Cell surface engineering to control cellular interactions

Cell surface composition determines all interactions of the cell with its environment, thus cell functions such as adhesion, migration and cell–cell interactions can potentially be controlled by engineering and manipulating the cell membrane. Cell membranes present a rich repertoire of molecules, therefore a versatile ground for modification. However the complex and dynamic nature of the cell surface is also a major challenge for cell surface engineering that should also involve strategies compatible with cell viability. Cell surface engineering by selective chemical reactions or by the introduction of exogenous targeting ligands can be a powerful tool for engineering novel interactions and controlling cell function. In addition to chemical conjugation and modification of functional groups, ligands of interest to modify the surface of cells include recombinant proteins, liposomes or nanoparticles. Here, we review recent efforts to perform changes to cell surface composition. We focus on the engineering of the cell surface with biological, chemical or physical methods to modulate cell functions and control cell–cell and cell–microenvironment interactions. Potential applications of cell surface engineering are also discussed

High-throughput evaluation of interactions between biomaterials, proteins and cells using patterned superhydrophobic substrates

We propose a new low cost platform for high-throughput analysis that permits screening the biological performance of independent combinations of biomaterials, cells and culture media. Patterned superhydrophobic flat substrates with controlled wettable spots are used to produce microarray chips for accelerated multiplexing evaluation.This work was partially supported by Fundação para a Ciência e Tecnologia (FCT) under project PTDC/FIS/68517/2006

Chitosan membranes for spatially controlled cell adhesion and specific cell recruitment

We propose a concept of biomaterials that are able to fix specific cell types onto their surface when in contact with a mix population of cells. Adipose tissue has shown to be an interesting source of stem cells with therapeutic potential. However only a small amount of the heteroge- neous mixture of the cells extracted from lipoaspirates are stem cells, and within stem cells there are different populations with different capabilities to differentiate through a lineage. We studied the ability of immobilized antibodies on chitosan surfaces to capture specific types of cells with a spatial micrometer resolution.Antibodies were covalently immobilized onto chitosan membranes using bis[sulfosuccinimidyl] su- berate (BS3). X-ray photoelectron spectroscopy (XPS) was used to chemically characterize the surface and quartz crystal microbalance (QCM) to calculate the amount of adsorbed and/or immobilized anti- body. Data shown greater immobilization when BS3 was used com- pared to simple adsorption. Specific antibodies covalently immobilized in a surface, kept their bioactivity and controlled the type of cell that attached on the chitosan surface. Microcontact printing permitted to covalently immobilize antibodies in patterns allowing a spatial control in cell attachment. Cell sorting experiments performed using a mixture of adipose stem cells and osteoblast like cells shown that chitosan sur- faces were able to capture a specific phenotype depending on the immobilized antibody

Functional cell microcarriers: a new platform for cell separation and expansion

Publicado em "Journal of Tissue Engineering and Regenerative Medicine", vol. 7, supp. 1 (2013)The success of many stem cell applications in the biomedical field is highly dependent on the development of separation techniques for isolation and purification of cells with a very high yield and purity. Despite all the achievements made in the field over the past several years, new systems for effective cell separation are still needed. Previous work from our group demonstrated that functional chitosan films grafted with antibodies promote selective cell adhesion. 1 Herein we developed chitosan microparticles able to capture a specific cell types based in the concept of antibody coating for cell sorting. Our goal was to create new biomaterial surfaces capable of recruit a specific cell population within a mixture, reducing cell manipulation and time-consuming allowing at the same time cell expansion. Such system acts as a microcarrier for cell expansion of a specific cell target. Microcarrier culture system offers the advantage of providing a larger surface area for the growth of anchorage-dependent cells in a suspension culture system. Chitosan was chosen due to the excellent biocompatibility, gel forming properties, chemistry surface and low cell adhesion. This allows the modification with specific biochemical cues, for a controllable cell attachment. Here we develop functional biotinylated microparticles, such system allows tailoring microparticles to a variety of functional biomolecules. Here we tested the immobilization of antibodies to target specific cell types, CD31 for endothelial cells and CD90 for adipose stem cells. Primarily designed for an application in tissue engineering, two main challenges are accomplished with the herein presented microparticles: separation and scale-up expansion of specific cell type. The herein developed polymeric microparticles can also be used for directly deliver cells in vivo to repair and regenerate tissues

Functional chitosan microcarriers for selective cell attachment and expansion

The success of many stem cell applications in the biomedical field is highly dependent on the development of reliable techniques either for isolation or selection of specific cell populations with a very high yield and purity.1 In this work we propose the use of chitosan microparticles (μPs) to capture a specific cell type based in the concept of antibody-antigen binding. Our goal was to create new biomaterials capable of selecting within a heterotypic cell suspension, a specific sub-population, and supporting subsequent cell expansion. Such system simultaneously allows the selection and acts as a microcarrier for a specific target, thus reducing cell manipulation and time-consumption

