipains, cysteine proteases, degraded CXADR at R145 and K235. CXADR degradation by P. gingivalis caused increased permeability to lipopolysaccharide and peptidoglycan through gingival epithelial tissues.

Development of Highly Sensitive Molecular Blocks at Cancer Microenvironment for Rapid Cancer Cell Death.

Improving the efficiency and selectivity of drug delivery systems (DDS) is still a major challenge in cancer therapy. Recently, the low transport efficiency of anticancer drugs using a nanocarrier due to the elimination of the carriers from the blood circulation and the blocking by tumor stromal tissues surrounding cancer cells has been reported. Furthermore, multiple steps are required for their intracellular delivery. We recently reported a cancer microenvironment-targeting therapy termed molecular block (MB) which induced cancer cell death by a pH-driven self-aggregation and cell membrane disruption at tumor microenvironment. The MB were designed to disperse as nanoscale assemblies in the bloodstream for efficient circulation and penetration through the stromal tissues. When the MBs reach the tumor site, they self-assembled in microscale aggregates on the cancer cell surfaces in response to the cancer microenvironment and induced cancer cell death. However, in vivo study in mice showed that the MB could not efficiently accumulate at the tumor site because slight hydrophobic aggregations in the bloodstream might potentially be the reason for the off-target accumulation. In this study, we optimize the hydrophilic-hydrophobic balance of MB for avoiding the off-target accumulation and for gaining higher sensitivity to the cancer microenvironment at weak acid condition. Copper-free click reaction with propiolic acid was used to reduce the hydrophobicity of the main chain and obtain higher responsive MB at cancer microenvironment for rapid cell killing. The optimized MB can be considered as a promising approach for an improved cancer cell targeting.

Biomacromolecule-Fueled Transient Volume Phase Transition of a Hydrogel.

A metabolic cycle-inspired hydrogel which exhibits the biomacromolecule-fueled transient volume phase transition is reported. This hydrogel has the affinity and digestive capacity for a fuel α-poly-L-lysine by incorporating acrylic acid and trypsin. The hydrogel captured fuel and transiently shrank owing to the construction of electrostatic cross-linkages. This process was inherently connected with the digestion of these cross-linkages and the release of oligo-lysine as waste, which induced the reswelling of the hydrogel at equilibrium. The transient volume change of the hydrogel realized the fuel-stimulated transient release of a payload. This study provides a strategy for engineering materials with biomacromolecule-fueled dynamic functions under the out-of-equilibrium condition.

Measurement of low-grade inflammation of the esophageal mucosa with electrical conductivity shows promise in assessing PPI responsiveness in patients with GERD.

A device that can easily measure electrical impedance might be a helpful tool for investigating the pathophysiology of gastroesophageal reflux disease. The first aim of this study was to validate our newly developed bioelectrical admittance measurement (BAM) through in vitro experimentation. The second aim was to investigate whether evaluation of BAM by this measurement differed between patients with heartburn according to their response to proton pump inhibitor (PPI) therapy. Caco-2 cell monolayers and three-dimensional tissues were examined by BAM using a frequency response analyzer. BAM was also used to measure the impedance through cell layers. Subsequently, BAM was performed during endoscopy in 41 patients experiencing heartburn without esophageal mucosal breaks. After 2-wk administration of 20-mg rabeprazole twice daily, patient responses to PPI were classified as "good" or "poor" according to their clinical course. In each patient, histological alterations and gene expression levels of inflammation mediators and tight junction proteins were evaluated. Impedance profiles indicated that monolayer Caco-2 cells on top of eight-layered normal human dermal fibroblasts had the highest magnitude of impedance over the range of frequencies. In vivo results revealed that patients with good responses to PPI displayed significantly higher admittance. Severity of low-grade inflammation was significantly associated with esophageal wall admittance. Moreover, esophageal wall admittance may be more closely related to basal zone hyperplasia than dilatation of intercellular spaces. Thus, BAM may be able to detect abnormalities in the subepithelial layer of the esophagus.NEW & NOTEWORTHY Bioelectrical admittance measurement is a new method to evaluate esophageal mucosal permeability vertically during upper gastrointestinal endoscopy. Measurement of low-grade inflammation of the esophageal mucosa with electrical conductivity shows promise in assessing proton pump inhibitor responsiveness in patients with gastroesophageal reflux disease. As various gastrointestinal diseases are associated with changes in mucosal permeability, bioelectrical admittance measurement is expected to be clinically applied to therapeutic decision-making for these diseases in the future.

Label-Free Cancer Stem-like Cell Assay Conducted at a Single Cell Level Using Microfluidic Mechanotyping Devices.

