Comparison between Platelet Lysate, Platelet Lysate Serum, and Fetal Bovine Serum as Supplements for Cell Culture, Expansion, and Cryopreservation.

As cell culture supplements, human platelet lysate (PL) and human platelet lysate serum (PLS) are alternatives to fetal bovine serum (FBS) due to FBS-related issues such as ethical concerns, variability between batches, and the possible introduction of xenogenic contaminants. This study compared the composition and efficacy of PL, PLS, and FBS as supplements in the culture and cryopreservation of human dermal fibroblasts, Wharton's jelly-derived mesenchymal stem cells (WJ-MCS), and adipose tissue (AdMSC). Biochemical components, some growth factors, and cytokines present in each of them were analyzed; in addition, the cells were cultured in media supplemented with 5% PL, 5% PLS, and 10% FBS and exposed to different freezing and thawing solutions with the supplements under study. Biochemical parameters were found to be similar in PL and PLS compared to FBS, with some differences in fibrinogen and calcium concentration. Growth factors and cytokines were higher in PL and PLS compared to FBS. Cell proliferation and morphology showed no significant differences between the three culture media. Regarding the cryopreservation and thawing of cells, better results were obtained with PLS and FBS. In conclusion, PL and PLS are an excellent choice to replace the standard supplement of animal origin (FBS) in the media used for the culture and cryopreservation of fibroblasts, WJ-MSC, and AdMSC.

Biological response of degradation products of PEO-modified magnesium on vascular tissue cells, hemocompatibility and its influence on the inflammatory response.

Cardiovascular stenting is the most widely used therapy to treat coronary artery disease caused by partial or total obstruction of the artery due to atherosclerotic plaque formation, with potentially fatal effects. There are different types of stents: bare metal stents, drug-eluting stents, bioabsorbable stents and dual therapy stents. However, they can lead to long-term complications, such as in-stent restenosis and late thrombosis. To reduce these adverse effects, research has focused on biodegradable metallic stents, since they retain the mechanical properties necessary to contain the injured artery while it is being repaired and, once their function has been fulfilled, the stent degrades without altering the system or compromising the patient's health. In this work we have evaluated the biological response of the degradation products of a bare Mg based biomaterial surface-modified by the plasma electrolytic oxidation (PEO) method on vascular tissue cells, hemocompatibility and inflammatory response. The results obtained are compatible with a biosafe material for future use as a cardiovascular implant, but it is necessary to continue with in vivo and mechanical properties tests to ensure and guarantee its use. SIGNIFICANCE STATEMENT: The development of fully bioresorbable stents is a promising alternative for the management of coronary artery disease without causing long-term problems at the implantation site. In this work, the hematological and immunological biocompatibility of bare Mg modified superficially by plasma electrolytic oxidation (PEO-Mg) was evaluated by in vitro and ex vivo assays. PEO-Mg was found to be compatible with blood and immune components surrounding the implantation site with no signs of toxicity to endothelial cells, macrophages, and arterial tissue. In addition, degradation products of PEO-Mg are eliminated by phagocytosis. However, an in-depth study of the physical and mechanical properties and in vivo biocompatibility must be carried out for its future use as a biomedical implant.

Mesenchymal Stem Cells: Their Role in the Tumor Microenvironment.

Mesenchymal stem cells (MSCs) have been seen for years as great candidates for treating different diseases and an alternative to embryonic stem cells due to their differentiation capacity in vitro. More recent research has focused on their ability to modulate the immune response and regeneration at sites associated with inflammation, activities attributable to the release of trophic factors into the extracellular medium, a set of components known as the secretome. It has been possible to demonstrate the presence of these cells within the tumor microenvironment, which is associated with their tropism for sites of inflammation; however, their role here needs to be clarified. In different investigations, the feasibility of using MSCs or their secretome to treat cancer has been sought, with these results being ambiguous. It has been described that MSCs can be activated and present various phenotypes, which could explain the divergence in their action; however, these activation mechanisms and the different phenotypes still need to be well known. This review explores MSCs and their use in regenerative medicine with a targeted approach to cancer. Impact Statement This text addresses the diverging findings on the role of mesenchymal stem cells in the tumor microenvironment and discrepancies on the use of these cells as cancer treatment, separating the direct use of the cells from the use of the secretome. Multiple authors refer equally to the cells and their secretome to conclude on the positive or negative outcome, without taking into consideration how the cells are affected by their surroundings.