Layer-by-layer assembly of chitosan and recombinant biopolymers into biomimetic coatings with multiple stimuli-responsive properties

In this work, biomimetic smart thin coatings using chitosan and a recombinant elastin-like recombinamer (ELR) containing the cell attachment sequence arginine–glycine–(aspartic acid) (RGD) are fabricated through a layer-by-layer approach. The synthetic polymer is characterized for its molecular mass and composition using mass spectroscopy and peptide sequencing. The adsorption of each polymeric layer is followed in situ at room temperature and pH 5.5 using a quartz-crystal microbalance with dissipation monitoring, showing that both polymers can be successfully combined to conceive nanostructured, multilayered coatings. The smart properties of the coatings are tested for their wettability by contact angle (CA) measurements as a function of external stimuli, namely temperature, pH, and ionic strength. Wettability transitions are observed from a moderate hydrophobic surface (CAs approximately from 62° to 71°) to an extremely wettable one (CA considered as 0°) as the temperature, pH, and ionic strength are raised above 50 °C, 11, and 1.25 m, respectively. Atomic force microscopy is performed at pH 7.4 and pH 11 to assess the coating topography. In the latter, the results reveal the formation of large and compact structures upon the aggregation of ELRs at the surface, which increase water affinity. Cell adhesion tests are conducted using a SaOs-2 cell line. Enhanced cell adhesion is observed in the coatings, as compared to a coating with a chitosan-ending film and a scrambled arginine–(aspartic acid)–glycine (RDG) biopolymer. The results suggest that such films could be used in the future as smart biomimetic coatings of biomaterials for different biomedical applications, including those in tissue engineering or in controlled delivery systems.EUJCyL - VA034A09, VA030A08Fundação para a Ciência e Tecnologia (FCT) - SFRH/BD/61126/2009, SFRH/BD/61390/2009MICINN - MAT 2007-66275-C02-01, MAT 2007-61604, MAT 2009-14195-C03-03, PSE-300100-2006-1European regional development fund (ERDF)Junta de Castilla y LeonNetwork Center of Regenerative Medicine and Cellular Therapy of Castilla and LeónCIBER-BBN (project CB06-01-0003

Multilayered membranes with tuned well arrays to be used as regenerative patches

Membranes have been explored as patches in tissue repair and regeneration, most of them presenting a flat geometry or a patterned texture at the nano/micrometer scale. Herein, a new concept of a flexible membrane featuring well arrays forming pore-like environments to accommodate cell culture is proposed. The processing of such membranes using polysaccharides is based on the production of multilayers using the layer-by-layer methodology over a patterned PDMS substrate. The detached multilayered membrane exhibits a layer of open pores at one side and a total thickness of 38±2.2µm. The photolithography technology used to produce the molds allows obtaining wells on the final membranes with a tuned shape and micro-scale precision. The influence of post-processing procedures over chitosan/alginate films with 100 double layers, including crosslinking with genipin or fibronectin immobilization, on the adhesion and proliferation of human osteoblast-like cells is also investigated. The results suggest that the presence of patterned wells affects positively cell adhesion, morphology and proliferation. In particular, it is seen that cells colonized preferentially the well regions. The geometrical features with micro to sub-millimeter patterned wells, together with the nano-scale organization of the polymeric components along the thickness of the film will allow to engineer highly versatile multilayered membranes exhibiting a pore-like microstructure in just one of the sides, that could be adaptable in the regeneration of multiple tissues

Immobilization of biomolecules into biodegradable polymeric based substrates for selective recruitment and adhesion of cells for tissue engineering applications