The mechanical phenotype of cells is an intrinsic property of individual cells. In fact, this property could serve as a label-free, non-destructive, diagnostic marker of the state of cells owing to its remarkable translational potential. A microfluidic device is a strong candidate for meeting the demand of this translational research as it can be used to diagnose a large population of cells at a single cell level in a high-throughput manner, without the need for off-line pretreatment operations. In this study, we investigated the mechanical phenotype of the human colon adenocarcinoma cell, HT29, which is known to be a heterogeneous cell line with both multipotency and self-renewal abilities. This type of cancer stem-like cell (CSC) is believed to be the unique originators of all tumor cells and may serve as the leading cause of cancer metastasis and drug resistance. By combining consecutive constrictions and microchannels with an ionic current sensing system, we found a high heterogeneity of cell deformability in the population of HT29 cells. Moreover, based on the level of aldehyde dehydrogenase (ALDH) activity and the expression level of CD44s, which are biochemical markers that suggest the multipotency of cells, the high heterogeneity of cell deformability was concluded to be a potential mechanical marker of CSCs. The development of label-free and non-destructive identification and collection techniques for CSCs has remarkable potential not only for cancer diagnosis and prognosis but also for the discovery of a new treatment for cancer.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of junctional adhesion molecule 1.

Porphyromonas gingivalis is a major pathogen in severe and chronic manifestations of periodontal disease, which is one of the most common infections of humans. A central feature of P. gingivalis pathogenicity is dysregulation of innate immunity at the gingival epithelial interface; however, the molecular basis underlying P. gingivalis-dependent abrogation of epithelial barrier function remains unknown. Gingival epithelial cells express junctional adhesion molecule (JAM1), a tight junction-associated protein, and JAM1 homodimers regulate epithelial barrier function. Here we show that Arg-specific or Lys-specific cysteine proteases (gingipains) secreted by P. gingivalis can specifically degrade JAM1 at K134 and R234 in gingival epithelial cells, resulting in permeability of the gingival epithelium to 40 kDa dextran, lipopolysaccharide (LPS), and proteoglycan (PGN). A P. gingivalis strain lacking gingipains was impaired in degradation of JAM1. Knockdown of JAM1 in monolayer cells and a three-dimensional multilayered tissue model also increased permeability to LPS, PGN, and gingipains. Inversely, overexpression of JAM1 in epithelial cells prevented penetration by these agents following P. gingivalis infection. Our findings strongly suggest that P. gingivalis gingipains disrupt barrier function of stratified squamous epithelium via degradation of JAM1, allowing bacterial virulence factors to penetrate into subepithelial tissues.

Resolution of 3D bioprinting inside bulk gel and granular gel baths.

Three-dimensional (3D) bioprinting has rapidly developed in the last decade, playing an increasingly important role in applications including pharmacokinetics research, tissue engineering, and organ regeneration. As a cutting-edge technology in 3D printing, gel bath-supported 3D bioprinting enables the freeform construction of complex structures with soft and water-containing materials, facilitating the in vitro fabrication of live tissue or organ models. To realize in vivo-like organs or tissues in terms of biological functions and complex structures by 3D printing, high resolution and fidelity are prerequisites. Although a wide range of gel matrices have recently been developed as supporting materials, the effect of bath properties and printing parameters on the print resolution is still not clearly understood. This review systematically introduces the decisive factors for resolution in both bulk gel bath systems and granular microgel bath systems, providing guidelines for high-resolution 3D bioprinting based on bath properties and printing parameters.

The Cell Line-Dependent Diversity in Initial Morphological Dynamics of Pancreatic Cancer Cell Peritoneal Metastasis Visualized by an Artificial Human Peritoneal Model.

BACKGROUND: Pancreatic ductal adenocarcinoma is considered as one of the most malignant types of cancer with rapid metastasis and invasion of the cancer cells, having peritoneal metastasis (PM) as a dominant factor of poor prognosis. Although the prevention of peritoneal dissemination would result in the inhibition of the initial metastatic process and contribute in improving the poor prognosis of the pancreatic cancer, the initial dynamics of PM are still unclear because of the lack of adequate models in studying the morphological and molecular details of pancreatic cancer cells. MATERIALS AND METHODS: The artificial human peritoneal tissue (AHPT) that can be applied in studying for the spatial dynamics of cancer PM in vitro has been established previously. In this study, the initial dynamics of the three pancreatic cell lines, undifferentiated carcinoma MIA PaCa-2, poorly differentiated adenocarcinoma Panc-1, and moderately differentiated adenocarcinoma BxPC3 on AHPT are examined. RESULTS: In a morphological analysis using light and electron microscopy, MIA PaCa-2 cells spread on the mesothelial layer with disruption of the sheet structure and infiltrated into the stroma-like tissue in AHPT. On the other hand, BxPC3 cells changed shapes from round into flat ones with rapid proliferation and formed sheet structure at the surface of the tissue replacing the mesothelial layer without vertical invasion into the tissue. Panc-1 cells demonstrated the intermediate characteristics of MIA PaCa-2 and BxPC3 on AHPT. These diverse morphological characteristics were verified by the correspondence with the results in a mouse model and were reflected by the profile of secreted oncogenic proteins of the three pancreatic cell lines. CONCLUSIONS: The initial dynamics in the peritoneal dissemination of these pancreatic cancer cell lines were demonstrated by AHPT, showing the morphological and molecular diversity depending on the degree of differentiation or the properties of oncogenic protein secretion.