Polyplexes System to Enhance the LL-37 Antimicrobial Peptide Expression in Human Skin Cells.

Inefficient autologous tissue recovery in diverse skin injuries increases the susceptibility of patients to infections caused by multiresistant microorganisms, resulting in a high mortality rate. Nonviral transfection is an attractive alternative for these patients, where genetically modified cells incorporated into skin substitutes could release additional antimicrobial agents into the native skin. In this work, we have modulated the conditions of using a nonviral system for transfection of primary human keratinocytes and fibroblasts, consisting of a polymer/plasmid DNA (pDNA) complex called polyplex and its effects on the expression of LL-37 antimicrobial peptide. Linear and branched polyethylenimine (PEI) polymers in different weight concentrations were varied for evaluating the formation and colloidal characteristics of the polyplexes. The PEI/pDNA polyplexes with 19 nitrogen/phosphate ratio are nanometric particles (400 and 250 nm with linear and branched PEI, respectively) exhibiting positive surface (+30 ± 2 mV). Both kinds of polyplexes allowed the expression of a reporter gene and increased the human cathelicidin antimicrobial peptide gene expression in transfected keratinocytes and fibroblasts; however, greater cytotoxicity was observed when polyplexes formed with branched PEI were used. Moreover, cell culture supernatants from transfected cells with linear PEI/pDNA polyplexes showed enhanced antimicrobial activity (decrease of bacterial growth in 95.8%) against a Staphylococcus aureus strain in vitro. The study of the PEI/pDNA polyplexes formation allowed us to develop an improved transfection strategy of skin cells, promoting the production of LL-37 antimicrobial peptide. In the future, this strategy could be used for the construction of skin substitutes to prevent, reduce, or eliminate bacterial infections. Impact statement The results of this study contribute to the understanding of the polyplexes system in the genetic modification of skin cells and its effects on the expression of the LL-37 antimicrobial peptide. In the future, three-dimensional skin substitutes built with these cells could be an efficient way to decrease bacterial growth and prevent the infections in skin wounds.

Polyplex System Versus Nucleofection for Human Skin Cell Transfection and Effect of Internal Ribosome Entry Site Sequence.

Nonviral transfection has important implications on gene therapy because of its safety. In particular, polyfection and nucleofection are two widely used systems for nonviral gene delivery. Their potential depends on the transfection efficiency achieved, which is influenced in turn by the type of cells transfected and by the plasmid that carries the gene of interest. The efficiency of transfection by polyfection or nucleofection in human fibroblasts and keratinocytes was evaluated in this study. Transfections were performed with plasmids containing a gene of interest (human cathelicidin antimicrobial peptide) and two reporter genes (red or green fluorescent protein) that included or not an internal ribosome entry site (IRES). The efficiency was measured by flow cytometry in terms of percentage of cells expressing the reporter gene; viability of transfected cells was also evaluated. It was found that nucleofection was more efficient than polyplexes for transfecting fibroblasts, while no significant differences were found between both systems of transfection when applied to keratinocytes. Regarding the viability of fibroblasts after transfection, values were high in both systems. In contrast, keratinocytes were more sensitive to nucleofection. It was also noted that both types of cells decreased reporter gene expression when IRES sequence was located upstream of the reporter gene, suggesting a negative effect on the expression of this gene. These results confirm that the transfection efficiency depends on the type of cells and the system used.