Tese de doutoramento do Programa Doutoral em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisDevelopment of bioactive materials with the ability to stimulate specific cellular responses is a topic of high interest in the fabrication and development of biomaterials. The proper presentation of these biochemical regulatory signals within a biomaterial is required for tissue regeneration. Furthermore, a better understanding of cell-biomolecule-material interactions is a key step toward the creation of multicellular and hierarchically organized tissues. Therefore the major focus of the present thesis was on developing new instructive polymeric materials by immobilized specific biochemical signals. To this end, chitosan films and microparticles were functionalized with biomolecules of interest yielding biomaterials with specific chemical signals to modulate cellular performance. An important feature that a material should display for functionalization with specific biochemical cues it is the availability of chemical groups for covalent modification. The chemical nature of chitosan provides many possibilities for covalent modifications which offer several possibilities for derivatization and immobilization of biologically active species. In a first study it was pointed out the importance of the covalent immobilization method of bioactive moieties, namely fibronectin, to enhance the rate of cell adhesion and proliferation on chitosan substrates. Chitosan membranes were functionalized with fibronectin through carbodiimide chemistry. Biological results showed that the chemical functionalization with fibronectin stimulated cell attachment when compared to simple adsorption of the protein. The functionalization of chitosan substrates with molecules that target a specific cell type could be a promising strategy to isolate and recruit cells of interest to regenerate a particular tissue. Additionally microtechnologies offer unprecedented means to generate precise patterns of surface-tethered biomolecules. In this thesis the spatial control of antibodies was achieved by two different strategies: (1) microcontact printing for covalent immobilization using bis[sulfosuccinimidyl] suberate as a crosslinker; (2) photopatterning by using surfaces functionalized with photoprotected biotin that upon irradiation through a mask generate well defined patterns of activated biotin. Such substrates were then used to immobilized biotinylated antibodies via streptavidin. Different cell types, namely adipose stem cells and endothelial cells were seeded on the functional surfaces. Results from both experiments showed that cell attachment and proliferation could be easily manipulated depending on the immobilized antibody. Finally moving up to three-dimensional systems, a novel “bottom-up” approach based on the assembly of functional microparticles was developed. In chapter VII chitosan microparticles were functionalized anti-human platelet-derived growth factor (PDGF) antibody using carbodiimide chemistry. The functional particles were then investigated as a method to recruit specific growth factors from a highly enriched concentrate of growth factors – Platelet Lysates – an autologous and easily obtained source of bioactive agents capable of providing high concentrations of several proteins. Results showed that antibody-functionalized particles allowed selective recruitment of a specific growth factor from a complex mixture. It was hypothesized that stem cells can crosslink polymeric microparticles, if they contain specific ligands capable of binding to receptors on the cell surface leading to the formation of stable and robust three-dimensional structures. In vitro tests revealed that microparticles aggregate due to cell connecting points, giving rise to 3D constructs. In chapter VIII chitosan microparticles functionalized with antibodies, were explored as a method for cell separation and expansion. Results showed that the microparticles could selectively capture a particular cell type from a mixed cell population. Moreover particles were also suitable for cell expansion of the target cell type. For clinical applications, injectable/moldable hydrogels are often needed to fill defects of irregular geometries. To test the capacity of the functional microparticles as an injectable system, functional microparticles were seeded with stem cells and injected into a mold with a tubular shape. After three days of incubation in vitro, a robust piece with a tubular shape was obtained. The works developed in chapters VII and VIII reported a novel approach for the production of instructive 3D constructs based on the assembly of functional micro building blocks. These versatile microgels can be a promising way to produce injectable system for non-invasive tissue engineering applications with additional control over cellular function by creating specific microenvironments for cell growth.O desenvolvimento de materiais biologicamente ativos com a capacidade de estimular respostas celulares específicas é um tópico de grande interesse na fabricação e no desenvolvimento de biomateriais. A apresentação adequada desses sinais reguladores bioquímicos dentro de um biomaterial é necessária para a regeneração de tecidos. Além disso, uma melhor compreensão das interações célula - biomolécula -material é um passo fundamental para a criação de tecidos multicelulares e hierarquicamente organizados. O principal objetivo desta tese consistiu no desenvolvimento de novos materiais poliméricos instrutivos através da imobilização sinais bioquímicos específicos. Membranas e micropartículas de quitosano foram funcionalizadas com biomoléculas de específicas de modo a desenvolver materiais que permitem um controlo sobre o desempenho celular. Uma característica importante que um material deve exibir de modo a ser funcionalizado com sinais bioquímicos é a disponibilidade de grupos químicos que permitam modificação. A natureza química do quitosano oferece varias possibilidades de modificação covalente para a imobilização de espécies biologicamente ativas. No primeiro trabalho desta tese, foi analisada a importância do método de funcionalização para a modificação com fibronectina de modo a aumentar a taxa de adesão e proliferação celular em substratos de quitosano. Membranas de quitosano foram funcionalizados com fibronectina por adsorção ou conjugação usando uma carbodiimida. Os resultados biológicos mostraram que a funcionalização química com carbodiimida aumenta a adesão e proliferação celular quando comparada com a modificação por adsorção. A funcionalização de substratos de quitosano com moléculas que têm como alvo um tipo específico de célula pode ser uma estratégia promissora para isolar e recrutar células de interesse para a regeneração de tecidos . Além disso diferentes técnicas de microfabricação permitem criar padrões bem definidos de biomoléculas em diferentes substratos. Nesta tese o controlo espacial de anticorpos foi conseguida usando duas estratégias diferentes: (1) impressão por microcontato para imobilização covalente utilizando bis [sulfosuccinimidil] suberato como agente de ligação, (2 ) fotolitografia usando superfícies funcionalizadas com biotina fotossensível que irradiadas através de uma máscara permitem a criação de padrões bem definidos de biotina ativada. Esses substratos foram então usados para imobilização de anticorpos biotinilados via estreptavidina. Diferentes tipos de células , nomeadamente células estaminais do tecido adiposo e células endoteliais foram cultivadas sobre as superfícies funcionais . Os resultados de ambas as experiências mostraram que a ligação de células e proliferação poderia ser facilmente manipulada dependendo do anticorpo imobilizado . Numa última fase desta tese foram explorados sistemas tridimensionais, através de uma abordagem “bottom-up”, que combina o uso de micropartículas funcionais. No capítulo VII micropartículas de quitosano foram funcionalizadas com um anticorpo. anti- fator de crescimento derivado de plaquetas humano, usando uma carbodiimide. As partículas funcionais foram então testadas como um método para o recrutamento de fatores de crescimento específicos a partir de uma solução enriquecida de múltiplos fatores de crescimento - Lisados de Plaquetas- uma fonte autóloga e de fácil obtenção de agentes bioativos, capazes de disponibilizar elevadas concentrações de várias proteínas. Os resultados mostraram que as partículas funcionalizadas com anticorpo permitem o recrutamento seletivo de um fator de crescimento específico a partir de uma mistura complexa. Foi hipotetizado que células estaminais podem reticular micropartículas poliméricas funcionalizadas com ligandos específicos capazes de se ligarem aos recetores na superfície celular levando à formação de estruturas tridimensionais. Testes in vitro revelaram que as micropartículas agregam devido a pontos de ligação celulares , dando origem a estruturas tridimensionais, estáveis e robustas. No capítulo VIII micropartículas de quitosano funcionalizadas com anticorpos, foram testadas para a separação e expansão de células. Os resultados obtidos revelaram que as micropartículas permitem capturar seletivamente um tipo de célula de uma população celular heterogénea. Além disso, as partículas foram também usadas para a expansão das células isoladas. Para aplicações clínicas, hidrogéis injetáveis/moldáveis são muitas vezes necessários para preencher defeitos de geometrias irregulares. Para testar a capacidade das micropartículas funcionais como um sistema injetável, micropartículas funcionais foram cultivadas com células estaminais e injetadas num molde de forma tubular. Após três dias de incubação in vitro, foi obtida uma estrutura sólida de forma tubular. Os trabalhos desenvolvidos nos capítulos VII e VIII exploraram uma nova abordagem para a produção de estruturas tridimensionais com sinais bioquímicos integrados através da combinação de microblocos funcionais. Estes microgeis de elevada versatilidade podem vir a traduzir-se no desenvolvimento de sistemas de elevado desempenho para a produção de sistema injetáveis para aplicações em engenharia de tecidos, criando microambientes específicos para o crescimento celular