Effects of radiofrequency and ultrasound on the turnover rate of skin aging components (skin extracellular matrix and epidermis) via HSP47-induced stimulation.

Skin aging cannot be escaped, being due to both intrinsic and extrinsic stimuli. They lead to a reduced extracellular collagen matrix in the dermis, along with a higher degradation by metalloproteases (MMPs) activity, as well as a lower differentiation and function of epidermis keratinocytes, characterized by wrinkling and loss of skin elasticity. One of the recent technology to overcome this skin aging process is the use of radiofrequency (RF) and ultrasound (US) technologies which use thermal stimulation to induce neocollagenesis in the skin. But no explanations exist on the involved pathways. Our hypothesis is that RF-US generated heat increases the collagen formation via the heat shock protein 47 (HSP47) induction, a heat sensitive protein related to the collagen expression. To confirm this hypothesis, normal human skin substitutes were subjected to RF-US treatment and results were monitored after 24 and 44 h. RNA sequencing showed a significant induction for the genes related to the epidermis differentiation processes. Almost all keratin genes were thus found upregulated from 2 to 15 times, while collagen type XVII and collagen type IV were increased 12 and 5 times respectively. In parallel, most of MMP genes were observed downregulated. RF-US treatment significantly increased levels of HSP47 proteins, while collagen XVII proteins showed a tendency to be increased and glycosaminoglycans were found 1.4 times significantly enhanced. Finally, histology assessment showed a higher expression of cytokeratins 10 and 14 which can testify a possible reactivation of the skin proliferative state as a rejuvenation strategy.

Interstitial flow regulates in vitro three-dimensional self-organized brain micro-vessels.

Cell culture under medium flow has been shown to favor human brain microvascular endothelial cells function and maturation. Here a three-dimensional in vitro model of the human brain microvasculature, comprising brain microvascular endothelial cells but also astrocytes, pericytes and a collagen type I microfiber - fibrin based matrix, was cultured under continuous medium flow in a pressure driven microphysiological system (10 kPa, in 60-30 s cycles). The cells self-organized in micro-vessels perpendicular to the shear flow. Comparison with static culture showed that the resulting interstitial flow enhanced a more defined micro-vasculature network, with slightly more numerous lumens, and a higher expression of transporters, carriers and tight junction genes and proteins, essential to the blood-brain barrier functions.

Fabrication of Artificial Nanobasement Membranes for Cell Compartmentalization in 3D Tissues.

In recent decades, tissue engineering techniques have attracted much attention in the construction of 3D tissues or organs. However, even though precise control of cell locations in 3D has been achieved, the organized cell locations are easily destroyed because of the cell migration during the cell culture period. In human body, basement membranes (BMs) maintain the precise cell locations in 3D (compartmentalization). Constructing artificial BMs that mimic the structure and biofunctions of natural BMs remains a major challenge. Here, a nanometer-sized artificial BM through layer-by-layer assembly of collagen type IV (Col-IV) and laminin (LM), chosen because they are the main components of natural BMs, is reported. This multilayered Col-IV/LM nanofilm imitates natural BM structure closely, showing controllable and similar components, thickness, and fibrous network. The Col-IV/LM nanofilms have high cell adhesion properties and maintain the spreading morphology effectively. Furthermore, the barrier effect of preventing cell migration but permitting effective cell-cell crosstalk between fibroblasts and endothelial cells demonstrates the ability of Col-IV/LM nanofilms for cell compartmentalization in 3D tissues, providing more reliable tissue models for evaluating drug efficacy, nanotoxicology, and implantation.

In Situ Cross-Linking of Artificial Basement Membranes in 3D Tissues and Their Size-Dependent Molecular Permeability.

In the human body, highly organized tissues rely on the compartmentalization effect of basement membranes (BMs) that separate different types of cells. We recently reported an artificial basement membrane (A-BM) composed of type-IV collagen and laminin (Col-IV/LM), which are the main components of natural BMs, for cell compartmentalization in three-dimensional (3D) tissues. However, such compartmentalized structures can be maintained only for 3 days, probably due to the degradation issues. In this study, a robust A-BM was fabricated by in situ cross-linking the Col-IV/LM layer-by-layer (LbL) nanofilms in 3D tissues by transglutaminase. The effects of molecular size and configuration on the permeability of obtained A-BMs were comprehensively studied using polystyrene nanoparticles (PS NPs) and dextran with various hydrodynamic diameters, as well as albumin. The findings agreed well with the known size-selective behavior of the glomerular basement membrane. Cross-linked Col-IV/LM nanofilms demonstrate improved stability and a more powerful barrier effect to maintain cell compartmentalization for organized 3D tissues. This in vitro A-BM exhibit great potentials for the design of more complex compartmentalized 3D tissues, for understanding the unique cell-cell cross talk through BMs, and for providing a more reliable 3D tissue model for new drug screening and other in vitro physiological studies.