Evaluación del método químico-enzimático de descelularización para la obtención de matrices extracelulares de tráquea en el modelo porcino / Avaliação do método químico-enzimático de descelularização para a obtenção de matrizes extracelulares de traqueia no modelo suíno / Evaluation of the chemical-enzymatic decellularization method for obtaining extracellular trachea matrixes in a porcine model

Diferentes enfermedades afectan la tráquea y deterioran la calidad de vida. La ingeniería tisular es una alternativa terapéutica para los pacientes con esas enfermedades: matrices de tráquea descelularizadas y sembradas con células del receptor no generan respuesta inmune y pueden prevenir incluso el rechazo de zootrasplantes. Objetivo: evaluar un método de descelularización para obtener matrices extracelulares de tráquea en el modelo porcino. Materiales y métodos: a partir de 5 tráqueas porcinas se formaron dos grupos de estudio, controles y tratados con un método químico-enzimático. Se hizo análisis histológico con hematoxilina-eosina, coloración tricrómica de Masson y safranina O. Se evaluaron las propiedades biomecánicas de ambos grupos, mediante la determinación del módulo de Young, la fuerza máxima y el porcentaje de deformación. Resultados: en las muestras tratadas se observó una disminución del 66 % del contenido celular en comparación con los controles. Se preservó el colágeno y se detectó reducción de los glucosaminoglucanos. Las pruebas biomecánicas revelaron una diferencia estadísticamente significativa del porcentaje de deformación, sin alteración de los demás parámetros. Conclusiones: el método evaluado demostró ser eficiente para descelularizar tráqueas de cerdo, con una disminución importante en el costo y el tiempo de tratamiento, por lo que podría ser una buena opción en las condiciones socioeconómicas de Colombia...

Different diseases may affect the trachea and, therefore, the quality of life. Tissue engineering may be a therapeutic alternative for patientswith such diseases, using decellularized trachea matrixes seeded with cells from the recipient, which do not generate immune response and may even prevent rejection of zoo-transplants. Objective: To evaluate a decellularization method to obtain tracheal extracellular matrixes in the porcine model. Materials and methods: Two study groups, treated and control, were obtained from 5 porcine tracheas.A chemical-enzymatic method for decellularization was used. Histological analyses were performed with hematoxylin-eosin, Masson’s trichromic stain and safranin O. Biomechanical properties of both groups were evaluated by determining the Young modulus, maximum strength and deformation rate. Results: Compared to the controls, there was a 66 % decrease in the cell content in the treated specimens. Collagen was preserved and a reduction of glycosaminoglycans was detected. Biomechanical tests revealed a significant difference in the percentage of deformation, with no alteration of the remaining parameters. Conclusions: The evaluated decellularization method proved to be efficient to reduce the cellular content of porcine tracheas, with a considerable decrease in cost and production time. These advantages could make it a good option for the socio-economic Colombian conditions...

Diferentes doenças afetam a traqueia e deterioram a qualidade de vida. A engenheira tisular é uma alternativa terapêutica para os pacientes com essas doenças: matrizes de traqueia descelularizadas e semeadas com células do receptor não geram resposta imune e podem prevenir incluso a rejeição de zoo-transplantes. Objetivo: avaliar um método de descelularização para obter matrizes extracelulares de traqueia no modelo suíno. Materiais e métodos: a partir de 5 traqueias suínas se formaram dois grupos de estudo, controles e tratados com um método químico-enzimático. Se fez análise histológico com hematoxilina-eosina, corante tricrómica de Masson e safranina O. Se avaliaram as propriedades biomecânicas de ambos grupos, mediante a determinação do módulo de Young, a força máxima e a porcentagem de deformação. Resultados: Nas amostras tratadas se observou uma diminuição de 66 % do conteúdo celular em comparação com os controles. Se preservou o colágenoe se detectou redução dos glucosaminoglucanos. As provas biomecânicas revelaram uma diferença estatisticamente significativa da porcentagem de deformação, sem alteração dos demais parâmetros. Conclusões: O método avaliado demostrou ser eficiente para descelularizar traqueias de porco, com uma diminuição importante no custo e o tempo de tratamento, pelo que poderia ser uma boa opção nas condições socioeconómicas da Colômbia...