Perinatal tissues and cells in tissue engineering and regenerative medicine

Perinatal tissues are an abundant source of human extracellular matrix proteins, growth factors and stem cells with proved potential use in a wide range of therapeutic applications. Due to their placental origin, these tissues possess unique biological properties, including being angiogenic, anti-inflammatory, anti-fibrotic, anti-microbial and immune privileged. Additionally, as a temporary organ, placenta is usually discarded as a medical waste, thus providing an easily available, cost effective, 'unlimited' and ethical source of raw materials. Although some of these tissues, such as the amniotic membrane and umbilical cord, have been used in clinical practices, most of them continue to be highly under explored. This review aims to outline the most relevant applications of perinatal tissues as a source of biomaterials and stem cells in the exciting fields of tissue engineering and regenerative medicine (TERM), as well as highlight how these solutions can be used to overcome the shortage of adequate scaffolds and cell sources that currently hampers the translation of TERM strategies towards clinical settings. STATEMENT OF SIGNIFICANCE: Stem cells and extracellular matrix derived from perinatal tissues such as placenta and umbilical cord, have drawn great attention for use in a wide variety of applications in the biomedical field. Due to their origin, these tissues possess unique biological properties, including being angiogenic, anti-inflammatory, anti-fibrotic, anti-microbial and immune privileged. Also they are typically considered medical waste, thus providing an easily available, cost effective, 'unlimited' and ethical source of raw materials. This work aims to present and discuss the most relevant applications of perinatal tissues as a source of biomaterials and stem cells in the exciting fields of tissue engineering and regenerative medicine (TERM).publishe

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.publishe