Photocrosslinkable microgels derived from human platelet lysates: injectable biomaterials for cardiac cell culture.

Cardiovascular diseases are a major global cause of morbidity and mortality, and they are often characterized by cardiomyocytes dead that ultimately leads to myocardial ischemia (MI). This condition replaces functional cardiac tissue with fibrotic scar tissue compromising heart function. Injectable systems for the in situ delivery of cells or molecules to assist during tissue repair have emerged as promising approaches for tissue engineering, particularly for myocardial repair. Methacryloyl platelet lysates (PLMA) have been employed for constructing full human-based 3D cell culture matrices and demonstrated potential for xeno-free applications. In this study, we propose using PLMA to produce microparticles (MPs) serving as anchors for cardiac and endothelial cells and ultimately as injectable systems for cardiac tissue repair. The herein reported PLMA MPs were produced by droplet microfluidics and showed great properties for cell attachment. More importantly, it is possible to show the capacity of PLMA MPs to serve as cell microcarriers even in the absence of animal-derived serum supplementation in the culture media.

Designing highly customizable human based platforms for cell culture using proteins from the amniotic membrane.

In the past few years researchers have witnessed a paradigm shift in the development of biomaterials for drug discovery, tissue engineering, and regenerative medicine. After the great advances resulting from the transition of the 2D to the 3D, the new focus has been to increase the clinical relevance of such systems, as well as avoid the use of animals, by developing platforms that better replicate the human physiology in vitro. In this sense, we envisage the use of human matrices extracted from ethically sourced and readily available tissues as an optimal and promising alternative to currently used approaches. Hereupon, we report for the first time the chemical modification of human ECM proteins from the amniotic membrane (AM) with photoresponsive groups to produce bioinks and hydrogel precursors to engineer customizable platforms that are representative of native tissues and capable of supporting long-term cell culture. Our results demonstrated an efficient decellularization, liquefaction and functionalization of AM-derived ECM with methacryloyl domains (AMMA), with production of stable and versatile hydrogels. Mechanical characterization evidenced an increased compression strength as a function of methacrylation degree and decellularized ECM concentration. Three-dimensional (3D) stem cell culture in the AMMA hydrogels resulted in viable and proliferative cells up to 7 days; moreover, the mouldable character of the hydrogel precursors permits the processing of patterned hydrogel constructs allowing the control over cellular alignment and elongation, or microgels with highly tunable shape.

Human Protein-Based Porous Scaffolds as Platforms for Xeno-Free 3D Cell Culture.

Extracellular matrix and protein-based biomaterials emerge as attractive sources to produce scaffolds due to their great properties regarding biocompatibility and bioactivity. In addition, there are concerns regarding the use of animal-derived supplements in cell culture not only due to the risk of transmission of xenogeneic contaminants and antigens but also due to ethical issues associated with collection methods. Herein, a novel human protein-derived porous scaffold produced from platelet lysates (PL) as platform for xeno-free 3D cell culture has been proposed. Human PL are chemically modified with methacryloyl groups (PLMA) to make them photocrosslinkable and used as precursor material to produce PLMA-based sponges. The herein reported human-based sponges have highly tunable morphology and mechanical properties, with an internal porous structure and Young's modulus dependent on the concentration of the polymer. Human adipose-derived stem cells (hASCs) are cultured on top of PLMA sponges to validate their use for 3D cell culture in xeno-free conditions. After 14 days hASCs remained viable, and results show that cells are able to proliferate during time even in the absence of animal-derived supplementation. This study reveals for the first time that such scaffolds can be promising platforms for culture of human cells avoiding the use of any animal-derived supplement.

Human Platelet Lysates-Based Hydrogels: A Novel Personalized 3D Platform for Spheroid Invasion Assessment.

Fundamental physiologic and pathologic phenomena such as wound healing and cancer metastasis are typically associated with the migration of cells through adjacent extracellular matrix. In recent years, advances in biomimetic materials have supported the progress in 3D cell culture and provided biomedical tools for the development of models to study spheroid invasiveness. Despite this, the exceptional biochemical and biomechanical properties of human-derived materials are poorly explored. Human methacryloyl platelet lysates (PLMA)-based hydrogels are herein proposed as reliable 3D platforms to sustain in vivo-like cell invasion mechanisms. A systematic analysis of spheroid viability, size, and invasiveness is performed in three biomimetic materials: PLMA hydrogels at three different concentrations, poly(ethylene glycol) diacrylate, and Matrigel. Results demonstrate that PLMA hydrogels perfectly support the recapitulation of the tumor invasion behavior of cancer cell lines (MG-63, SaOS-2, and A549) and human bone-marrow mesenchymal stem cell spheroids. The distinct invasiveness ability of each cell type is reflected in the PLMA hydrogels and, furthermore, different mechanical properties produce an altered invasive behavior. The herein presented human PLMA-based hydrogels could represent an opportunity to develop accurate cell invasiveness models and open up new possibilities for humanized and personalized high-throughput screening and validation of anticancer drugs.

An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection.

A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens.IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.

Photopolymerizable Platelet Lysate Hydrogels for Customizable 3D Cell Culture Platforms.

3D cell culture platforms have emerged as a setting that resembles in vivo environments replacing the traditional 2D platforms. Over the recent years, an extensive effort has been made on the development of more physiologically relevant 3D cell culture platforms. Extracellular matrix-based materials have been reported as a bioactive and biocompatible support for cell culture. For example, human plasma derivatives have been extensively used in cell culture. Despite all the promising results, in most cases these types of materials have poor mechanical properties and poor stability in vitro. Here plasma-based hydrogels with increased stability are proposed. Platelet lysates are modified by addition of methacryloyl groups (PLMA) that polymerize in controlled geometries upon UV light exposure. The hydrogels could also generate porous scaffolds after lyophilization. The results show that PLMA materials have increased mechanical properties that can be easily adjusted by changing PLMA concentration or modification degree. Cells readily adhere, proliferate, and migrate, exhibiting high viability when encapsulated in PLMA hydrogels. The innovation potential of PLMA materials is based on the fact that it is a complete xeno-free solution for human cell culture, thus an effective alternative to the current gold standards for 3D cell culture based on animal products.