Tratamiento de la enfermedad arterial periférica de las extremidades inferiores con células mononucleares de médula ósea autólogas: reporte de seguimiento a un año / Experience of autologous bone marrow mononuclear cells implantation as a treatment in patients with peripheral arterial disease: one year follow-up

Introducción: las células mononucleares de la médula ósea son efectivas para el tratamiento de pacientes con enfermedad arterial periférica (EAP). Los factores de crecimiento que ellas sintetizan se usan para inducir angiogénesis. Objetivo: establecer en Colombia la seguridad, factibilidad y resultado del tratamiento de la EAP utilizando células autólogas, para evitar su progreso a estadios más avanzados. Métodos: previo consentimiento, se hizo aspirado de médula ósea bajo anestesia local. Las células obtenidas se concentraron y se inyectaron intramuscularmente en los gastrocnemios de las extremidades isquémicas. El grupo control retrospectivo no recibió células. La evaluación clínica incluyó varios parámetros. Las células se analizaron por citometría de flujo. Resultados: edad media de los pacientes: 69 ± 11 años; viabilidad celular: 99,15% ± 0,76%; número de células inyectadas: 9,2 x 108 ± 6,2 x 108. Los análisis angiográficos postratamiento mostraron formación de vasos colaterales nuevos en las extremidades afectadas, con mínimo engrosamiento. Se observaron aumento en la distancia caminada libre de dolor y mejoría del dolor en reposo. La obtención y aplicación de las células no se asoció con ninguna complicación. Conclusiones: este estudio preliminar demostró que la terapia celular autóloga es segura, factible y cambia positivamente la historia natural de la EAP. Se sugiere el estudio de un número mayor de pacientes antes de establecer este tratamiento en Colombia.

Introduction: Autologous bone marrow mononuclear cells have been shown to be safe and effective for treatment of patients with peripheral arterial disease (PAD). Angiogenesis can also be induced by growth factors synthesized by them. Objective: To determine in Colombia the feasibility, safety and outcome of the afore-mentioned treatment. Methods: After informed consent, bone marrow was obtained by aspiration under local anesthesia; mononuclear cells were concentrated and their number and viability were established. They were suspended in saline solution and implanted by intramuscular injection into the gastrocnemius muscles of ischemic legs. Control patients were left untreated. Clinical evaluation included several parameters. Flow cytometry was used for cell analysis. Results: Mean age of patients: 69 ± 11 years; cell viability: 99.15 ± 0.76%; total number of injected cells: 9.2 x 108 ± 6.2 x 108. After treatment, angiographic studies showed the formation of new collateral vessels in all patients, with minimal thickness increase. There were no complications from bone marrow aspiration and intramuscular administration of cells. All treated patients experienced increase in the walking distance and improvement of rest pain. Conclusions: These preliminary results demonstrate that autologous cell therapy is safe, feasible and positively changes the natural history of patients with advanced peripheral arterial disease. In order to establish this treatment as a current practice in Colombia, we suggest the study of a larger number of patients.

Sustitutos cutáneos desarrollados por ingeniería de tejidos / Skin substitutes developed by tissue engineering

La piel es un tejido complejo vulnerable a procesos que alteran su estructura, integridad y funcionalidad como, por ejemplo, quemaduras, heridas crónicas y diversas enfermedades congénitas. Los avances tecnológicos en la fabricación de biomateriales y en el cultivo de células han permitido la producción de sustitutos cutáneos que han sido una alternativa terapéutica para algunas de estas complicaciones. Esta revisión pretende actualizar los aspectos generales, composición, perspectivas futuras y de aplicación de los principales sustitutos cutáneos que se ofrecen actualmente en el mercado internacional. Igualmente, presentará algunas experiencias del Grupo de Ingeniería de Tejidos y Terapias Celulares (GITTC) de la Facultad de Medicina de la Universidad de Antioquia.

The skin is a complex tissue vulnerable to different processes that may alter its structure, integrity and functionality, among them: burns, chronic wounds and various congenital diseases. Technological advances in biomaterials manufacture and cell culture have allowed the production of skin substitutes thus providing an alternative therapy for some of these complications. This review aims to update the general aspects, composition, future prospects and implementation of the most common skin substitutes currently available in the international market. Some experiences of the Tissue Engineering and Cellular Therapy Group (GITTC), at the University of Antioquia, Faculty of Medicine, in Medellín, Colombia, are also presented.